Halophilic Microorganisms and Their Environments

HALOPHILIC MICROORGANISMS AND THEIR ENVIRONMENTS

Cellular Origin and Life in Extreme Habitats Volume 5

Series Editor: Joseph Seckbach Hebrew University of Jerusalem, Israel

Halophilic Microorganisms and their Environments by

Aharon Oren The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, Israel

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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0-306-48053-0 1-4020-0829-5

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DEDICATION This book is dedicated to the memory of Donn J. Kushner, one of the founding fathers of halophile microbiology, who passed away on September 15, 2001, at the age of 74. My first meeting with Donn took place at the 1983 ASM meeting in New Orleans. At the time I was a newcomer in the field of halophile science, and I clearly remember how thrilled I was to meet one of the 'big' names in the field. It quickly appeared that we shared not only an interest in science, but in music as well. Donn was an accomplished player of the violin and the viola. We have since performed chamber music together at scientific meetings in Jerusalem (1987), in Alicante (1989, together with Morris Kates playing the violin and Masamichi Kohiyama on 'cello), in Williamsburg (1992, again with Morris Kates' violin and Larry Hochstein's clarinet), and once more in Jerusalem (1997, again joined by Larry Hochstein). I will always remember these informal concerts with colleague halophile scientists as highlights in my career. I hope that the following pages will form a fitting tribute to Donn’s many contributions to our understanding of halophilic microorganisms.

Aharon Oren

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TABLE OF CONTENTS DEDICATION

v

TABLE OF CONTENTS

vii

FOREWORD BY JOSEPH SECKBACH

xiii

PREFACE

xv

COMMENTS ON PROKARYOTE NOMENCLATURE AND SALT CONCENTRATION UNITS

xix

SECTION 1. AN HISTORICAL SURVEY

1

CHAPTER 1. HALOPHILIC MICROORGANISMS IN THEIR NATURAL ENVIRONMENT AND IN CULTURE - AN HISTORICAL INTRODUCTION 1.1. Red brines - early observations and explanations 1.2. The first records of the isolation of halophilic Archaea and Bacteria 1.3. Dunaliella and other halophilic algae 1.4. Ecological studies: the Great Salt Lake 1.5. Alkaline soda lakes 1.6. The Dead Sea 1.7. The study of biogeochemical processes in hypersaline environments 1.8. The beginning of the modern era of halophile research 1.9. References

3

8 9 10 11

SECTION 2. HALOPHILIC MICROORGANISMS AND THEIR PROPERTIES

19

INTRODUCTION

21

CHAPTER 2. TAXONOMY OF HALOPHILIC MICROORGANISMS: ARCHAEA, BACTERIA AND EUCARYA 2.1. The place of halophiles within the microbial world 2.2. The halophilic Archaea 2.3. The halophilic and halotolerant Bacteria 2.4. The halophilic and halotolerant Eucarya 2.5. References

23

vii

3 5

11 13 14

23 25 36 56 57

viii CHAPTER 3. THE CELLULAR STRUCTURE OF HALOPHILIC MICROORGANISMS 3.1. Cellular structures of halophilic Archaea 3.2. Cellular structures of halophilic Bacteria 3.3. Cellular structrues of halophilic Eucarya 3.4. References CHAPTER 4. CELLULAR METABOLISM AND PHYSIOLOGY OF HALOPHILIC MICROORGANISMS 4.1. Physiology of halophilic Archaea 4.2. Physiology of halophilic Bacteria 4.3. Physiology of halophilic Eucarya 4.4. Metabolic diversity among the halophiles: a bioenergetic approach 4.5. References CHAPTER 5. PIGMENTS OF HALOPHILIC MICROORGANISMS

5.1. Algal carotenoids 5.2. Pigments of oxygenic and anoxygenic photosynthetic Bacteria 5.3. Carotenoids of aerobic heterotrophic Archaea and Bacteria 5.4. The retinal pigments: bacteriorhodopsin, halorhodopsin, and sensory rhodopsins 5.5. The photoactive yellow protein of Halorhodospira and other halophilic purple bacteria 5.6. References

69 69 99 110 111 125 125 146 152 153 157 173 174 178 179 183 196 198

CHAPTER 6. INTRACELLULAR SALT CONCENTRATIONS AND ION METABOLISM IN HALOPHILIC MICROORGANISMS 6.1. Introduction 6.2. Methods used to estimate intracellular ionic concentrations in halophilic microorganisms 6.3. Ion metabolism in the Halobacteriaceae 6.4. Ion metabolism in aerobic halophilic Bacteria 6.5. Ion metabolism in the Halanaerobiales 6.6. Ion metabolism in Dunaliella 6.7. References

207

CHAPTER 7. PROPERTIES OF HALOPHILIC PROTEINS 7.1. Introduction 7.2. Halophilic behavior of enzymes from halophilic Archaea 7.3. Purification of halophilic proteins 7.4. Salt relationships of selected proteins from halophilic Archaea

233 233 235 240 249

207 208 210 214 223 223 227

ix 7.5. Halophilic behavior of enzymes from the aerobic halophilic Bacteria 7.6. Halophilic behavior of enzymes from the anaerobic halophilic Bacteria 7.7. Halophilic behavior of enzymes from the halophilic Eucarya 7.8. References

253 264 264 267

CHAPTER 8. ORGANIC COMPATIBLE SOLUTES 8.1. Organic osmotic solutes and their distribution 8.2. Compatible solutes in the domain Archaea 8.3. Compatible solutes in the domain Bacteria 8.4. Compatible solutes in the domain Eucarya 8.5. The mode of action of compatible solutes 8.6. References

279 279 284 286 294 297 299

CHAPTER 9. HALOPHILIC BACTERIOPHAGES AND HALOCINS 9.1. Bacteriophages of halophilic microorganisms 9.2. Halocins 9.3. References

307 307 316 319

CHAPTER 10. GENETICS AND GENOMICS OF HALOPHILIC ARCHAEA AND BACTERIA 10.1. Genetics of halophilic microorganisms - an historical survey 10.2. Genetics of halophilic Archaea 10.3. Genetics of halophilic Bacteria 10.4. References

323 323 324 343 348

CHAPTER 11. BIOTECHNOLOGICAL APPLICATIONS AND POTENTIALS OF HALOPHILIC MICROORGANISMS 11.1. Introduction 11.2. Applications of halophlic Archaea 11.3. Applications of halophlic Bacteria 11.4. Applications of halophlic Eucarya 11.5. References

357

SECTION 3.

391

HYPERSALINE ENVIRONMENTS AND THEIR BIOTA

357 359 367 375 380

INTRODUCTION

393

CHAPTER 12. GREAT SALT LAKE, UTAH 12.1. The lake and its setting 12.2. The microbial communities of the Great Salt Lake 12.3. Microbial isolates and their properties 12.4. Biogeochemical processes 12.5. References

395 395 400 407 409 415

x CHAPTER 13. THE DEAD SEA 13.1. The lake and its setting 13.2. Early studies on the biota of the Dead Sea 13.3. Dynamics of microbial blooms in the Dead Sea 13.4. Microbial isolates and their properties 13.5. Adaptations of the Dead Sea biota to the ionic composition of the lake 13.6. The nature of the species dominant in the archaeal blooms 13.7. Anaerobic processes in the Dead Sea sediments 13.8. Biology of the Dead Sea - expected future developments 13.9. References

419 419 423 424 431

CHAPTER 14. SOLAR SALTERNS 14.1. The saltern environment and its biota 14.2. Benthic microbial mats in the evaporation ponds 14.3. Microbial isolates and their properties 14.4. Approaches toward the identification of the dominant Archaea in saltern crystallizer ponds 14.5. Salinibacter and other halophilic Bacteria in saltern ponds 14.6. The red pigments in salten crystallizer ponds 14.7. Dynamics of archaeal and bacterial communities in saltern ponds 14.8. The importance of the saltern biota in the production of solar salt 14.9. References

441 441 444 448

CHAPTER 15. ALKALINE HYPERSALINE LAKES IN AFRICA AND ASIA 15.1. The Wadi Natrun lakes 15.2. Lake Magadi and other East-African soda lakes 15.3. Hypersaline soda lakes in Asia - chemical and biological characteristics 15.4. References

471 472 478

CHAPTER 16. MONO LAKE, CALIFORNIA, AND BIG SODA LAKE, NEVADA 16.1. Hypersaline lakes in the Great Basin of North America 16.2. Mono Lake, California 16.3. Big Soda Lake, Nevada 16.4. References

495 495 496 508 512

433 434 434 435 436

451 453 454 455 459 462

489 491

xi CHAPTER 17. MISCELLANEOUS HABITATS OF HALOPHILIC MICROORGANISMS – FROM ANTARCTIC LAKES TO HYDROTHERMAL VENTS 17.1. Cold and hypersaline: Antarctic hypersaline lakes 17.2. Hot and hypersaline: Solar Lake (Sinai) and other warm brines 17.3. Seawater, deep sea brines and hydrothermal vents 17.4. Halophilic microorganisms in oil field brines 17.5. Halophiles in salt marshes, hypersaline lagoons and miscellaneous lakes 17.6. Hypersaline springs 17.7. Hypersaline soils 17.8. Wall paintings 17.9. Desert plants and animals 17.10. References

517 517 519 525 528 528 531 532 532 533 533

SECTION 4. EPILOGUE

541

CHAPTER 18. EPILOGUE: EVOLUTION OF HALOPHILES AND SURVIVAL OF HALOPHILES ON EARTH AND IN SPACE 18.1. The evolutionary origin of halophiles 18.2. Long-term survival of halophiles in ancient salt crystals 18.3. Halophiles in space? 18.4. References

543

SECTION 5. SUPPLEMENT

553

METHODS FOR CULTIVATION AND HANDLING OF HALOPHILIC ARCHAEA AND BACTERIA

555

GLOSSARY OF LIMNOLOGICAL TERMS

559

ABOUT THE AUTHOR

561

ORGANISM INDEX

563

GEOGRAPHICAL INDEX

567

SUBJECT INDEX

571

543 545 548 549

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FOREWORD

"This water" he told me, "runs out to the eastern region, and flows into the Arabah; and when it comes into the sea, into the sea of foul waters [i.e., the Dead Sea], the water will become wholesome. Every living creature that swarms will be able to live wherever this stream goes; the fish will be very abundant once these waters have reached there. It will be wholesome, and everything will live wherever this stream goes. Fishermen shall stand beside it all the way from En-gedi to En-eglaim; it shall be a place for drying nets; and the fish will be of various kinds [and] most plentiful, like the fish of the Great Sea." Ezekiel’s prophecy (Ezekiel 47: 8-10) for revival and purification of the Dead Sea waters

This new book on "Halophilic Microorganisms and their Environments" is the fifth volume in the COLE series (Cellular Origin and Life in Extreme Habitats (see: http://www.wkap.nl/prod/s/COLE). In the previous books we covered aspects of enigmatic microorganisms, microbial diversity, astrobiology, and symbiosis, so this book on halophilic microbes adds a fitting link to the rest of series' books. Since ancient times hypersaline habitats have been considered extreme environments, and some were thought not to sustain life at all. Yet, every organism requires salt for its existence. Salty places have been compared to an environment of extinction (e.g., the Dead Sea). In the Bible, Lot's wife was converted into a pillar of salt as a punishment for disobeying the request to not glance backward (Genesis 19:26). Moses warned the people that: "all its soil devastated by sulfur and salt, beyond sowing and producing, no grass growing in it, just like the upheaval of Sodom and Gomorrah...which the Lord overthrew in His fierce anger" (Deuteronomy 29:22). Abimelech razed the town of Shechem and as an act of desolation he sowed it with salt in order to punish that rebellious city (Judges 9:45). But, in the Bible too, salt also has some curative power, as the prophet Elisha cures bad water by pouring salt into it (Kings II 2:19-22). The prophet Ezekiel recognizes the universal need for salt and promises that some areas (following the purification of the Dead Sea – see above) will still remain saline (Ezekiel 47:11). Hypersaline habitats like the Dead Sea have been considered a "dead" environment devoid of life. Over a half century ago, however, Ben Volcani (1936) demonstrated that the Dead Sea is rather alive and sustains active life of microorganisms. Since then, our knowledge of the biota of the Dead Sea and of other hypersaline environments has increased tremendously. This current volume enriches our information about various aspects of the hypersaline world.

xiii

xiv This volume describes the highly diverse groups of microorganisms growing in hypersaline environments all over the earth. Topics covered include taxonomy, physiology, ecology, osmoregulation, as well as other properties of halophiles. Special chapters are devoted to the hypersaline lakes such as the Dead Sea (between Israel and Jordan) and the Great Salt Lake (Utah, USA), both as halo-extremophilic environments. This book is addressed to researchers and students in the fields of microbiology, marine biology, ecological biology, workers in the field of extreme environments, and to every "knowledge-thirsty" reader interested in biology. The author, Aharon Oren, a well-known expert in the area of halophilic microorganisms, took up the challenge to review the halophiles. He is an active board member of this series. On this broad subject he has published numerous journal articles and contributed several chapters to books. Dr. Oren organized an international research workshop on "Microbiology and Biogeochemistry of Hypersaline Environments" (1997) and edited its proceedings (1999). Other volumes in this series "Cellular Origin and Life in Extreme Habitats" contain additional chapters on the halophiles and the Dead Sea, first in Enigmatic Microorganisms and Life in Extreme Environments (http://www.wkap. nl/prod/b/0-79235492-3) and then in Journey to Diverse Microbial Worlds (http://www.wkap.nl/prod/b/0-7923-6020-6). In these lines, I wish to thank the author, Professor Oren, for his swift completion of the project of writing this book, and his full cooperation with the editor. Much appreciation is due to the Kluwer Academic Publishers team and specifically to Mrs. Claire van Heukelom and Dr. Frans van Dunne for their constant interest, collaboration and assistance in the "making" of this new volume. Last but not least, I am grateful to my wife, Fern Seckbach for encouragement to proceed with this book series. The Hebrew University of Jerusalem [email protected] Chief Editor of COLE Book Series April 2002

Joseph Seckbach

PREFACE

How surprising it is that any creatures should be able to exist in a fluid, saturated with brine, and that they should be crawling among crystals of sulphate of soda and lime! (Charles Darwin, 1839).

Halophilic microorganisms - organisms that prefer to take their habitat "cum grano salis", with a grain of salt - inhabit hypersaline environments with salt concentrations up to NaCl saturation. Halophiles are found in all three domains of life: Archaea, Bacteria and Eucarya. They may occur in large numbers in hypersaline lakes and in other habitats characterized by salt concentrations approaching saturation, and they often have a strong impact on the ecosystems in which they thrive. Halophilic microorganisms also present the biochemist/physiologist with interesting questions on how organisms cope with the high osmotic pressures exerted by the highly salty medium. The study of the halophiles has shown that the microbial world has developed a variety of adaptive strategies to enable growth in brines that are hostile to other forms of life. I was first introduced to the fascinating world of the halophiles in 1980, when the late Prof. Moshe Shilo invited me to join a study of the biology of the Dead Sea. In fact I had worked with halophilic microorganisms before: my Ph.D. thesis work dealt with physiological and biochemical aspects of a cyanobacterium named Oscillatoria limnetica, isolated from the hypersaline Solar Lake, Sinai (see also Section 17.2). I used to grow this organism at a salt concentration twice that of seawater, but it tolerates much higher salinities. At the time, however, I did not devote much thought to aspects of its salt tolerance, salt requirement and salt adaptation. The variety of halophiles encountered in high numbers in the Dead Sea in 1980, a year in which unusually dense blooms of the alga Dunaliella and of red halophilic Archaea developed in the lake (see Chapter 13), aroused my interest in these forms of life and in the ways they cope with the challenges presented by their environment. The biology of halophilic microorganisms has been the subject of a number of earlier monographs. These include the two-volume "Halophilic Bacteria" edited by Francisco Rodríguez-Valera (CRC Press, 1988), and "The Biology of Halophilic Bacteria", edited by Russ Vreeland and Larry Hochstein (CRC Press, 1993). There are also a number of books published as meeting proceedings: "Energetics and Structure of Halophilic Microorganisms", edited by Roy Caplan and Margaret Ginzburg (Elsevier, 1978), General and Applied Aspects of Halophilic Microorganisms (edited by Francisco Rodríguez-Valera, Plenum Press, 1991), and "Microbiology and Biogeochemistry of Hypersaline Environments", a book I edited as the proceedings of a symposium on

xv

xvi halophilic microorganisms I had the pleasure of organizing in 1997 (CRC Press, 1999). These volumes all give a good overview of many aspects of the life of halophilic microorganisms. A number of excellent review articles and chapters have also been written in the course of the years. The comprehensive overview of all aspects of the ecology, physiology and biochemistry of halophiles presented by Donn Kushner in 1978 in a 52-page chapter in the book he edited on "Microbial Life in Extreme Environments" (Academic Press), a chapter that beautifully summarized the knowledge available at the time, is still relevant today. The writing of this book was inspired to a large extent by the exemplary monograph written by Barbara Javor on "Hypersaline Environments - Microbiology and Biogeochemistry" (Brock/Springer, 1989). That book covered the state of the art of halophile microbiology at the time, with a strong emphasis on the biogeochemical aspects, but without neglecting in-depth information on the taxonomy, physiology and biochemistry of those organisms that inhabit hypersaline environments. I can only hope that the present volume will provide an overview of the current state of the science as thorough and as well-balanced as Javor's book. Tremendous progress has been made in the last decades. The greatest breakthrough in the past few years came of course from the field of genomics. The first complete genome sequence of a halophile was published in 2000; more such sequences are expected to become available in the near future. New questions will be asked and deeper insights will be obtained than thought possible until even a few years ago. To some extent this volume presents an overview of halophilic science in the "pregenomic" era, while providing glimpses of the first exciting results obtained from whole genome analyses. With all the advances in the understanding of the properties of individual model organisms we should not forget the place of the halophilic microorganisms in their natural habitats. Also here significant breakthroughs have been made in recent years, especially thanks to the application of methods derived from molecular biology, including culture-independent techniques that allow us to obtain information on the structure of the microbial community. This book contains four sections. It opens with a historical overview, showing how the ideas about the nature of the halophilic microorganisms have evolved over the centuries. The second section provides an overview of our understanding of the halophilic microorganisms themselves, including aspects of their taxonomy, physiology, genetics, and biotechnological applications. The third section is ecosystem-centered. A number of representative hypersaline environments are discussed and information is reviewed on their community structure and on the activities of the different halophilic microorganisms. The volume concludes with some thoughts about the evolution of halophiles and their longevity, addressing questions about the possibility of survival of halophilic prokaryotes within salt crystals for hundreds of millions of years, and the possible existence of halophiles elsewhere in the Universe. References to methods used for the cultivation and handling of the halophiles, as well as a glossary of limnological terms are given in the supplement.

xvii While preparing the chapters for this book the author was confronted with the question how to define a halophile and a hypersaline environment: how salty should an environment be and how much salt should be required or tolerated by a microorganism to be included in this volume? As microorganisms present us with a continuum of adaptations, from freshwater to salt saturation, and as many organisms easily adapt to a wide range of often rapidly changing salt concentrations, there is no obvious answer to such a question. Any attempt to classify the halophiles into categories according to their salt requirement and salt tolerance is to a large extent artificial, and many species defy classification within such schemes. I arbitrarily chose the value of salt as the boundary. I have attempted to include those environments in which the salt concentration exceeds salt and those microorganisms that are able to grow well above that value, even if their salinity optimum may sometimes be in the lower range. Mono Lake (California) and Big Soda Lake (Nevada) (Chapter 16) are borderline cases: with salt concentrations up to 98 and respectively, these lakes have salinities just below the above-defined boundary. However, both these lakes have many interesting properties, and their biota have been the subject of many in-depth studies. A discussion of their properties therefore appears warranted. Each chapter contains a large number of references. These cover not only the most recent articles relevant to the field, but I intentionally included citations of old papers, often going back to the first pioneering studies that opened up the field. In the course of the time I have been involved with the halophiles, I have come across many forgotten publications that demonstrate that earlier generations of scientists knew much more than we are likely to admit nowadays. Hopefully this book will therewith become a valuable source of references, including papers that have been published many decades or even more than a century ago. The large number of literature citations may give a false impression of completeness. Even lists of references twice as long as those presented here cannot cover the literature in full. This is especially true in the case of bacteriorhodopsin and the other retinal pigments of halophilic Archaea. A search for articles on bacteriorhodopsin in the ISI Web of Science, covering the period from 1988 to 2001, yielded over 3,500 entries. In such cases a rigid selection was needed. Special emphasis has then been placed on coverage of those papers published in the early days, during which the function of the pigment has become clear, and on the major breakthroughs achieved in recent years that have led to an exact picture of the functioning of the retinal proteins. I thus hope that the book provides a fair coverage of all relevant aspects of the biology of halophilic microorganisms and our current understanding of what halophilic microorganisms are and how they function in nature. I have attempted to include all relevant material that was published before the end of January 2002. A book of this scope and size will undoubtedly contain omissions, inaccuracies and errors. Comments are welcome at all times. I have learned much while working on this book and I hope to learn even more from the comments by my colleagues.

xviii I want to thank Joseph Seckbach for his invitation to contribute a volume to his book series on "Cellular Origin and Life in Extreme Habitats". Thanks also to Claire van Heukelom and her colleagues at Kluwer Academic Publishers who helped me to produce this volume within a relatively short time. I finally would like to thank those colleagues who have reviewed the different chapters of this book and have suggested many corrections and improvements: Mike Dyall-Smith (Melbourne. Australia), Erwin Galinski (Bonn, Germany), Bill Grant (Leicester, UK), Nina GundeCimerman (Ljubljana, Slovenia), Tim Hollibaugh (Athens, GA, USA), Kjeld Ingvorsen (Aarhus, Denmark), Bob Jellison (Mammoth Lakes, CA, USA), Martin Kessel (Bethesda, MD, USA), Carol Litchfield (Manassas, VA, USA), Dominique Madern (Grenoble, France), Volker Müller (München, Germany), Dieter Oesterhelt (Martinsried, Germany), Ron Oremland (Menlo Park, CA, USA), Francisco RodríguezValera (Alicante, Spain), and Russ Vreeland (West Chester, PA, USA).

Jerusalem, April 2002 Aharon Oren

The Institute of Life Sciences, and the Moshe Shilo Minerva Center for Marine Biogeochemistry The Hebrew University of Jerusalem E-mail address: [email protected]

COMMENTS ON PROKARYOTE NOMENCLATURE AND SALT CONCENTRATION UNITS

Taxonomy is a living science, and the nomenclature of microorganisms has seen many changes as our views on microbial taxonomy have developed. The increasing understanding of the phylogenetic relationships between different microorganisms, as enabled by 16S or 18S rRNA sequence analysis, has revolutionized microbial taxonomy in general, and the taxonomy of prokaryotes in particular. As a result, many names of halophilic microorganisms, Archaea as well as Bacteria, have changed over the years. To give a few examples: some Archaea formerly classified in the genus Halobacterium have later been transferred to genera such as Haloferax, Haloarcula, and Halorubrum. Moreover, the former species Halobacterium salinarium, Halobacterium halobium, and Halobacterium cutirubrum have been unified into a single species, Halobacterium salinarum. In the bacterial domain, Vibrio costicola became Salinivibrio costicola. The Dead Sea isolate Ba1, an organism that has been the subject of many physiological studies, was first named Halomonas israelensis, and has recently been renamed Chromohalobacter israelensis. To further complicate the matter, an isolate (DSM 3043 = ATCC 33174) formerly classified as a strain of Halomonas elongata has now been removed from the genus Halomonas and renamed Chromohalobacter salexigens. This particular strain as been used in a large number of genetic studies, and so has the type strain of Halomonas elongata. A considerable extent of confusion may result from such nomenclatural changes. This book has attempted to use the currently approved nomenclature, as given in the lists that periodically appear in the International Journal of Systematic and Evolutionary Microbiology ("Notification that new names and new combinations have appeared in volume …, part …, of the IJSEM", and "Validation of publication of new names and new combinations previously effectively published outside the IJSEM"), even when this may lead to some confusion. The prokaryote nomenclature web site ("List of bacterial names with standing in nomenclature") maintained by Jean Euzéby (www.bacterio.cict.fr) is also a reliable source of updated information. Nomenclatural information, including former species designations (basonyms), has been included in the description of the different halophilic microorganisms in Chapter 2. When performing experiments with different species of Bacteria or Archaea, the importance of the proper designation of the strain used and the desirability of using the designated type strain whenever possible cannot be sufficiently stressed. Communication between physiologists, molecular biologists and geneticists at one side and taxonomists on the other side is often poor or altogether absent. There are still too many articles, even those published in recent years, in which the source of the strain used was not clarified beyond statements such as: "Halobacterium halobium was a gift from colleague xyz". The case of Halobacterium salinarum - Halobacterium salinarium - Halobacterium halobium - Halobacterium cutirubrum is a particularly

xix

xx interesting one. Complete genome sequences of two strains (Halobacterium strain NRC-1 and Halobacterium salinarum [halobium] strain R1) are now available – at the time of writing the only genomes of halophilic Archaea sequenced – and none of these is the type strain of the species. In fact, the source of strain NRC-1 is obscure. The authors of the publication describing the genome (Ng et al., 2000) state: "The precise relationships among [H. halobium. H. cutirubrum, H. salinarium, and H. salinarum] and Halobacterium sp. strain NRC-1 are not entirely clear. Strain NRC-1 was a gift from W.F. Doolittle, Dalhousie University, Halifax, Canada. The strain has been deposited with the American Type Culture Collection, Manassas, VA (reference no. ATCC 700922)." Halobacterium strain R1 is a mutant laboratory strain often used in physiological experiments. Tindall (1992) has presented an excellent overview of the source and history of many of the old Halobacterium isolates that circulate in culture collections and laboratories. In this book all isolates designated Halobacterium salinarium, Halobacterium halobium or Halobacterium cutirubrum, including the strains NRC-1 and R1, have been named Halobacterium salinarum. Details of the particular strain used or its culture collection accession number (if available at all) have not been specified in most cases. For additional details on the identity and the source of the strain used readers should therefore refer to the original publications as cited. There are probably even cases in the literature in which strains designated as "Halobacterium sp." may not actually belong to that genus. Caution should therefore be exerted when comparing data published on "Halobacterium salinarum". In the case of the cyanobacteria matters of nomenclature are also exceedingly confusing. There are many cases in which the same organism has become known under two, three and more different names in the literature. More information on the nomenclatural problems with halophilic and halotolerant cyanobacteria can be found in Chapter 2 and in a book chapter on cyanobacteria in hypersaline environments (Oren, 2000). Taxonomic issues dealing with the halophilic Archaea of the family Halobacteriaceae are discussed by the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halobacteriaceae. Such a subcommittee also exists for the photosynthetic prokaryotes, a group that also contains a considerable number of halophiles. A subcommittee that will deal with the taxonomy of the Halomonadaceae is expected to become established in the near future. The minutes of the meetings of these subcommittees, as published in the International Journal of Systematic and Evolutionary Microbiology (see e.g. Oren and Ventosa, 2002) contain much relevant information on the taxonomy and nomenclature of halophiles. In this book the word "Bacteria" (with a capital B) is reserved for describing organisms in a phylogenetic sense, i.e. as members of the domain Bacteria. In contrast, the word "bacteria" is used to refer to prokaryotes in general, both Bacteria and Archaea. Salt concentrations can be expressed in a variety of units. These include molar units , molal units , weight per volume - as % salt = g per 100 ml solution or or on a weight per weight basis - as salinity (g per kg). While oceanographers and limnologists generally prefer the salinity unit to describe the salt

xxi concentrations of saline and hypersaline water bodies, the composition of growth media is nearly always given in units of gram or mol salt per liter. While reviewing the literature during the preparation of this book it became clear that the term "salinity" is often (mis)used to describe salt concentrations in terms of grams of salt per liter rather than grams of salt per kg. Especially at high salt concentrations these terms are not equivalent. Rather than attempting to elucidate what units had been used in each article reviewed, I have assumed that the term salinity when expressed as % or referred to salt concentrations in units of gram per 100 ml or gram per liter, respectively. Errors may possibly have been introduced while doing so. To achieve consistency throughout the book I have converted salt concentration data to units of as much as possible. Only in Chapters 6-8, where the concentrations and the effects of different ions are compared, have I used molar units, and occasionally also molal units.

REFERENCES Ng, W.V., Kennedy, S.P., Mahairas, G.G., Berquist, B., Pan, M., Shukla, H.D., Lasky, S.R., Baliga, N.S., Thorsson, V., Sbrogna, J., Swartzell, S., Weir, D., Hall, J., Dahl, T.A., Welti, R., Goo, Y.A., Leithauser, B., Keller, K., Cruz, R., Danson, M.J., Hough, D.W., Maddocks, D.G., Jablonski, P.E., Krebs, M.P., Augevine, CM., Dale, H., Isenberger, T.A., Peck, R.F., Pohlschroder, M., Spudich, J.L., Jong, K.-H., Alam, M., Freitas, T., Hou, S., Daniels, C.J., Dennis, P.P., Omer, A.D., Ebhardt, H., Lowe, T.M., Liang, P., Riley, M., Hood, L., and DasSarma, S. 2000. Genome sequence of Halobacterium species NRC-1 Proc. Natl. Acad. Sci. USA97: 12176-12181. Tindall, B.J. 1992. The family Halobacteriaceae, pp. 768-808 In: Balows, A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer, K.-H. (Eds.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. Vol. I. Springer-Verlag, New York. Oren, A. 2000. Salts and brines, pp. 281-306 In: Whitton, B.A., and Potts, M. (Eds.), Ecology of cyanobacteria: their diversity in time and space. Kluwer Academic Publishers, Dordrecht. Oren, A., and Ventosa, A. 2002. International Committee on Systematics of Prokaryotes. Subcommittee on the Taxonomy of Halobacteriaceae. Minutes of the Meetings, 24 September 2001, Sevilla, Spain. Int. J. Syst. Evol. Microbiol. 52: 289-290.

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SECTION 1

AN HISTORICAL SURVEY

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CHAPTER 1 HALOPHILIC MICROORGANISMS IN THEIR NATURAL ENVIRONMENT AND IN CULTURE - AN HISTORICAL INTRODUCTION

An embankment is made and ditches to draw clear sea water. It is left for a long time until the color becomes red. If the south wind blows with force during the summer and autumn the salt may grain over night. If the south wind does not come all the profits are lost.

This translation from the Peng-Tzao-Kan-Mu, a work that may date back to about 2,700 B.C., as presented by Baas-Becking (1931), describes salt production from sea water in the Chinese province of Yai-cheau. This quotation is one of the few early reports of the occurrence of red brines, now known to be caused by dense communities of microorganisms adapted to life at salt concentrations at or approaching saturation. This chapter presents an historical overview of observations of halophilic life over many centuries, from the first descriptions of colored brines to the early attempts to obtain an understanding of the nature of the microorganisms that cause the red coloration of concentrated salt solutions and of other phenomena associated with the presence and activity of halophilic and halotolerant microorganisms.

1.1. RED BRINES – EARLY OBSERVATIONS AND EXPLANATIONS Red brines have been the subject of attention since ancient times. It has been suggested that the first Plague of Egypt (Exodus 7: 17-25), in which the water of the Nile turned into blood, refers to such red waters. Waters colored red still occur in the Wadi Natrun lakes in Egypt (Jannasch, 1957): Besonders auffallend war seit jeher die Rotfärbung von stehenden Gewässern meist höheren Salzgehaltes, die oft als Blutseen bezeichnet geworden sind. Auf einen derartigen Salzsee stießen wir während einer limnologischen Untersuchungsreise in Ägypten (1954), das schon in de mosaischen Berichten mit der Erscheinung des blutigen Wassers, eine der "sieben Plagen Ägyptens", genannt wird. [Especially prominent has since long been the red coloration of stagnant waters of mostly high salt content, that have often been designated as blood lakes. We arrived at such a salt lake in the course of our limnological expedition to Egypt, where the appearance of bloody waters, one of the seven [sic] Plagues of Egypt, had already been mentioned in the Mosaic Scriptures.]

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One of these alkaline hypersaline Wadi Natrun lakes is named "el-Hamrah" or "Hamara" (see also Section 15.1), or "the red" (Schweinfurth and Lewin, 1898): Hieran anschließsend sei noch erwähnt, daß gerade einer der Seen, der noch im letzten Jahrzehnt ausgebeutet wurde, "el-Hamrah", d.h. der rote benannt wird. [In this respect it should also be mentioned that precisely one of the lakes, which was still exploited in the last decade, is named "el-Hamrah", i.e. the red one.]

The biblical reference to red waters, possibly in the Dead Sea, in Kings II 3.22: And they rose up early in the morning and the sun shone upon the water, and the Moabites saw the water on the other side as red as blood. And they said, 'This is blood'

may as well refer to blooms of halophilic microorganisms (Bloch, 1976). Also in modern times the Dead Sea has seen blooms of red halophilic Archaea, sufficiently dense to impart a reddish color to the lake (Oren, 1983; Oren and Gurevich, 1995; see also Chapter 13). According to Bloch (1976) this passage may also refer to red salt pans that may have been operative near Sodom at the time. The mosaic map of the Dead Sea area at Madaba in Jordan, dating from about 550 A.D., shows two ships sailing on the Dead Sea, one loaded with a cargo of reddish material and one with a grey payload. One interpretation has been that the red cargo consisted of solar salt from the evaporation ponds, the whitish-grey material being rock salt from the quarry at Mount Sodom (Bloch, 1962, 1976; see also Oren and Litchfield, 1998). A truly admirable description of the colors of a salt lake, with a for the time surprisingly exact analysis of the causes of these colors, was given by Charles Darwin in his account of his travels on H.M.S. Beagle (1839). About his visit in the area of the mouth of the Rio Negro near the town of El Carmen in Patagonia in August 1833 he wrote: One day I rode to a large salt lake, or Salina, which is distant fifteen miles from the town. During the winter it consists of a shallow lake of brine, which in summer is converted into a field of snow-white salt. This layer near the margin is from four to five inches thick, but toward the centre its thickness increases. The lake was two and a half miles long, and one broad. Others occur in the neighbourhood many times larger, and with a floor of salt, two and three feet in thickness, even when under water during the winter. One of these brilliantly-white and level expanses, in the midst of the brown and desolate plain, offers and extraordinary spectacle. A large quantity of salt is annually drawn from the salina; and great piles, some hundred tons in weight, were lying ready for exportation. It is singular that the salt, although well crystallized, and appearing quite pure, does not anwer so well for preseving meat as sea salt from the Cape de Verd Islands. Although the latter is necessarily much dearer, it is constantly imported and mixed with the salt procured from these salinas. A merchant at Buenos Ayres told me that he considered the Cape de Verd salt worth fifty per cent. more than that from the Rio Negro. The season for working the salinas forms the harvest of Patagones; for on it, the prosperity of the place depends. Nearly the whole population encamps on the banks of the river, and the people are employed in drawing out the salt in bullock-wagons. The border of the lake is formed of mud: and in this numerous large crystals of gypsum, some of which are three inches long, lie embedded; whilst on the surface, others of sulphate of magnesia lie scattered about. The Gauchos call the former the "Padre del sal, " and the latter the "Madre"; they state that these progenitive salts always occur on the border of the salinas, when the water begins to evaporate. The mud is black, and has a fetid odour. I could not, at first, imagine the cause of this, but I afterwards perceived that the froth, which

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the wind drifted on shore was coloured green, as if by confervæ: I attempted to carry home some of this green matter, but from an accident failed. Parts of the lake seen from a short distance appeared of a reddish colour, and this, perhaps, was owing to some infusorial animalcula. The mud in many places was thrown up by numbers of some kind of annelidous animal. How surprising it is that any creatures should be able to exist in a fluid, saturated with brine, and that they should be crawling among crystals of sulphate of soda and lime! And what becomes of these worms when, during a long summer, the surface at least is hardened into a solid layer of salt? Flamingoes in considerable numbers inhabit this lake; they breed here, and their bodies are sometimes found by the workmen, preserved in the salt. I saw several wading about in search of food,- probably for the worms which burrow in the mud; and these latter, perhaps, feed on infusoria or confervæ. Thus we have a little world within itself, adapted to these little inland seas of brine.

More than sixty years passed since Darwin's account until Schweinfurth and Lewin (1898) presented a description of the biology of a salt lake as detailed as the one above! It is in fact surprising that the red coloration of saltern crystallizer ponds is not mentioned much more often in the older literature. Throughout the many recorded descriptions of the technology of salt production (see e.g. Figure 1) there have been only scattered references to changes in the color of the salines, and only occasionally have writers referred to the importance of this color for the successful economic production of salt. Thus, the medieval descriptions of French and Italian solar salt production facilities do not mention any red coloration. It has been suggested that it was such an accepted fact that it was not considered worthy of mention (Litchfield, 1991). The red color of salt-saturated brines was, however, mentioned in the Encyclopédie of Diderot and d'Alembert (Diderot, 1765): On connaît que le sel se forme quand l’eau rougit; c’est en cet état qu’étant réchauffé par le soleil & par le vent, il se crême de l’épaisseur du verre: alors on le casse, il va au fond. [One knows that the salt is forming when the water turns red; it is in this state that, when being heated by the sun and by the wind, it has a surface layer the thickness of glass. When one breaks it, it sinks to the bottom.]

Additional reports on red brines are found in the ancient Chinese literature. One example was given in the beginning of this chapter. Another such record, dating from the seventeenth century states (Ying-Hsing, 1966): This step of the operation (letting in the brine) should be done in the spring. After a period the brine will turn red, until late summer or early autumn when the south wind blows hard across this area, so that the salt crystallizes overnight.

1.2. THE FIRST RECORDS OF THE ISOLATION OF HALOPHILIC ARCHAEA AND BACTERIA There is an account, dating from 1771, of the development of a red color on meat salted with salt from Tripoli (Lybia), possibly due to the use of salt contaminated with red halophilic Archaea (Monro, 1771): There are great mines of sea salt in the country of Tripoli, the salt of which should seem to contain a large proportion of this natron; for, I am told, that all the meat salted with it acquired a red colour.

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The first definitive description of red bacteria in brines, now known to belong to the archaeal domain, was probably made in 1914 by Pierce. By that time the reddening of salted hides and salted cod had become an important economic issue in North America. It had already been known for hundreds of years that foods salted with "bay salt" (obtained by solar evaporation of sea water) often became red and spoiled. It may be assumed that the allegedly low quality of the salt produced in the Patagonian saltern described by Darwin – see above – was also due to a high content of such spoilage bacteria. Pierce isolated red bacteria which would grow in saturated salt solutions, and he correctly attributed the green and reddish colors of the concentrator ponds of the San Francisco salt works to the presence of algae and brine shrimp(Artemia) and the red coloration in the crystallizer basins to the massive appearance of bacteria. The first accurate description of Halobacterium was made by Klebahn (1919), who isolated "Bacillus halobius ruber" from red discolorations of salted fish. Unfortunately, none of Klebahn's isolates have survived. Subsequently, Harrison and Kennedy (1922)

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isolated a variety of bacteria from salted fish, and described Pseudomonas salinaria, presented later under the name Serratia salinaria in the first edition of Bergey's Manual (1923). The name Halobacterium was formally introduced in 1957 by Elazari-Volcani in the edition of this handbook (Elazari-Volcani, 1957). Tindall (1992) provides a critical discussion of these early isolates and the subsequent fate of the cultures. Another early paper of interest on the isolation of halophilic bacteria is the report by Browne (1922) on the characterization of the red coloration developing on salted fish in the summer months. He states that the coloration is due to two types of halophiles unable to grow at salt concentrations below Spirochaeta halophilica" and "Bacterium halophilica", which are both present in the solar evaporated sea salt with which the fish are cured. Much of the work on halophilic bacteria in the late 1920s - early 1930s was connected with the Delft school of microbiology (Baas Becking, 1928; Petter, 1931, 1932; Hof, 1935; see also Larsen, 1973). Studies performed under the guidance of Albert Jan Kluyver resulted in Helena Petter's thesis "Over roode en andere bacterieën van gezouten visch" (On red and other bacteria of salted fish) and the thesis of Trijntje Hof: "Investigations concerning bacterial life in strong brines". Petter studied different isolates of halophilic bacteria, most of them red colored members of the Halobacteriaceae, from environments such as salted fish and "Trapani" salt. Among her isolates were red rod-shaped bacteria and red and colorless sarcina-shaped organisms obtained from salted herring, a red rod isolated from dried cod, and an orange rod from Trapani salt obtained from a cannery in Bergen, Norway. She gave an accurate description of "Bacterium halobium" (now known as Halobacterium salinarum), including the presence of gas vesicles within its cells (Figure 2). Petter also performed the first characterization of the red pigment of these organisms, and the name bacterioruberin, still in use, was first proposed by her. Another organism studied in the Delft laboratory during this period was the motile halophilic rod that caused a purple discoloration of salted beans, named by Hof Pseudomonas beijerinckii in honor of Martinus Beijerinck, the founding father of the Delft school of microbiology.

The first isolation of an obligatory anaerobic halophilic bacterium can probably be found in the report by Baumgartner (1937) who obtained an organism designated "Bacteroides halosmophilus" from salted anchovies. This strain grew optimally at salt concentrations between 125 and and no growth was observed below

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salt. The strain is no longer extant, but it probably had much resemblance to the anaerobic fermentative halophile Halanaerobium praevalens, found to be abundant in the bottom sediments of the Great Salt Lake (Zeikus et al., 1983), and recently isolated also from canned Swedish fermented herrings ("Surströmming") (Kobayashi et al., 2000).

1.3. DUNALIELLA AND OTHER HALOPHILIC ALGAE Unicellular green halophilic algae, probably of the genus now known as Dunaliella, were first described by Dunal (1838), who reported a red unicellular organism designated as Haematococcus salinus and another salt alga named Protococcus (sec also Turpin, 1839). Dunal's description challenged the assumption, widespread at the time, that chemical and physical parameters are responsible for the red color of brines this in spite of the fact that, as shown above. Darwin (1839) had already correctly identified the presence of microorganisms as the cause of the coloration of highly saline waters. Another concept common during the period was that brine shrimp (Artemia) caused the red coloration of the waters. Other opinions brought forward arc for example the idea that not Artemia itself but the flagellate algae eaten by it, both alive in the brines and within the Artemia intestine, are the true cause of the coloration of the waters (Joly, 1840). The first in-depth descriptions of Dunaliella appeared in the first decade of the 20th century. Hamburger (1905) summarized the at the time the still ongoing debate on the true cause of the red coloration in salt pans, and gave a detailed account of the red unicellular algae abundant in these environments (Figure 3). A formal description of these algae was published almost simultaneously by Teodoresco (1905, 1906), who named the new genus Dunaliella after Dunal who had described similar forms almost seventy years earlier.

It is interesting to note that Hamburger (1905) already presented details on the location of the red pigment, now known to be and to be located as globules between the thylacoids of the single chloroplast. Hamburger's description has at least in part withstood the test of time:

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Er tritt in Form kleiner Tröpfchen auf, und ist, wie mir sicher scheint, nur der äußeren Alveolarschicht des Plasmas eingelagert, während das Chromatophor Träger des grünen Farbstoffes ist. Die bemerkung Teodoresco's "hématochrome imprégnant non seulement le chromatophore, mais encore tout le corps des individus âgés", stimmt mit meinen Beobachtungen nicht überein. [It occurs in the form of small droplets, and is, as seems sure to me, only deposited in the outer alveolar layer of the plasma, while the chromatophore [= chloroplast] is the bearer of the green pigment. The remark by Teodoresco "the blood pigment that impregnates not only the chromatophore, but also the whole body of adult individuals" does not correspond with my observations.]

Baas-Becking (1931) correctly stated that the orange pigment is located in the plastid.

1.4. ECOLOGICAL STUDIES: THE GREAT SALT LAKE The first biological research on the Great Salt Lake was done by Packard (1879). He reported the algae Polycystis packardii, Rhizoclonium sp. and Ulva marginata to be present in the lake. Other early biological studies on the microbial communities in the Great Salt Lake dealt with protozoa. Vorhies (1917) recognized two or three varieties of protozoa in the lake, the most common being an organism named Amoeba limax. Two years later, Pack (1919) described two ciliates from the lake, Uroleptus packii and Prorodon utahensis (= Chilophrya utahensis). The first bacteriological studies performed in the Great Salt Lake were probably those by Daniels (1917), who counted viable bacteria on agar plates containing 250 salt. He reported numbers of colony forming bacteria between 200 and 625 The colonies obtained displayed different colors: yellow, orange, and violet. Little or no additional studies were performed during the following twenty years. The existence of a rich autochthonous community of bacteria in the Great Salt Lake was unequivocally proven by Claude ZoBell, often considered the founding father of marine microbiology, and his student Whitney Smith (Smith, 1936; Smith and ZoBell, 1937a, 1937b, 1937c). Using a technique developed by A.T. Henrici a few years earlier, glass microscope slides were immersed in the lake and in jars filled with Great Salt Lake water, and microorganisms that became attached to the slides were examined microscopically. Even after periods as short as six hours, bacteria were seen attached to the glass. After 24 hours of incubation between 40 and 1,100 microcolonies were found, depending on the site in the lake where the slides had been immerged. At least nine morphological varieties of bacteria were observed, most of them being Gramnegative rods. About one-third of the bacteria observed had capsules, some contained structures resembling endspores, and less than 10% stained Gram-positive. Smith and ZoBell (1937a) also set up enrichment cultures with Great Salt Lake water (336 total dissolved salts), and they estimated that 50% of the Great Salt Lake bacteria required at least 70 g salt to grow, and that 96% of the community could not grow without salt. About at the same time a renewed study was made of the algae of Great Salt Lake (Kirkpatrick, 1934).

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1.5. ALKALINE SODA LAKES Surprisingly, an exotic, difficult to access environment like the alkaline hypersaline lakes of the Wadi Natrun in Egypt has been the subject of a thorough microbiological examination very early in the history of microbiology. A detailed geochemical and biogeochemical account of the brines, the salt crusts and the sediments of these lakes was presented by Schweinfurth and Lewin in 1898. This apparently little known paper deserves far more attention than it has received thus far, and therefore the relevant passages are quoted here extensively: In der Seen und in der nähe der Quellen entwickelt sich ein reiches Leben niederster Pflanzen. Die rote Farbe der Seen war von jeher den Besuchern derselben aufgefallen. Andréossy schreibt sie einer "substance végéto-animale" zu. Sickenberger läßt in ihnen eine Oscillarie, Conferven, an anderer Stelle einen Micrococcus vorkommen. [In the lakes and close to the springs a rich life of lower plants develops. The red color of the lakes was noted by each of the visitors there. Andréossy attributes it to a vegetoanimal substance. Sickenberger claims that Oscillatoria and algae occur in them, elsewhere a Micrococcus.) Da nun alle Seen Natron zu enthalten scheinen und manche nicht rot sind, so ist es nicht gerade wahrscheinlich, daß nur sauerstoffbedürftige niederste Pflanzen die Entstehung desselben veranlassen. Überhaupt werden dort die roten Mikroorganismen vielleicht ganz anderen Pflanzenklassen angehören, als den vermuteten Spaltpilzen, eher den Spaltalgen und den Diatomeen, die im Gegenteil Sauerstoff ausscheiden. [As all lakes appear to contain soda and some are not red, it is not very probably that only oxygen-requiring lower plants are responsible for its formation. Indeed the red microorganisms there may belong to completely different classes of plants than the presumed bacteria, but rather to the cyanobacteria and diatoms, which in contrast excrete oxygen.] Ein frischeres Stück des roten Salzes aus einem Natron-See – auch bei Alexandria in der Lagune der Saline kommt desartiges rotes Salz vor – zeigte nach unseren Untersuchungen folgende Beschaffenheit: Heller und dunkler rote, stellenweis tief burgunderfarbene Partieen wechselten an demselben ab. Der rote, in Wasser und Alkohol lösliche Farbstoff schwand beim Erhitzen des Salzes. Weder die Behandlung mit Zink und Schwefelsäure, noch mit Ätzalkalien änderte wesentlich seine Intensität. Die mikroskopische Untersuchung ließs eigentümlich aggregierte, dunkle Körperchen erkennen, deren Natur nicht festzustellen war, die aber vielleicht Pilzsporen sind. [a fresher piece of the red salt from a soda lake – also near Alexandria in the laguna of the saltern such red salt is found – showed according to our investigations the following properties: brighter and darker red, partwise deep burgundy-colored areas alternated. The red, in water and alcohol soluble pigment, disappeared upon heating of the salt. Neither treatment with zinc and sulfuric acid, nor with strong bases, changed its intensity significantly. The microscopical examination showed dark bodies, aggregated in a characteristic way, whose nature it was impossible to establish, but which may be fungal spores.]

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Additional quotations from the Schweifurth and Lewin 1898 paper will be presented below.

1.6. THE DEAD SEA Compared to the above early studies in the Great Salt Lake, solar salterns, and the alkaline brines of the Wadi Natrun, the scientific study of the Dead Sea started relatively late. This is to some extent unexpected in view of the large biblical interest in the properties of this unusual lake. The first report of the isolation of a living organism from the Dead Sea does not at all deal with halophiles, but with non-halophilic pathogenic clostridia instead. M.L. Lortet, a microbiologist from the university of Lille, France, performed in 1891 a microbiological examination of water and sediment samples obtained from the Dead Sea with the intention to exploit its alleged hostility to life and use the water and mud as an aseptic substance. To his surprise he was able to culture pathogenic species of the genus Clostridium that caused the symptoms of tetanus and gas gangrene in guinea-pigs and donkeys (Lortet, 1892). This observation is not too surprising as these clostridia may survive extreme environmental conditions in the form of dormant endospores. Only upon suspension into low-salt media will these spores germinate (Oren, 1991). Being unable to grow and multiply at the high salt concentration of the Dead Sea water, these clostridia cannot be considered part of the indigenous life in the Dead Sea. The first report of the existence of a varied microbial community in the Dead Sea appeared as late as 1936 (Wilkansky, 1936). Inspired by the studies by Hof, Petter, and others who had found extremely halophilic microorganisms in a variety of salt-saturated environments (see above), Benjamin Elazari-Volcani (Wilkansky) set up a series of enrichment cultures with water and sediments sampled from the Dead Sea. The lake was found to harbor a varied microbial community, consisting of different aerobic and anaerobic bacteria, Dunaliella and possibly other unicellular algae as primary producers, and even several types of protozoa (Elazari-Volcani, 1940; Volcani, 1944; Wilkansky, 1936).

1.7. THE STUDY OF BIOGEOCHEMICAL PROCESSES IN HYPERSALINE ENVIRONMENTS When one realizes that the first true understanding of biogeochemical processes involved in the cycles of elements such as nitrogen and sulfur was achieved only in the last two decades of the century - mainly thanks to the pioneering work of Sergei Winogradsky and Martinus Beijerinck - it is again surprising to note that the newly obtained concepts were so quickly applied to the hypersaline alkaline environment of the Wadi Natrun lakes in Egypt. The following quotations from the account by Schweinfurth and Lewin (1898) provide ample illustration: Das aus den Quellen austretende Wasser beginnt nach kurzer Strecke seines Verlaufes Schwefelwasserstoff zu entwickeln. Die grünen Algen verschwinden, und es erscheint etwas weiter durch Zersetzung der grünen Algen eine schlammige rote und dann schwarze Masse. Die letztere sei schwarzes Schwefeleisen. Mit der Zunahme des letzteren wachse

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auch die alkalische Reaktion des Wassers. Der rote und der schwarze Schlamm entwickele Kohlensäure durch große Mengen eines Micrococcus. [The water that emerges from the springs starts to develop hydrogen sulfide after a short distance from its sourse. The green algae disappear, and somewhat further on, as a result of degradation of the green algae, a muddy red and then black mass appears. The last is black iron sulfide. With the increase in the latter also the alkaline reaction of the water increases. The red and the black mud develop carbonic acid by large amounts of a Micrococcus.]

The accounts by Jannasch (1957) and Imhoff et al. (1979), written sixty to eighty years later, fully confirm and extend these observations, as does the recognition of the correlation between sulfate reduction and alkalinity in the Wadi Natrun by Abd-elMalek and Rizk (1963). The early work even points to the presence of trimethylamine in the environment: Die ganze Salzmasse roch, besonders an frischen Bruchstellen, stark nach Trimethylamin. An ein präfermiertes Vorhandensein dieser Base im Salz is nicht zu denken. Die Annahme liegt näher, daßs sich dieselbe aus Cholin bildet. Dieses u. a. in höheren und niedersten Pilzen vorkommende Alkaloid kann auch bei der Zersetzung von eiweiß- und lecithinhaltigem Material entstehen und liefert, mit Alkalien behandelt, seinerseits Trimethylamin. [The whole salt mass had a strong smell of trimethylamine, particularly at fresh fracture planes. A preformed presence of this base in the salt is unimaginable. It should rather be assumed that this compound is formed from choline. This alkaloid, which occurs e.g. in higher and lower fungi, can be formed during degradation of protein and lecithine-containing material, and yields upon treatment with bases trimethylamine.]

Today, with our increased understanding of the microbiological and biogeochemical processes occurring in hypersaline environments, we would probably explain the observation of the formation of trimethylamine not from choline, but by the degradation of glycine betaine. This compound is accumulated as an osmotic stabilizer by many halophilic prokaryotes, including members of the genus Halorhodospira and different halophilic cyanobacteria, organisms that abound in the Wadi Natrun lakes (Imhoff et al., 1979; Jannasch, 1957). The use of glycine betaine as a compatible solute by halophilic microorganisms was not recognized until the early 1980s (Galinski and Trüper, 1982), and was first discovered in the Wadi Natrun isolate Halorhodospira halochloris (Imhoff and Trüper, 1977). Upon degradation, glycine betaine often yields trimethylamine (Oren, 1990). Schweinfurth and Lewin fully realized that the microbiology of the Wadi Natrun lakes may have many more surprises to offer, and they encouraged further investigations: Einer besondren Besprechung bedürfen die besonders interessanten Einwirkungen denen in manchen Seen die Salze durch die pflanzlichen Lebeweisen ausgesetzt sind. In welchem Umfang diese sich aber abspielen, das kann sich erst nach langwierigen Untersuchungen an Ort und Stelle unter Anwendung der verschiedenartigsten Kulturmedien ergeben. [The very interesting influences that the plant plant forms exert on the salts in many lakes deserve a special discussion. To what extent these do occur can, however, only be established after prolonged investigations at the site, while using a variety of culture media.]

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This statement is then followed by an in-depth discussion of the then current microbiological literature, with ample citations from the contemporary works by Winogradsky, Beijerinck, and others. Further studies of biogeochemical processes connected with the cycles of carbon, nitrogen, and sulfur were subsequently performed in the late 1920s and early 1930s by a group of Russian investigators who worked in hypersaline lakes and lagoons in the region near Odessa. Among the processes assessed were the anaerobic degradation of cellulose, the bacterial breakdown of urea, nitrification (up to about 150 salt), and chemolithotrophic oxidation of reduced sulfur compounds, found to occur up to 240 g salt (Issatchenko and Salimowska, 1929; Rubentschik, 1926a, 1926b, 1929, 1933). Dissimilatory sulfate reduction was observed up to a salt concentration of 300 the highest rates surprisingly being found at the highest salt concentrations examined (Rubentschik, 1946; Saslawsky, 1928; Saslawsky and Chait, 1929).

1.8. THE BEGINNING OF THE MODERN ERA OF HALOPHILE RESEARCH It is not surprising that many of the first in-depth studies on the nature of halophilic microorganisms were performed in northern countries such as Canada and Norway. In these areas salted fish is an important economic resource, and spoilage of salted fish and meat products by halophilic bacteria has long since been a problem there. The fact that the first true descriptions of red halophilic Archaea deal with isolates obtained from salted fish (Harrison and Kennedy, 1922; Klebahn, 1919) also bear witness to this fact. We owe a great part of our knowledge of the biology of halophilic microorganisms to the work of Norman Gibbons (from the 1950s) and his student Donn Kushner in Canada (from the 1960s) and of Helge Larsen and his student Ian Dundas in Norway (from the 1960s). The work of these eminent scientists has opened the way toward a true understanding of the diversity of salt-adapted microorganisms that exist in hypersaline environments and the unique adaptations these organisms have developed to live at near-saturating salt concentrations. It is especially noteworthy that Helge Larsen was honored to deliver the fourth A.J. Kluyver memorial lecture in Delft in 1972. This lecture was entitled "The halobacteria’s confusion to biology" (Larsen, 1973), the title paraphrasing the famous essay by Kluyver and van Niel (who had been one of Larsen’s teachers) "The microbe's contribution to biology" (Kluyver and van Niel, 1956). It commemorated the contribution of the Delft school of microbiology to the study of halophilic bacteria, and summarized in an admirable way the state of the field in 1972 and the progress made since the pioneering studies of Helena Petter and Trijntje Hof. An earlier review paper by Larsen presented an overview of the understanding of the biochemistry of halophilic microorganisms at the time (Larsen, 1967). The following chapters in this book are a tribute to the efforts of all those investigators, in the past and in the present, who have contributed to our understanding of the biology of halophilic and halotolerant microorganisms and of their impact on the environments in which they live.

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REFERENCES Abd-el-Malek, Y., and Rizk, S.G. 1963. Bacterial sulphate reduction and the development of alkalinity. III. Experiments under natural conditions in the Wadi Natrun. J. Appl. Bacteriol. 26: 20-26. Agricola, G. 1556. De re metallica. J. Froben and N. Episcopus, Basel. Translation (1950) by Hoover, H.C., and Hoover, L.H. Dover Publications, New York Bergey, D.H., Harrison, F.C., Breed, R.S., Hammer, B.W., and Huntoon, F.M. (Eds.). 1923. Bergey's manual of determinative bacteriology, ed. Williams & Wilkins, Baltimore. Baas Becking, L.G.M. 1928. On organisms living in concentrated brine. Tijdschr. Ned. Dierkund. Ver. Ser. III. 1: 6-9. Baas Becking, L.G.M. 1931. Historical notes on salt and salt-manufacture. Scientific Monthly 32: 434-446. Baumgartner, J.G. 1937. The salt limits and thermal stability of a new species of anaerobic halophile. Food Res. 2: 321-329. Bloch, M.R. 1962. Red salt and grey salt. Mada 6: 3-8 (in Hebrew). Bloch, M.R. 1976. Salt in human history. Interdisc. Sci. Rev. 4: 336-352. Browne, W.W. 1922. Halophilic bacteria. Proc. Soc. Exp. Biol. Med. 19: 321-322. Daniels, L.L. 1917. On the flora of Great Salt Lake. American Naturalist 51: 499-506. Darwin, C. 1839. Journal of researches into the geology and natural history of the various countries visited by H.M.S. Beagle, under the command of Captain Fitzroy, R.N. from 1832 to 1836. Henry Colburn, London. Diderot, D. 1765. Encyclopédic, or dictionnaire raisonné des sciences, des arts et des métiers, par une societé de gens de lettres. Tome quatorzieme, pp. 544-546. Samuel Faulche & Co., Neufchastel (facsimile edition: Friedrich Frommann Verlag, Stuttgart, 1967). Dunal, F. 1838. Extrait d’un mémoire sur les Algues qui colorent en rouge certains eaux des marais salants méditerranéens. Ann. Sc. Nat. 2 Sér. Tom. 9 Bot. Paris. Elazari-Volcani, B. 1940. Studies on the microflora of the Dead Sea. Ph.D. thesis, The Hebrew University of Jerusalem. Elazari-Volcani, B. 1957. Genus XII. Halobacterium Elazari-Volcani, 1940, pp. 207-212 In: Breed, R.S., Murray, E.G.D., and Smith, N.R. (Eds.), Bergey's manual of determinative bacteriology, ed. Williams & Wilkins, Baltimore. Galinski, E.A., and Trüper, H.G. 1982. Betaine, a compatible solute in the extremely halophilic phototrophic bacterium, Ectothiorhodospira halochloris. FEMS Microbiol. Lett. 13: 357-360. Hamburger, C. 1905. Zur Kenntnis der Dunaliella salina und einer Amöbe aus Salinenwasser von Cagliari. Arch. f. Protistenkd. 6: 111-131. Harrison, F.C., and Kennedy, M.E. 1922. The red discoloration of cured codfish. Trans. Roy. Soc. Canad. Sct. III 16: 101-152. Hof, T. 1935. Investigations concerning bacterial life in strong brines. Rec. Trav. Bot. Neerl. 32: 92-173 (also published as Ph.D. thesis, University of Leiden). Imhoff, J.F., and Trüper, H.G. 1977. Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch. Microbiol. 114: 115-121. Imhoff, J.F., Sahl, H.G., Soliman, G.S.H., and Trüper, H.G, 1979. The Wadi Natrun: Chemical composition and microbial mass developents in alkaline brines of eutrophic desert lakes. Geomicrobiol. J. 1: 219-234. Issatchenko, B.L., and Salimowska. 1929. Die Charakteristik der bakteriologischen Prozesse in Schwarzen und Azowschen Meeren. Proceedings of the International Congress of Plant Science 1: 211-220. Jannasch, H.W. (1957). Die bakterielle Rotfärbung der Salzseen des Wadi Natrun (Ägypten). Arch. f. Hydrobiol. 53: 425-433. Joly, M. 1840. Histoire d'un petit crustacé (Artemia salina) auquel on a faussement attribué la coloration en rouge des marais salants méditerranéens, suivie de recherches sur la cause réelle de cette coloration. Ann. Sc. Nat. 2 Sér Zoologie Bd. 13. Paris. Kirkpatrick, R. 1934. The life of Great Salt Lake, with special reference to the algae. M.Sc. thesis, Utah University, Salt Lake City. Klebahn, H. 1919. Die Schädlinge des Klippfisches. Mitt. Inst. Allg. Bot. Hamburg 4: 11-69. Kluyver, A.J., and van Niel, C.B. 1956. The microbe's contribution to biology, p. 5, John M. Prather Lectures, Harvard University, 1954. Harvard University Press, Cambridge, MA. Kobayashi, T., Kimura, B., and Fujii, T. 2000. Strictly anaerobic halophiles isolated from canned Swedish fermented herrings (Surströmming). Int. J. Food Microbiol. 54: 81-89. Larsen, H. 1967. Biochemical aspects of extreme halophilism. Adv. Microb. Physiol. 1: 97-132. Larsen, H. 1973. The halobacteria's confusion to biology. Antonie van Leeuwenhoek 39: 383-396.

AN HISTORICAL INTRODUCTION

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Litchfield, C.D. 1991. Red – the magic color for solar salt production, pp. 403-412 In: Hocquet, J.-C., and Palme, R. (Eds.), Das Salz in der Rechts- und Handelsgeschichte. Berenkamp, Hall in Tirol. Lortet, M.L. 1892. Researches on the pathogenic microbes of the mud of the Dead Sea. Pales. Expl. Fund 1892: 48-50. Monro, D. 1771. All account of a pure native crystalised natron, or fossil alkaline salt, which is found in the country of Tripoli in Barbary. Trans. R. Soc. London 61: 567-573. Oren, A. 1983. Population dynamics of halobacteria in the Dead Sea water column. Limnol. Oceanogr. 28: 1094-1103. Oren, A. 1990. Formation and breakdown of glycine betaine and trimethylamine in hypersaline environments. Antonie van Leeuwenhoek 58: 291-298. Oren, A. 1991. Tetanus bacteria and other pathogens in the Dead Sea? Salinet 6: 84-85. Oren, A., and Gurevich, P. 1995. Dynamics of a bloom of halophilic archaea in the Dead Sea. Hydrobiologia 315: 149-158. Oren, A., and Litchfield, C.D. 1998. Early salt production at the Dead Sea and the Mediterranean coast of the Holy Land. J. Salt History 6: 7-17. Pack, D.A. 1919. Two ciliates of Great Salt Lake. Biol. Bull. 36: 273-282. Packard, A.S., Jr. 1879. The sea weeds of Great Salt Lake. American Naturalist 13: 701-703. Petter, H.F.M. 1931. On bacteria of salted fish. Proc. Kon. Akad. Wetensch. Ser. B 34: 1417-1423. Petter, H.F.M. 1932. Over roode en andere bacterieën van gezouten visch. Ph.D. thesis, University of Utrecht. Pierce, G.J. 1914. The behavior of certain micro-organisms in brine. Carnegie Institution of Washington Publication no. 193: 49-69. Rubentschik, L. 1926a. Über die Einwirkung von Salzen auf die Lebenstätigkeit der Urobakterien. Zentralbl. f. Bakteriol. II 67: 167-194. Rubentschik, L. 1926b. Über einige neue Urobakterienarten. Zentralbl. f. Bakteriol. II 66: 161-180. Rubentschik, L. 1929. Zur Nitrifikation bei hohen Salzkonzentrationen. Centralbl. Bakteriol. Parasitenk. Infektionskr. Abt. 2. 77: 1-18. Rubentschik, L. 1933. Zur anaeroben Zellulosezersetzung in Salzseen. Zentralbl. f. Bakteriol. II 88: 182-186. Rubentschik, L. 1946. Sulfate-reducing bacteria. Microbiology (USSR) 15: 443-456. Saslawsky, A.S. 1928. Zur Frage der Wirkung hoher Salzkonzentrationen auf die biochemischen Prozesse im Limanschlamm. Zentralbl. f. Bakteriol. Abt. 2. 73: 18-28. Saslawsky, A.S. and Chait, S.S. 1929. Über den Einsfluß des Natriumchlorids auf einige biochemische Prozesse in den Limanen. Centralbl. Bakteriol. Parasitenk. Infektionskr. Abt. 2. 77: 18-21. Schweinfurth, G. and Lewin, L. 1898. Beiträge zur Topographie und Geochemie des ägyptischen NatronThals. Zeitschr. d. Ges. f. Erdk. 33: 1-25. Smith, W.W. 1936. Evidence of a bacterial flora indigenous to the Great Salt Lake. M.Sc. Thesis, University of Utah. Smith, W.W., and ZoBell, C.E. 1937a. An autochthonous bacterial flora in Great Salt Lake. J. Bacteriol. 33: 118. Smith, W.W., and ZoBell, C.E. 1937b. Direct microscopic evidence of an autochthonous bacterial flora in Great Salt Lake. Ecology 18: 453-458. Smith, W.W., and ZoBell, C.E. 1937c. Direct microscopic evidence of an indigenous bacterial flora in Great Salt Lake. J. Bacteriol. 33: 87. Teodoresco, E.C. 1905. Organisation et développement du Dunaliella, nouveau genre de VolvocacéePolyblepharidée. Beih. Z. Bot. Centralbl. Bd. XVIII. Teodoresco, E.C. 1906. Observations morphologiques et biologiques sur le genre Dunaliella. Revue Generale de Botanique 18: 353-371. Tindall, B.J. 1992. The family Halobacteriaceae, pp. 769-808 In: Balows, A., Trüper, H.G., Dworkin, M., Harder, W., and Schleifer, K.-H. (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. ed., Vol. I. Springer-Verlag, New York. Turpin. 1839. Queques observations nouvelles sur les Protococcus, qui colorent en rouge les eaux des marais salants. Comp. Rend. 1939 (as cited by Hamburger, 1905). Volcani, B. 1944. The microorganims of the Dead Sea, pp. 71-85 In: Papers collected to commemorate the 70th anniversary of Dr. Chaim Weizmann. Collective Volume, Daniel Sieff Research Institute, Rehovoth. Vorhies, C.T. 1917. Notes on the fauna of Great Salt Lake. American Naturalist 51: 494-499. Wilkansky, B. 1936. Life in the Dead Sea. Nature 138: 467.

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Ying-Hsing, S. 1966. Salt. In: T’ien-Kung K’ai-Wu Chinese technology in the seventeenth century (1637), translated by E-Tu Zen Sun and Shiou-Chuan Sun. Pennsylvania State University Press, University Park and London, pp. 109-123. Zeikus, J.G., Hegge, P.W., Thompson, T.E., Phelps, T.J., and Langworthy, T.A. 1983. Isolation and description of Haloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophile common to Great Salt Lake sediments. Curr. Microbiol. 9: 225-234.

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SECTION 2

HALOPHILIC MICROORGANISMS AND THEIR PROPERTIES

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INTRODUCTION

The following chapters present an overview of the halophilic microorganisms, the ways they cope with the high salinity in their environment and other aspects of their physiology, a summary of our increasing understanding of their genetics and genomics, and an account of useful applications of the halophiles. Halophiles are found in all three domains of life. The properties of the halophilic Archaea, Bacteria and Eucarya are discussed in separate sections in each chapter, while at the same time an attempt is made to compare the ways the halophiles in each domain cope with the challenges created by the presence of high concentrations of salt. Following a taxonomic overview of the halophiles (Chapter 2) and a discussion of the special properties of their cell walls, membranes, and intracellular structures (Chapter 3), the general physiology and biochemistry of different groups of halophiles is presented with special emphasis on their metabolic potential (Chapter 4). Many halophiles are pigmented, and some of their pigments have proven of great interest, ecologically, biochemically, as well as and biotechnologically. Chapter 5 is therefore dedicated to a description of the different pigments present and their functions. The retinal pigments (bacteriorhodopsin, halorhodopsin, and the sensory rhodopsins) of Halobacterium salinarum have attracted tremendous interest, and probably more articles have been published on these pigments in the past thirty years than on all other aspects of halophilic microorganisms combined. The following chapters present the specific mechanisms of adaptation of microorganisms to life at high salt concentrations. Chapter 6 discusses the intracellular ionic concentrations within different types of halophiles and the way these concentrations are regulated. Some types of halophiles contain extremely high intracellular ionic concentrations, and their proteins are functional in concentrated salt solutions. Extracellular enzymes of halophiles are always exposed to high salt, independent of strategy the organism uses to osmotically adjust its cytoplasm with the high salinity outside. The special properties of such "halophilic" proteins are presented in Chapter 7. Many halophiles accumulate organic "compatible" solutes while keeping intracellular salt concentrations at a low level. A great diversity of such compatible solutes exists, and we are now starting to obtain some insight in the way these compounds function (Chapter 8). Halophilic Archaea and Bacteria can be killed by bacteriophages, and it this respect they are no different from other prokaryotes. Halophilic Archaea are also susceptible to attack by specific "halocins", a group of archaeocins consisting of protein antibiotics excreted by closely related organisms. The properties of the halophilic phages and the halocins are presented in Chapter 9. Genetic systems for the manipulation of many halophiles have become available in recent years. The first complete genome of a halophilic archaeon has been published in 2000, and efforts to sequence additional halophiles are now in progress. Chapter 10 summarizes the present state of genetics and genomics of different types of halophilic microorganisms.

21

22 The section concludes with an overview of the biotechnological applications of the halophiles that are already being exploited, as well as their potential applications in the future (Chapter 11).

CHAPTER 2 TAXONOMY OF HALOPHILIC MICROORGANISMS: ARCHAEA, BACTERIA, AND EUCARYA

…… "Is there no other creature on your list?" Little Vishnik asked. The Archangel looked at his list again, rolling it up from the clouds below. "None of them seems suitable." "You'll have to make a new one, then," said Little Vishnik firmly. The Archangel cheered up. "Why not?" "It should ask questions we can't answer." Said Little Vishnik. "Absolutely!" The Archangel keyed this into his computer. "And it should like salt." "Very much," Little Vishnik agreed. "But it should be very small." The Archangel pushed more keys. "Now it's all arranged." The two heavenly creatures beamed at each other, well pleased with the new creature they had designed. They were especially pleased that it had been programmed into the celestial computer, whose circuits cannot be altered ("How the first halophilic microorganism was created" – a tale by Donn Kushner) (from Kushner, 1991)

2.1. THE PLACE OF THE HALOPHILES WITHIN THE MICROBIAL WORLD Halophilic microorganisms are found in all three domains of life: Archaea, Bacteria, and Eucarya (Oren, 1999). In each of these domains we encounter representatives that can grow up to the highest salt concentrations. In NaCl-saturated environments such as the Dead Sea and saltern crystallizer ponds we find both halophilic Archaea of the family Halobacteriaceae and unicellular eukaryotic algae of the genus Dunaliella. The recently discovered Salinibacter ruber (domain Bacteria) also lives in saltern crystallizer ponds (Antón et al., 2002), and alkaliphilic anoxygenic photosynthetic Bacteria of the genus Halorhodospira abound in soda lakes at salt concentrations exceeding In this volume a halophilic microorganism is operationally defined as an organism that shows considerable growth at salt concentrations higher than Figure 2.1 shows the universal tree of life, highlighting those branches that contain halophilic microorganisms that conform to this definition. The sections below provide taxonomic and nomenclatural information on the halophiles described to date (updated to January

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TAXONOMY

OF

HALOPHILES

25

2002 with a few later additions). Many of the organisms included are unable to grow at saturated salt concentrations, and most do not show the exceedingly high salt requirement of Halobacterium and its relatives. However, the moderate halophiles play important roles in many hypersaline environments. The variety in physiological properties and modes of osmotic adaptation encountered is as great as their phylogenetic diversity (Trüper et al., 1991). Different aspects of the taxonomy of these moderate halophiles have been reviewed on several occasions in the past (Ventosa, 1988, 1994; Ventosa et al., 1998). Halophilic microorganisms are often interspersed between non-halophilic relatives within the phylogenetic tree. There are three notable cases of phylogenetically coherent groups that are composed entirely or almost entirely of halophiles: the order Halobacteriales, family Halobacteriaceae in the archaeal domain, consisting of mostly red, extremely halophilic aerobic microorganisms, and in the domain Bacteria the order Halanaerobiales, the representatives of which are obligate anaerobes, and the family Halomonadaceae, in which most species are moderately halophilic aerobies or facultative anaerobes. The following sections present an overview of the phylogenetic diversity of halophiles in all three domains: Archaea, Bacteria, and Eucarya. The taxonomic treatment of the prokaryotic halophiles is to a large extent based on that followed by the last edition of Bergey's Manual of Systematic Bacteriology (Boone and Castenholz, 2001; Garrity and Holt, 2001). Useful information can also be found in "The Prokaryotes" (Dworkin et al., Eds.), an electronically published handbook that is periodically updated with the latest information on all groups of prokaryotic microorganisms. Reliable updated information on the nomenclature of Archaea and Bacteria is available online in a web site maintained by Jean Euzéby: http://www.bacterio.cict.fr.

2.2. THE HALOPHILIC ARCHAEA Within the domain Archaea halophilic microorganisms occur in three families: the Halobacteriaceae, the Methanospirillaceae, and the Methanosarcinaceae. The Methanospirillaceae and the Methanosarcinaceae contain non-halophilic representatives as well as organisms that are adapted to seawater salinity and to hypersaline conditions. Some of these can grow at salt concentrations up to The order Halobacteriales with a single family, the Halobacteriaceae, consists entirely of halophiles. The most salt-requiring and salt-tolerant of all microorganisms are found in this family. Most extensive studies have been made of the family Halobacteriaceae, which may be considered as the halophiles par excellence because of their strict dependence on high salt concentrations for growth and structural stability. Twenty years ago the diversity within this group was considered low, being limited to rod-shaped, Halobacterium-type cells and cocci of the genus Halococcus (Colwell et al., 1979).

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Since that time many new representatives have been discovered, and these display a Considerable morphological and physiological diversity. The question: 'Are extreme halophiles actually "bacteria"?' (Magrum et al., 1978) was answered negatively when it was recognized in the late 1970s that Halobacterium salinarum and related halophiles belong to the newly recognized domain Archaea. The order Halobacteriales (Grant and Larsen, 1989) forms a branch within the Euryarchaeota, branching off close to the Methanomicrobiales/Methanosarcinales. A single family is recognized within the order: the Halobacteriaceae. Presently this family is divided into 15 genera with 44 species (as of January 2002). Figure 2.2 presents a phylogenetic tree of the family, based on 16S rDNA sequence comparisons. The order Halobacteriales contains a variety of morphological types, from rods and cocci to flat extremely pleomorphic cells (Mullakhanbhai and Larsen, 1975) and triangular and trapezoid cells such as displayed by Haloarcula japonica (Takashina et al., 1990) (see Section 3.1.1). The yet uncultured perfectly square flat cells encountered in some hypersaline environments (Walsby, 1980) also belong to this family, as has been ascertained on the basis of 16S rRNA sequence analysis (Antón et al., 1999). Classification of the species belonging to the family Halobacteriaceae is currently based on a polyphasic approach (Oren et al., 1997a), which includes the evaluation of properties such as cell morphology, growth characteristics, chemotaxomic traits (notably the presence or absence of specific polar lipids), and nucleic acid sequence data. Examination of the polar lipids present (see also Section 3.1.4) has proven extremely useful for the rapid characterization of isolates, as many genera have a distinctive polar lipid signature (Kamekura, 1998, 1999; Torreblanca et al., 1986). In recent years the comparison of 16S rDNA sequences has contributed much to the classification of the species within the Halobacteriaceae. New genera have been created, existing genera have been split into a number of new genera, and in some cases have species that were earlier classified in separate genera been unified in a single genus (see e.g. Kamekura, 1998, 1999; Kamekura et al., 1997; Oren, 2001a). Also the 23S rRNA genes have been used in taxonomic studies, and the analysis of the available data suggested that the halobacterial genes diverged over a relatively short time interval (Lodwick et al., 1994). Phylogenetic trees based on 23S rRNA sequences may have a completely different topology from those based on 16S rRNA sequences (Briones and Amils, 2000). Caution should thus be exerted when basing conclusions on the phylogeny of the halophilic Archaea on 16S rRNA sequence comparisons only. 16S rRNA nucleotide sequence comparisons have led to the insight that the alkaliphilic members within the family do not form a separate phylogenetic group: many alkaliphilic species are interspersed between their neutrophilic relatives in the phylogenetic tree. Most Haloarcula species contain more than one heterologous 16S rRNA gene. This fact sometimes complicates the use of 16S rRNA sequences for the construction of phylogenetic trees. The two rRNA operons of Haloarcula marismortui have been characterized (Mevarech et al., 1989). It was recently shown by fluorescence in situ hybridization of single cell isolations that both rRNA genes are expressed within the same cell (Amann et al., 2000).

TAXONOMY OF HALOPHILES

27

Techniques such as the characterization of cellular protein patterns by gel electrophoresis (Hesselberg and Vreeland, 1995; Zvyagintseva et al., 1999) and antigenic fingerprinting (Conway de Macario et al., 1986) have also proved useful in taxonomic characterizations, but they have not been extensively used. Antigenic fingerprinting showed that strains that had been classified at the time as Halobacterium

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halobium, Halobacterium salinarium, and Halobacterium cutirubrum – species now unified in a single species, Halobacterium salinarum (Oren and Ventosa, 1996) – showed distinct antigenic differences (Conway de Macario et al., 1986). There has been considerable confusion in the past with regard to the identity of certain strains, and there have been many cases of improper strain designations. Tindall (1992) has presented a critical assessment of strain histories and identities of those strains, and many of the old controversies have now been resolved. The importance of the use of type strains in taxonomic and other (e.g. biochemical and genetic) studies should be stressed here once more. Molecular biological techniques now provide easy tests for the authenticity of strains. Random amplified polymorphic DNA analysis using oligonucleotide probes of arbitrary sequence was used to compare closely related strains (Martinez-Murcia and Rodriguez-Valera, 1994). The method proved to be a powerful tool to sort out problems of strain identity (Martínez-Murcia et al., 1995). There are a number of "species incertae sedis", with names that appear in the literature and/or in the GenBank database, that are still awaiting to be described either as new species or as representatives of recognized taxa. These include "Haloarcula aidinensis", "Haloarcula californiae", "Haloarcula sinaiiensis", "Halobacterium dachaidanensis", "Halococcus dachaidanensis", "Halorubrum hochsteinium", "Haloferax alicantei" (to be described as Haloferax lucentensis), "Haloalcalophilum atacamensis", "Natronobacterium innermongoliae", "Natronobacterium wudunaoensis", and "Natronococcus xinjangense" (Oren and Ventosa, 2002). The list below, arranged according to the classification presented in the latest edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001) and updated to January 2002 according to the most recent literature, presents the archaeal halophiles described thus far. A few taxonomic and physiological characteristics are given for each species. More information can be found in the following chapters, as well as in the original species descriptions.

Domain Archaea, Phylum AII Euryarchaeota Class II Methanococci, Order II Methanomicrobiales, Family III Methanospirillaceae Genus Methanocalculus Methanocalculus halotolerans Reference: Ollivier et al., 1998 Habitat: oilfield, Alsace, France Salt range for growth: optimum DNA G+C content: 55 mol% Type strain: OCM 470

TAXONOMY OF HALOPHILES Order III Methanosarcinales, Family I Methanosarcinaceae Genus III Methanohalobium Methanohalobium evestigatum Originally named: Methanohalobium evestigatus Reference: Zhilina and Zavarzin, 1987 Habitat: saline lagoon, Lake Sivash, Crimea Salt range for growth: DNA G+C content: 37 mol% Type strain: DSM 3721 Genus IV Methanohalophilus Methanohalophilus mahii Reference: Paterek and Smith, 1988 Habitat: Great Salt Lake sediment Salt range for growth: optimum DNA G+C content: 48.5 mol% Type strain: ATCC 35705 Methanohalophilus halophilus Basonym: Methanococcus halophilus References: Wilharm et al., 1991; Zhilina, 1983 Habitat: Cyanobacterial mat, Shark Bay, Australia Salt range for growth: not reported; optimum DNA G+C content: 41-44 mol% Type strain: DSM 3094 Methanohalophilus portucalensis Reference: Boone et al., 1993 Habitat: Saltern, Portugal Salt range for growth: optimum DNA G+C content: 43-44 mol% Type strain: OCM 59 Methanohalophilus zhilinae References: Mathrani et al., 1988 Habitat: Alkaline brines of the Wadi Natun, Egypt Salt range for growth: optimum DNA G+C content: 38-40.1 mol% Type strain: DSM 4017 Methanohalophilus zhilinae has been renamed Methanosalsum zhilinae in the latest edition of Bergey’ s Manual (Boone and Baker, 2001), but the new name has not yet been validated Genus ... Halomethanococcus Halomethanococcus doii Reference: Yu and Kawamura, 1987 Habitat: Solar saltern, San Francisco Bay Salt range for growth: optimum DNA G+C content: 43.2 mol% Type strain: ATCC 43619 (lost) The type strain is no longer availabe, and the present status of the taxon is unclear. The species was probably highly similar to Methanohalophilus

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Class III Halobacteria, Order I Halobacteriales, Family I Halobacteriaceae Genus I Halobacterium Halobacterium salinarum Originally named Halobacterium salinarium. The species includes many strains previously assigned to Halobacterium halobium and Halobacterium cutrirubrum References: Harrison and Kennedy, 1922; Elazari-Volcani, 1957; Ventosa and Oren, 1996 Habitat: Salted cow hide; salt lakes Salt range for growth: optimum DNA G+C content: 66-70 mol% (major component), 57-60 mol% (minor component) Type strain: ATCC 33171 Genus II Haloarcula Haloarcula vallismortis Basonym: Halobacterium vallismortis References: Gonzalez et al., 1978; Torreblanca et al., 1986 Habitat: Salt pools, Death Valley, California Salt optimum for growth: DNA G+C content: 64.7 mol% Type strain: ATCC 29715 Haloarcula marismortui Basonym: Halobacterium marismortui References: Elazari-Volcani, 1957; Ginzburg et al., 1970; Oren et al., 1988, 1990 Habitat: the Dead Sea Salt range for growth: optimum DNA G+C content: 62 mol% (major component), 55 mol% (minor component) Type strain: ATCC 43049 Haloarcula hispanica Reference: Juez et al., 1986 Habitat: Saltern, Spain Salt range for growth: optimum DNA G+C content: 62.7 mol% Type strain: ATCC 33960 Haloarcula japonica Reference: Takashina et al., 1990 Habitat: Saltern, Japan Salt range for growth: optimum DNA G+C content: 63.3 mol% Type strain JCM 7785 Haloarcula argentinensis Reference: Ihara et al., 1997 Habitat: Salt flats, Argentina Salt range for growth: optimum DNA G+C content: 62 mol% Type strain: JCM 9737

TAXONOMY OF HALOPHILES

31

Haloarcula mukohataei Reference: Ihara et al., 1997 Habitat: Salt flats, Argentina Salt range for growth: optimum DNA G+C content: 65 mol% Type strain: JCM 9738 Significant differences exist with the other species within the genus Haloarcula, and reclassification in a new genus appears warranted (Oren and Ventosa, 2002) Haloarcula quadrata Reference: Oren et al., 1999 Habitat: Sabkha, Sinai, Egypt Salt range for growth: optimum DNA G+C content: 60.1 mol% Type strain: DSM 11927 Genus III Halobaculum Halobaculum gomorrense Reference: Oren et al., 1995 Habitat: the Dead Sea NaCl range for growth: DNA G+C content: 70 mol%

optimum Type strain: DSM 9297

requires high magnesium

Genus IV Halococcus Halococcus morrhuae References: Kocur and Hodgkiss, 1973; Montero et al., 1988 Habitat: Salted codfish Salt range for growth: optimum DNA G+C content: 61-66 mol% Type strain: ATCC 17082 Halococcus saccharolyticus Reference: Montero et al., 1989 Habitat: Saltern, Spain Salt range for growth: optimum DNA G+C content: 59.5 mol% Type strain: ATCC 49257 Halococcus salifodinae Reference: Denner et al., 1994 Habitat: Salt mine, Austria Salt range for growth: optimum DNA G+C content: 62 mol% Type strain: ATCC 51437 Genus V Haloferax Haloferax volcanii Basonym: Halobacterium volcanii References: Mullakhanbhai and Larsen, 1975; Torreblanca et al., 1986 Habitat: the Dead Sea Salt range for growth: optimum requires high magnesium DNA G+C content: 63.4 mol% (major component), 55.3 mol% (minor component) Type strain: ATCC 29605

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Haloferax gibbonsii Reference: Juez et al., 1986 Habitat: Saltern, Spain Salt range for growth: optimum DNA G+C content: 61.8 mol% Type strain: ATCC 33959 Haloferax denitrificans Basonym: Halobacterium denitrificans References: Tomlinson et al., 1986; Tindall et al., 1989 Habitat: Saltern, California Salt range for growth: optimum DNA G+C content: 64.2 mol% Type strain: ATCC 35960 Haloferax mediterranei Basonym: Halobacterium mediterranei References: Rodriguez-Valera et al., 1983; Torreblanca et al., 1986 Habitat: Saltern, Spain Salt range for growth: not reported; optimum DNA G+C content: 60 mol% Type strain: ATCC 33500 Genus VI Halogeometricum Halogeometricum borinquense Reference: Montalvo-Rodríguez et al., 1998 Habitat: Saltern, Puerto Rico Salt range for growth: optimum DNA G+C content: 59 mol% Type strain: ATCC 700274 Genus VII Halorhabdus Halorhabdus utahensis Reference: Wainø et al., 2000 Habitat: Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 64 mol% Type strain: DSM 12940 Genus VIII Halorubrum Halorubrum saccharovorum Basonym: Halobacterium saccharovorum References: Tomlinson and Hochstein, 1976; McGenity and Grant, 1995; Kamekura and Dyall-Smith, 1995 Habitat: Saltern, California Salt range for growth: optimum DNA G+C content: 69.1 mol% (major component), 54.8-56.5 mol% (minor component) Type strain: ATCC 29252 Halorubrum sodomense Basonym: Halobacterium sodomense References: Oren 1983a; McGenity and Grant, 1995; Kamekura and Dyall-Smith, 1995 Habitat: the Dead Sea NaCl range for growth: optimum requires high magnesium DNA G+C content: 68 mol% Type strain: ATCC 33755

TAXONOMY OF HALOPHILES

Halorubrum lacusprofundi Basonym: Halobacterium lacusprofundi References: Franzmann et al., 1988a; McGenity and Grant, 1995; Kamekura and Dyall-Smith, 1995 Habitat: Deep Lake, Antarctica Salt range for growth: optimum DNA G+C content: 65.3-65.8 mol% (major component), 54.6-56.5 (minor component) Type strain: ACAM 34 Halorubrum coriense References: Nuttall and Dyall-Smith, 1993; Kamekura and Dyall-Smith, 1995; Oren and Ventosa, 1996 Habitat: Saltern, Australia Salt range for growth: optimum DNA G+C content: not reported Type strain: ACM 3911 Halorubrum distributum Basonym: Halobacterium distributum, has also been described as Halorubrobacterium distributum References: Zvyagintseva and Tarasov, 1987; Kamekura and Dyall-Smith, 1995; Kostrikina et al., 1990; Oren et al., 1997b Habitat: Saline soil, USSR Salt range and optimum for growth: not reported DNA G+C content: 63.6-70.8 mol% Type strain: VKM B-1733 Halorubrum vacuolatum Basonym: Natronobacterium vacuolatum, originally described as Natronobacterium vacuolata References: Mwatha and Grant, 1993; Kamekura et al., 1997 Habitat: Lake Magadi, Kenya Salt range for growth: optimum DNA G+C content: 62.7 mol% Type strain: NCIMB 13189 Halorubrum trapanicum Basonym: Halobacterium trapanicum References: Elazari-Volcani, 1957; McGenity and Grant, 1995; Grant et al., 1998a Habitat: Solar Salt, Italy Salt range and optimum for growth: not reported DNA G+C content: 64.3 mol% Type strain: NCIMB 13488 Halorubrum tebequense References: Lizaba et al., 2000. Habitat: Lake Tebequiche, Chile Salt range for growth: DNA G+C content: 63.2 mol% Type strain: SEM 14210

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Genus IX Haloterrigena Haloterrigena turkmenica Basonym: Halococcus turkmenicus; the genus includes the former Halobacterium trapanicum JCM 9743 References: Zvyagintseva and Tarasov, 1987; Ventosa et al., 1999 Habitat: Alkaline soil, USSR Salt requirement for growth: at least DNA G+C content: 59.2-60.2 mol% Type strain: VKM B-1734 Haloterrigena thermotolerans Reference: Montalvo-Rodríguez et al., 2000 Habitat: Saltern, Puerto Rico Salt range for growth: at least required; optimum DNA G+C content: 63.3 mol% Type strain: DSM 11552 Genus X Natrialba Natrialba asiatica Reference: Kamekura and Dyall-Smith, 1995 Habitat: Beach sand, Japan Salt range for growth: at least optimum DNA G+C content: 60.3-63.1 mol% Type strain: JCM 9576 Natrialba taiwanensis Basonym: Natrialba asiatica (strain B1T) Reference: Hezayen et al., 2001 Habitat: Taiwanese solar salt Salt range for growth: at least required; optimum DNA G+C content: 62.3 mol% Type strain: JCM 9577 Natrialba aegyptia (aegyptiaca) Reference: Hezayen et al., 2001; Notification list, 2001. Habitat: Hypersaline soil, Egypt Salt range for growth: up to saturation; optimum DNA G+C content: 61.5 mol% Type strain: DSM 13077 The species was originally described as Natrialba aegyptiaca. This name was corrected in the Notification list to Natrialba aegyptia with no apparent reason. The issue of the final name of the species is still open (Oren and Ventosa, 2002) Natrialba magadii Basonym: Natronobacterium magadii References: Tindall et al., 1984; Kamekura et al., 1997 Habitat: Lake Magadi, Kenya Salt range for growth: optimum alkaliphilic DNA G+C content: 63.0 mol% (major component), 49.5 (minor component) Type strain: NCIMB 2190 Natrialba hulunbeirensis Reference: Xu et al., 2001 Habitat: Soda lake of the Hulunbeir prefecture, China Salt range for growth: up to saturation; optimum DNA G+C content: 64.3 mol% Type strain: JCM 10989

TAXONOMY OF HALOPHILES

Natrialba chahannaoensis Reference: Xu et al., 2001 Habitat: Chahannao soda lake, China Salt range for growth: up to saturation; optimum DNA G+C content: 63.7 mol% Type strain: JCM 10990 Genus XI Natrinema Natrinema pellirubrum The type strain was originally deposited as Halobacterium salinarum NCIMB 786 References: Formisano, 1962; McGenity et al., 1998 Habitat: Salted hide Salt range for growth: minimum; optimum DNA G+C content: 69.9 mol% (major component), 60.0 (minor component) Type strain: NCIMB 786 Natrinema pallidum The type strain was originally deposited as Halobacterium halobium NCIMB 777; the species also includes Halobacterium trapanicum NCIMB 784 References: Formisano, 1962; McGenity et al., 1998 Habitat: Salted cod Salt range for growth: minimum; optimum DNA G+C content: not reported Type strain: NCIMB 777 Natrinema versiforme Reference: Xin et al., 2000 Habitat: Salt lake, China Salt range for growth: minimum; optimum DNA G+C content: 64.2 mol% Type strain: JCM 10478 Genus XII Natronobacterium Natronobacterium gregoryi Reference: Tindall et al., 1984 Habitat: Lake Magadi, Kenya Salt range for growth: optimum alkaliphilic DNA G+C content: 65 mol% Type strain: DSM 339 Natronobacterium nitratireducens Reference: Xin et al., 2001 Habitat Chahannao soda lake, China Salt range for growth: minimum; optimum alkaliphilic DNA G+C content: 63.5-63.8 mol% Type strain: JCM 10879 Genus XIII Natronococcus Natronococcus occultus Reference: Tindall et al., 1984 Habitat: Lake Magadi, Kenya Salt range for growth: optimum alkaliphilic DNA G+C content: 64 mol% (major component), 55.7 mol% (minor component) Type strain: NCIMB 2192

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Natronococcus amylolyticus Reference: Kanai etal., 1995 Habitat: Lake Magadi, Kenya Salt range for growth: optimum alkaliphilic DNA G+C content: 63.5 mol% Type strain: JCM 9655 Genus XIV Natronomonas Natronomonas pharaonis Basonym: Natronobacterium pharaonis References: Soliman and Trüper, 1992; Tindall et al., 1984; Kamekura et al., 1997 Habitat: Wadi Natrun, Egypt Salt range for growth: optimum alkaliphilic DNA G+C content: 61.2 mol% (major component), 51.9 mol% (minor component) Type strain: DSM 2160 Genus XV Natronorubrum Natronorubrum bangense Reference: Xu et al., 1999 Habitat: Alkaline salt lake, Tibet Salt range for growth: optimum alkaliphilic DNA G+C content: 59.9 mol% Type strain: AS 1.1984 Natronorubrum tibetense Reference: Xu et al., 1999 Habitat: Alkaline salt lake, Tibet Salt range for growth: optimum alkaliphilic DNA G+C content: 60.1 mol% Type strain: AS 1.2123

2.3. THE HALOPHILIC AND HALOTOLERANT BACTERIA Halophiles are spread all over the phyla and the orders within the bacterial domain. Halophilic Bacteria vary widely in their physiological properties: we find aerobic and anaerobic chemoheterotrophs, photoautotrophic and photoheterotrophic species, as well as chemolithotrophs (Ollivier et al., 1994; Oren, 1999). A few taxonomic groups in which halophilic and halotolerant microorganisms abound deserve a special taxonomic discussion. Photosynthetic Bacteria, both oxygenic and anoxygenic, are found up to the very highest salt concentrations. Cyanobacterial mats are abundantly present in hypersaline lakes and in saltern evaporation ponds up to salinities of and higher. Although the occurrence of highly salt-tolerant cyanobacteria is thus well documented, the taxonomy of the group is still poorly developed. This is in part due to the fact that different classification schemes exist, alternatively based on the rules of the Bacteriological Code or those of the Botanical Code. The same organism may then be known under two or more different names, and different species have sometimes been designated with identical names. Only recently have the first attempts been made to rearrange the taxonomy of the halophilic cyanobacteria, using 16S rRNA sequence comparisons as one of the principal tools to obtain relevant phylogenetic information.

TAXONOMY OF HALOPHILES

37

The description of the genus Halospirulina with the type species Halospirulina tapeticola (Nübel et al., 2000) probably represents the first formal description of a halophilic cyanobacterial species in the framework of the Bacteriological Code. Comparative taxonomic studies have also been published for the cosmopolitan species Microcoleus chthonoplastes, a species that proved to be phylogenetically coherent worldwide (Garcia-Pichel et al., 1996) and for the Halothece cluster, a group of unicellular cyanobacteria from hypersaline environments. This cluster, which contains isolates designated in the past with names such as Cyanothece, Aphanothece, Chroococcidiopsis, Myxobactron, and others, appears to be monophyletic and coherent on basis of physiological properties (Garcia-Pichel et al., 1996). As no formal nomenclature revision has been made, the old names will still be used in this book as given by the authors of the respective studies to be discussed. The halophilic anoxygenic photosynthetic sulfur bacteria of the Halorhodospira Ectothiorhodospira group have been subject to in-depth taxonomic reevaluations in recent years (Imhoff, 2001). 16S rRNA sequence analyses of the members of the family Ectothiorhodospiraceae has led to the recognition of the more extremely halophilic alkaliphilic representatives of the group as members of a separate species, Halorhodospira (Imhoff and Süling, 1996). Fatty acid composition analysis of the cellular lipids provided further justification for the separation of the two genera (Thiemann and Imhoff, 1996). Amplified 16S rDNA restriction analysis (ARDRA) has been used to aid in the species delineation within the Ectothiorhodospiraceae (Ventura et al., 1999). Recently a comparative study was performed of a large number of isolates belonging to the genus Ectothiorhodospira, a genus which now only contains species with a salinity optimum below using techniques such as DNA-DNA hybridization and restriction length polymorphism of 16S/23 rDNA (ribotyping). Only four genospecies were thus found, corresponding with the species Ectothiorhodospira mobilis, Ectothiorhodospira shaposhnikovii, Ectothiorhodospira marina, and Ectothiorhodospira halalkaliphila. Ectothiorhodospira vacuolata was suggested to be a junior synonym of Ectothiorhodospira shaposhnikovii, and Ectothiorhodospira marismortui is a junior synonym of Ectothiorhodospira mobilis (Ventura et al., 2000). The family Halomonadaceae contains a large number of metabolically versatile aerobic moderate halophiles. Extensive taxonomic rearrangements have been made within this group in the last decade. First the genera Deleya and Halovibrio and the species Paracoccus halodenitrificans were reclassified within the genus Halomonas (Dobson et al., 1993; Franzmann et al., 1988b, see also Franzmann and Tindall, 1990). Later a number of species were removed from the genus Halomonas to the related genus Chromohalobacter on the basis of 16S rRNA similarity studies (Arahal et al., 200la, 2001b). A review of the phylogeny of the family Halomonadaceae, based on 23S and 16S rDNA sequence analyses, was presented recently (Arahal et al., 2002). The family Halomonadaceae contains only few non-halophilic representatives. The halophilic members of the Halomonadaceae have been extensively discussed in reviews by Ventosa et al. (1998) and Vreeland (1992). The fermentative obligatory anaerobic halophilic Bacteria form a phylogenetically coherent group (Rainey et al., 1995). Based on 16S rRNA gene sequences the

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halophilic anaerobic bacteria are classified in the domain Bacteria within the phylum Firmicutes (Patel et al., 1995; Rainey et al., 1995; Tourova et al., 1995). The phylogenetic affiliation of Halanaerobium praevalens with the Bacillus/Clostridium group was confirmed by the amino acid sequence of its ribosomal A-protein (Matheson et al., 1987). The deep branching justifies classification in a separate order, and thus the order Halanaerobiales (originally named Haloanaerobiales) was created to accommodate these halophilic anaerobes (Rainey et al., 1995). Thus far only very few fermentative halophilic anaerobes have been characterized that do not belong to this order. Exceptional cases are Clostridium halophilum (Fendrich et al., 1990) and Thermohalobacter berrensis (Cayol et al., 2000). As of January 2002, the order contained 24 species, classified in two families, the Halanaerobiaceae (Oren et al., 1984a; Rainey et al., 1995) and the Halobacteroidaceae (Rainey et al., 1995) (Figure 2.3). With the exception of the thermophilic Halothermothrix orenii which has a G+C content of 39.6 mol%, all species have G+C contents between 27 and 36.9 mol%. Reviews on the taxonomy of the Halanaerobiales and other aspects of the group were given by Oren (1992, 2001b), Rainey et al. (1995), and Tourova (2000). Some species of the Halanaerobiales produce heat-resistant endospores. These include Sporohalobacter lortetii (Oren, 1983b), Orenia marismortui (Oren et al., 1987), and Natroniella acetigena (Zhilina et al., 1996). When initially isolated, Acetohalobium arabaticum produced spores occasionally, but with continued cultivation sporulation was no longer observed (Zavarzin et al., 1994). Additional anaerobic halophiles that probably belong to the Halanaerobiales have been isolated from salt lakes in California and Nevada (Shiba, 1991; Shiba et al., 1989). Some of these have been obtained from aerobic surface sediments. These isolates are still awaiting taxonomic characterization. Many additional branches within the Bacteria contain halophilic representatives. Much additional information on the physiological and biochemical properties of the highly diverse group of halophilic Bacteria can be found in the following chapters. The list of halophilic and halotolerant species within the domain Bacteria presented below (updated to January 2002) aims to include all those species known to grow well at salt concentrations above A few taxonomic and physiological characteristics are given for each species. More information can be found in the following chapters and in the original species descriptions.

Domain Bacteria, Phylum BX Cyanobacteria, Class I Cyanobacteria, Subsection I, Form genus V Cyanothece Form genus VI Dactylococcopsis Subsection III Form genus VIII Microcoleus

TAXONOMY OF HALOPHILES

Genus Halospirulina (Form genus XIII Spirulina) Halospirulina tapeticola Earlier described under the names Spirulina subsalsa and Spirulina labyrinthiformis Reference: Nübel et al., 2000 Habitat: Saltern, Mexico Salt range for growth: DNA G+C content: not reported Type strain: Baja-95 Cl.2, available from the Pasteur Culture Collection, Paris

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Phylum BXII Proteobacteria, Class I " Alphaproteobucteria", Order I Rhodospirillales Family I Rhodospirillaceae Genus VII Rhodothalassium Rhodothalassium salexigens Basonym: Rhodospirillum salexigens References: Drews, 1981; Imhoff et al., 1998b Habitat: Salt water, Oregon coast Salt range for growth: optimum DNA G+C content: 64 mol% Type strain: DSM 2132 Genus VIII Rhodovibrio Rhodovibrio sodomensis Basonym: Rhodospirillum sodomense References: Mack et al., 1993; Imhoff et al., 1998b Habitat: Dead Sea shore Salt range for growth: optimum DNA G+C content: 66.2-66.6 mol% Type strain: ATCC 51195 Rhodovibrio salinarum Basonym: Rhodospirillum salinarum References: Nissen and Dundas, 1984; Imhoff et al., 1998b Habitat: Saltern, Portugal Salt range for growth: optimum DNA G+C content: 67.4 mol% Type strain: ATCC 35394 Order VI "Rhizobiales", Family VIII Hyohomicrobiaceae Genus IX Dichotomicrobium Dichotomicrobium thermohalophilum Reference: Hirsch and Hoffmann, 1989 Habitat: Solar Lake, Sinai Salt range for growth: optimum DNA G+C content: 62-64 mol% Type strain: DSM 5002 Class II "Betaproteobacteria", Order II "Hydrogenophilales", Family I "Hydrogenophilaceae" Genus ... Halothiobacillus Halothiobacillus halophilus Basonym: Thiobacillus halophilus References: Wood and Kelly, 1991; Kelly and Wood, 2000 Habitat: Hypersaline lake, Western Australia Salt range for growth: up to optimum DNA G+C content: 45 mol% Type strain: DSM 6132

TAXONOMY OF HALOPHILES

Halothiobacillus kellyi Reference: Sievert et al., 2000 Habitat: Aegean Sea hydrothermal vent Salt range for growth: optimum DNA G+C content: 62 mol% Type strain: DSM 13162 Class III "Gammaproteobacteria", Order I "Chromatiales", Family I Chromatiaceae Genus IV Halochromatium Halochromatium salexigens Basonym: Chromatium salexigens References: Caumette et al., 1988; Imhoff et al., 1998a Habitat: Saltern, France Salt range for growth: optimum DNA G+C content: 64.6 mol% Type strain: DSM 4395 Halochromatium glycolicum Basonym: Chromatium glycolicum References: Caumette et al., 1997; Imhoff et al., 1998a Habitat: Solar Lake, Sinai Salt range for growth: optimum DNA G+C content: 66.1-66.5 mol% Type strain: DSM 11080 Genus XVII Thiohalocapsa Thiohalocapsa halophila Basonym: Thiocapsa halophila References: Caumette et al., 1991a; Imhoff et al., 1998a Habitat: Saltern, France Salt range for growth: optimum DNA G+C content: 65.9-66.6 mol% Type strain: DSM 6210 Family II Ectothiorhodospiraceae Genus I Ectothiorhodospira Ectothiorhodospira mobilis Reference: Pelsh, 1936 Habitat: Salt lakes, salt flats Salt range for growth: DNA G+C content: 67.3-68.4 mol% Type strain: DSM 237 Ectothiorhodospira marismortui References: Oren et al., 1989; Ventura et al., 2000 Habitat: Sulfur spring on the Dead Sea shore Salt range for growth: optimum DNA G+C content: 65 mol% Type strain: DSM 4180 Suggested to be a junior synonym of Ectothiorhodospira mobilis

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Ectothiorhodospira haloalkaliphila Reference: Imhoff and Süling, 1996 Habitat: Soda lakes Salt range for growth: optimum alkaliphilic DNA G+C content: 62.2-63.5 mol% Type strain: ATCC 51935 Genus II Arhodomonas Arhodomonas aquaeolei Reference: Adkins et al., 1993 Habitat: Petroleum reservoir production fluid Salt range for growth: optimum DNA G+C content: 67 mol% Type strain: ATCC 49307 Genus III Halorhodospira Halorhodospira halophila Basonym: Ectothiorhodospira halophila References: Raymond and Sistrom, 1969; Imhoff and Süling, 1996 Habitat: Salt and soda lakes Salt range for growth: optimum alkaliphilic DNA G+C content: 68.4 mol% Type strain: DSM 244 Halarhodospira halochloris Basonym: Ectothiorhodospira halochloris References: Imhoff and Trüper, 1977; Imhoff and Süling, 1996 Habitat: Soda lakes, Egypt Salt range for growth: optimum alkaliphilic DNA G+C content: 50.5-52.9 mol% Type strain: DSM 1059 Halorhodospira abdelmalekii Basonym: Ectothiorhodospira abdelmalekii References; Imhoff and Trüper 1981; Imhoff and Süling, 1996 Habitat: Wadi Natrun, Egypt Salt range for growth: optimum alkaliphilic DNA G+C content: 63.3-63.8 mol% Type strain: DSM 2110 Genus ... Alcalilimnicola Alcalilimnicola halodurans Reference: Yakimov et al., 2001 Habitat: Lake Natron, Tanzania Salt range for growth: optimum alkaliphilic DNA G+C content: 65.6 mol% Type strain: DSM 13718

TAXONOMY OF HALOPHILES

43

Order VII "Oceanospirillales", Family II Halomonadaceae Reference: Franzmann et al., 1988 Genus I Halomonas Halomonas elongata Reference: Vreeland et al., 1980 Habitat: Saltern, Bonaire Salt range for growth: optimum DNA G+C content: 60.5 mol% Type strain: ATCC 33173 Halomonas subglaciescola Reference: Franzmann et al., 1987 Habitat: Antarctic saline lakes Salt range for growth: DNA G+C content: 60.9-62.9 mol% Type strain: UCM 2927 Halomonas halodurans Basonym: Pseudomonas halodurans Reference: Rosenberg, 1983; Hebert and Vreeland, 1987 Habitat: Estuarine water Salt range for growth: optimum DNA G+C content: 63.2 mol% Type strain: ATCC 29686 Halomonas halmophila Basonym: Flavobacterium halmophilum or Flavobacterium halmephilum References: Elazari-Volcani, 1940; Dobson et al., 1990 Habitat: the Dead Sea Salt range for growth: DNA G+C content: 63 mol% Type strain: ATCC 19717 Halomonas eurihalina Basonym: Volcaniella eurihalina References: Quesada et al., 1990; Mellado et al., 1995 Habitat: Saline soils, salterns Salt range for growth: optimum DNA G+C content: 59.1-65.7 mol% Type strain: ATCC 49336 Halomonas halophila Basonym: Deleya halophila References: Quesada et al., 1984; Dobson and Franzmann, 1996 Habitat: Saline soils Salt range for growth: optimum DNA G+C content: 66.7 mol% Type strain: CCM 3662 Halomonas salina Basonym: Deleya salina References: Valderrama et al., 1991; Dobson and Franzmami, 1996; see also Baumgarte et al., 2001 for a discussion of problems with the authenticity of deposited 16S rRNA sequences Habitat: Saline soils, salterns Salt range for growth: optimum DNA G+C content: 60.7-64.2 mol% Type strain: ATCC 49509

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Halomonas halodenitrificans Basonym: Paracoccus halodenitrificans References: Miller et al., 1994; Dobson and Franzmann, 1996 Habitat: Meat-curing brines Salt range for growth: optimum DNA G+C content: 64-66 mol% Type strain: ATCC 1 3 5 1 1 Halomonas variabilis Basonym: Halovibrio variabilis References: Fendrich, 1988; Dobson and Franzmann, 1996 Habitat: the Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 61 mol% Type strain: DSM 3051 Halomonas pantelleriensis Reference: Romano et al., 1996 (originally described as Halomonas pantelleriense) Habitat: Pantelleria island, Italy Salt range for growth: optimum alkaliphilic DNA G+C content: 65 mol% Type strain: DSM 9661 Halomonas magadiensis Reference: Duckworth et al., 2000 (originally described as Halomonas magadii) Habitat: East African soda lakes Salt range for growth: optimum alkaliphilic DNA G+C content: 62 mol% Type strain: NCIMB 13595 Halomonas desiderata Reference: Berendes et al., 1996 Habitat: Sewage treatment plant Salt range for growth: alkaliphilic DNA G+C content: 66 mol% Type strain: DSM 9502 Halomonas meridiana Reference: James et al., 1990 Habitat: Antarctic saline lakes Salt range for growth: optimum DNA G+C content: 58.8-59.1 mol% Type strain: ACAM 246 Halomonas campisalis Reference: Mormile et al., 1999 Habitat: Salt plane sediment of Alkali Lake, Washington Salt range for growth: optimum moderately alkaliphilic DNA G+C content: 66 mol% Type strain: ATCC 700597 Halomonas maura Reference: Bouchotroch et al., 2001 Habitat: Saltern, Morocco Salt range for growth: optimum DNA G+C content: 62.2-64.1 mol% Type strain: DSM 13445 Halomonas alimentaria Reference: Yoon et al., 2002 Habitat: Jeotgal (Korean fermented seafood) Salt range for growth: optimum DNA G+C content: 63 mol% Type strain: JCM 10888

TAXONOMY OF HALOPHILES Genus IV Chromohalobacter Chromohalobacter marismortui Basonym: Chromobacterium marismortui References: Elazari-Volcani, 1940; Ventosa et al., 1989a Habitat: The Dead Sea, salterns Salt range for growth: optimum DNA G+C content: 62.1-64.9 mol% Type strain: ATCC 17056 Chromohalobacter canadensis Basonym: Halomonas canadensis References: Huval et al., 1995; Arahal et al., 2001a Habitat: Unknown (isolated as a culture contaminant) Salt range for growth: optimum DNA G+C content: 54-57 mol% Type strain: ATCC 43984 Chromohalobacter israelensis Basonym: Halomonas israelensis References: Huval et al., 1995; Arahal et al., 2001a Habitat: The Dead Sea Salt range for growth: optimum DNA G+C content: 64 mol% Type strain: ATCC 43985 Chromohalobacter salexigens Previously classified within the species Halomonas elongata (strain DSM 3043; ATCC 33174) Reference: Arahal et al., 2001b Habitat: Solar salterns Salt range for growth: optimum DNA G+C content: 64-66 mol% Type strain: DSM 3043 Order VIII Pseudomonadales, Family I Pseudomonadaceae Genus I Pseudomonas Pseudomonas halophila Reference: Fendrich, 1988 Habitat: The Great Salt Lake, Utah Salt range for growth: DNA G+C content: 57 mol% Type strain: DSM 3050 Pseudomonas beijerinckii Reference: Hof, 1935 Habitat: Salted beans Salt range for growth: optimum DNA G+C content: not reported Type strain: ATCC 19372

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Order IX "Alteromonadales", Family I "Alteromonadaceae" Genus V Marinobacter Marinobacter hydrocarbonoclasticus Reference: Gauthier et al., 1992 Habitat: Mediterranean seawater Salt range for growth: DNA G+C content: 52.7 mol% Marinobacter aquaeolei Reference: Huu et al., 1999 Habitat: Oil well, Vietnam Salt range for growth: DNA G+C content: 55.7 mol%

optimum Type strain: ATCC 49840

optimum Type strain: DSM 11845

Order X "Vibrionales", Family I Vibrionaceae Genus VI Salinivibrio Salinivibrio costicola Basonym: Vibrio costicola References: Garcia et al., 1987; Mellado et al., 1996 Habitat: Cured meat and brines Salt range for growth: optimum DNA G+C content: 49.4-50.5 mol% Type strain: NCIMB 701 Subspecies: Salinivibrio costicola subsp. vallismortis Reference: Huang et al., 2000 Habitat: Death Valley, California Salt range for growth: optimum DNA G+C content: 50 mol% Type strain: DSM 8285 Class IV "Deltaproteobacteria", Order II "Desulfovibrionales", Family I "Desulfovibrionaceae" Genus I Desulfovibrio Desulfovibrio halophilus Reference: Caumette et al., 1991b Habitat: Solar Lake, Sinai Salt range for growth: optimum DNA G+C content: 60.7 mol% Type strain: DSM 5663 Desulfovibrio senezii Reference: Tsu et al., 1998 Habitat: Saltern, California Salt range for growth: optimum DNA G+C content: 62 mol% Type strain: DSM 8436 Desulfovibrio oxyclinae Reference: Krekeler et al., 1997 Habitat: Solar Lake, Sinai Salt range for growth: optimum DNA G+C content: Not reported. Type strain: DSM 11498

TAXONOMY OF HALOPHILES

"Desulfovibrio vietnamensis" Reference: Nga et al., 1996 Habitat: Oil production waters, Vietnam Salt range for growth: optimum DNA G+C content: 60.6 mol% Type strain: DSM 10520 The name of the species has not yet been validated Family III "Desulfohalobiaceae" Genus I Desulfohalobium Desulfohalobium retbaense Reference: Ollivier et al., 1991 Habitat: Lake Retba, Senegal Salt range for growth: DNA G+C content: 57.1 mol%

optimum Type strain: DSM 5692

Order III "Desulfobacterales", Family I "Desulfobacteraceae" Genus I Desulfobacter Desulfobacter halotolerans Reference: Brandt and Ingvorsen, 1997 Habitat: Sediment of the Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 49 mol% Type strain: DSM 11383 Genera of unknown affiliation: Genus... Desulfonatronovibrio Desulfonatronovibrio hydrogenovorans Reference: Zhilina et al., 1997b Habitat: Lake Magadi, Kenya Salt range for growth: optimum DNA G+C content: 48.8 mol% Type strain: DSM 9292 Genus... Desulfocella Desulfocella halophila Reference: Brandt et al., 1999 Habitat: Sediment of the Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 35 mol% Type strain: DSM 11763 Order VI "Bdellovibrionales" Highly halotolerant Bdellavibrio strains that grow up to NaCl have been isolated from saltern ponds in Spain (Sánchez Amat and Torrella, 1989). The isolates have never been formally described as a new species.

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Phvlum BXIII Firmicutes , Class I "Clostridia", Order I. Clostridiales Family I Clostridiaceae Genus I Clostridium Clostridium halophilum Reference: Fendrich et al., 1990 Habitat: Anoxic hypersaline and marine sediments Salt range for growth: optimum DNA G+C content: 26.9 mol% Type strain: DSM 5387 Family V. Peptococcaceae Genus XII Desulfotomaculum Desulfotomaculum halophilum Reference: Tardy-Jaquenod et al., 1998 Habitat: Oil field brine, France Salt range for growth: optimum DNA G+C content: 56.3 mol% Type strain: DSM 11559 Genus of unknown affiliation: Genus ... Thermohalobacter Thermohalobacter berrensis Reference: Cayol et al., 2000 Habitat: Solar saltern, France Salt range for growth: optimum thermophilic, grows up to 70 °C DNA G+C content: 33 mol% Type strain: CNCM 105955 Order III Halanaerobiales, Family I Halanaerobiaceae References: Rainey et al., 1996; Oren et al., 1984a, 2000 Genus I Halanaerobium Halanaerobium praevalens Originally described as Haloanaerobium praevalens; may be similar to "Bacteroides halosmophilus" from salted anchovies (Baumgartner, 1937); identical 16S rRNA sequences have recently been recovered from canned Swedish fermented herrings (Surströmming) (Kobayashi et al., 2000) References: Zeikus et al., 1983; Oren, 2000a Habitat: Sediment of the Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 27-28 mol% Type strain: DSM 2228 Halanaerobium alcaliphilum Originally described as Haloanaerobium alcaliphilum References: Tsai et al., 1995; Oren, 2000 Habitat: Sediment of the Great Salt Lake, Utah Salt range for growth: optimum slightly alkaliphilic DNA G+C content: 31 mol% Type strain: DSM 8275

TAXONOMY OF HALOPHILES

Halanaerobium acetethylicum Basonym: Halobacteroides acetoethylicus, subsequently renamed Haloanaerobium acetoethylicum References: Rengpipat et al., 1988; Patel et al., 1995, Rainey et al., 1995; Oren, 2000 Habitat: Offshore oil rig, Gulf of Mexico Salt range for growth: optimum DNA G+C content: 32 mol% Type strain: ATCC 43120 Halanaerobium salsuginis Earlier named Haloanaerobium salsugo, Haloanaerobium salsuginis References: Bhupathiraju et al., 1994; Trüper and de' Clari, 1998; Oren, 2000 Habitat: Petroleum reservoir, Oklahoma Salt range for growth: optimum DNA G+C content: 34 mol% Type strain: ATCC 51327 Halanaerobium saccharolyticum Halanaerobium saccharolyticum subsp. saccharolyticum Basonym: Haloincola saccharolyticus (originally described as Haloincola saccharolytica), then renamed Haloanaerobium saccharolyticum References: Zhilina et al., 1992a; Rainey et al., 1995; Euzéby, 1998; Oren, 2000 Habitat: Lake Sivash, Crimea Salt range for growth: optimum DNA G+C content: 31.3 mol% Type strain: DSM 6643 Halanaerobium saccharolyticum subsp. senegalensis Basonym: Haloincola saccharolyticus subsp. senegalensis (originally described as Haloincola saccharolytica subsp. senegalensis), then renamed Haloanaerobium saccharolyticum subsp. senegalensis References: Cayol et al., 1994a; Rainey et al., 1995; Euzéby, 1998; Oren, 2000 Habitat: Lake Retba, Senegal Salt range for growth: optimum DNA G+C content: 31.7 mol% Type strain: DSM 7379 Halanaerobium congolense First described as Haloanaerobium congolense References: Ravot et al., 1997; Oren, 2000 Habitat: Offshore oil well, Congo Salt range for growth: optimum DNA G+C content: 34 mol% Type strain: DSM 11287 Halanaerobium lacusrosei First described as Haloanaerobium lacusroseus References: Cayol et al., 1995; Oren, 2000 Habitat: Sediment of Lake Retba, Senegal Salt range for growth: optimum DNA G+C content: 32 mol% Type strain: DSM 10165 Halanaerobium kushneri First described as Haloanaerobium kushneri References: Bhupathiraju et al., 1999; Oren, 2000 Habitat: Petroleum reservoir fluid, Oklahoma Salt range for growth: optimum DNA G+C content: 32-37 mol% Type strain: ATCC 700103

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Halanaerobium fermentans First described as Haloanaerobium fermentans References: Kobayashi et al., 2000b; Oren, 2000 Habitat: Salted puffer fish ovaries Salt range for growth: optimum DNA G+C content: 33.3 mol% Type strain: JCM 10494 Genus II Halocella Halocella cellulosilytica First described as Halocella cellulolytica References: Simankova et al., 1993; Oren, 2000 Habitat: Sediment of Lake Sivash, Crimea Salt range for growth: optimum DNA G+C content: 29 mol% Type strain: DSM 7362 Genus III Halothermothrix Halothermothrix orenii Reference: Cayol et al., 1994b Habitat: Sediment of a hypersaline lake, Tunisia Salt range for growth: optimum thermophilic DNA G+C content: 39.6 mol% Type strain: OCM 544 Genus IV Natroniella Natroniella acetigena Reference: Zhilina et al., 1996 Habitat: Sediment of Lake Magadi, Kenya Salt range for growth: optimum alkaliphilic DNA G+C content: 31.9 mol% Type strain: DSM 9952 Family II Halobacteroidaceae Reference: Rainey et al. 1995 Genus I Halobacteroides Halobacteroides halobius Reference: Oren et al., 1984b Habitat: Sediment of the Dead Sea Salt range for growth: optimum DNA G+C content: 30.7 mol% Type strain: ATCC 35273 Halobacteroides elegans Originally described as a strain of Halobacteroides halobius Reference: Zhilina et al., 1997a Habitat: Cyanobacterial mat, Lake Sivash, Crimea Salt range for growth: optimum DNA G+C content: 30.5 mol% Type strain: DSM 6639

TAXONOMY OF HALOPHILES Genus II Acetohalobium Acetohalobium arabaticum Reference: Zhilina and Zavarzin, 1990 Habitat: Sediment of Lake Sivash, Crimea Salt range for growth: optimum DNA G+C content: 33.6 mol% Type strain: DSM 5501 Genus III Halanaerobacter Halanaerobacter chitinivorans Originally described as Haloanaerobacter chitinovorans References: Liaw and Mah, 1992; Oren, 2000 Habitat: Sediment of a saltern, California Salt range for growth: optimum DNA G+C content: 34.8 mol% Type strain: OCG 229 Halanaerobacter lacunarum Basonym: Halobacteroides lacunaris; later renamed Haloanaerobacter lacunaris References: Zhilina et al., 1992b; Rainey et al., 1995; Oren, 2000 Habitat: Lake Chokrak, Kerech Peninsula Salt range for growth: optimum DNA G+C content: 32.4 mol% Type strain: DSM 6640 Halanaerobacter salinarius Originally described as Haloanaerobacter salinarius References: Mouné et al., 1999; Oren, 2000 Habitat: Sediment of a saltern, France Salt range for growth: optimum DNA G+C content: 31.6 mol% Type strain: DSM 12146 Genus IV Orenia Orenia marismortui Basonym: Sporohalobacter marismortui References: Oren et al., 1987; Rainey et al., 1995 Habitat: Sediment of the Dead Sea Salt range for growth: optimum DNA G+C content: 29.6 mol% Type strain: ATCC 35420 Orenia salinaria Reference: Mouné et al., 2000 Habitat: Sediment of a saltern, France Salt range for growth: optimum DNA G+C content: 33.7 mol% Type strain: ATCC 700911 Orenia sivashensis Reference: Zhilina et al., 1999 Habitat: Cyanobacterial mat, Lake Sivash, Crimea Salt range for growth: optimum DNA G+C content: 28 mol% Type strain: DSM 12596

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Genus V Sporohalobacter Sporohalobacter lortetii Basonym: Clostridium lortetii References: Oren, 1983b; Oren et al., 1987 Habitat: Sediment of the Dead Sea Salt range for growth: optimum DNA G+C content: 31.5 mol% Type strain: ATCC 35059 Genus ... Selenihalanaerobacter Selenihalanaerobacter shriftii Reference: Switzer Blum et al., 2001 Habitat: Sediment of the Dead Sea Salt range for growth: optimum DNA G+C content: 31.2 mol% Type strain: ATCC BAA-73 Genus ... Halonatronum Halonatronum saccharophilum Reference: Zhilina et al., 2001 Habitat: Sediment from Lake Magadi, Kenya Salt range for growth: optimum DNA G+C content: 34.4 mol% Type strain: DSM 13868 Class III "Bacilli", Order I Bacillales, Family I Bacillaceae Genus I Bacillus Bacillus halophilus Reference: Ventosa et al., 1989b Habitat: Isolated from rotting wood from seawater Salt range for growth: optimum DNA G+C content: 51.5 mol% Type strain: ATCC 49085 Bacillus haloalkaliphilus Reference: Fritze, 1996 Habitat: Alkaline lakes of the Wadi Natrun, Egypt Salt range for growth: up to alkaliphilic DNA G+C content: 37-38 mol% Type strain: DSM 5271 Genus IV Gracilibacillus Gracilibacillus halotolerans Reference: Wainø et al., 1999 Habitat: The Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 38 mol% Type strain: DSM 11805 Gracilibacillus dipsosauri Basonym: Bacillus dipsosauri References: Lawson et al. 1996; Wainø et al., 1999 Habitat: Nasal glands of desert iguanas Salt range for growth: optimum KC1 DNA G+C content: 39.4 mol% Type strain: NCFB 3027

TAXONOMY OF HALOPHILES

Genus V Halobacillus Halobacillus halophilus Basonym: Sporosarcina halophila References: Claus et al., 1983; Farrow et al., 1992; Spring et al., 1996 Habitat: Salt marsh soils Salt range for growth: optimum DNA G+C content: 40.1-40.9 mol% Type strain: DSM 2266 Halobacillus litoralis Reference: Spring et al., 1996 Habitat: the Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 42 mol% Type strain: DSM 10405 Halobacillus trueperi Reference: Spring et al., 1996 Habitat: The Great Salt Lake, Utah Salt range for growth: optimum DNA G+C content: 43 mol% Type strain: DSM 10404 Halobacillus thailandensis Reference: Chaiyanan et al., 1999 Habitat: Fermented fish sauce Salt range for growth: optimum DNA G+C content: 41 mol% Type strain: unknown; the listing as DSM 10405 in Chaiyanan et al., 1999 is incorrect; The name of the species has not yet been validated Genus VII Salibacillus Salibacillus salexigens Basonym: Bacillus salexigens References: Garabito et al., 1997; Wainø et al., 1999 Habitat: Salterns, saline soils Salt range for growth: optimum DNA G+C content: 36.9-39.5 mol% Type strain: ATCC 700290 Salibacillus marismortui Basonym: Bacillus marismortui References: Arahal et al., 1999, 2000 Habitat: the Dead Sea Salt range for growth: optimum DNA G+C content: 39.0-42.8 mol% Type strain: DSM 12325 Genus ... Oceanobacillus Oceanobacillus iheyensis Reference: Lu et al., 2001 Habitat: Deep sea bottom, Iheya Ridge Salt range for growth: optimum alkaliphilic DNA G+C content: 35.8 mol% Type strain: JCM 11309 The names of the genus and the species have not yet been validated

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Family V "Staphylococcaceae" Genus IV Salinicoccus Salinicoccus roseus Reference: Ventosa et al., 1990 Habitat: Salterns Salt range for growth: optimum DNA G+C content: 51.2 mol% Type strain: ATCC 49258 Salinicoccus hispanicus Basonym: Marinococcus hispanicus References: Márquez et al., 1990; Ventosa et al., 1992 Habitat: Salterns, saline soils Salt range for growth: optimum DNA G+C content: 45.6-49.3 mol% Type strain: ATCC 49259 Family VI "Snorolactobacillaceae" Genus II Marinococcus Marinococcus halophilus Basonym: Planococcus halophilus References: Novitsky and Kushner, 1976; Hao et al., 1984; Márquez et al., 1992 Habitat: Salterns, saline soils Salt range for growth: optimum DNA G+C content: 46.4 mol% Type strain: ATCC 27964 Marinococcus albus Reference: Hao et al., 1984 Habitat: Salterns Salt range for growth: optimum DNA G+C content: 44.9 mol% Type strain: CCM 3517 Order II "Lactobacillales", Family IV "Enterococcaceae" Genus III Tetragenococcus Tetragenococcus halophilus Reference: Mees, 1934; Collins et al., 1990 Habitat: Anchovies, soy mash Optimum salt range for growth: DNA G+C content: 34-35 mol% Type strain: ATCC 33315 Tetragenococcus muraticus Reference: Satomi et al., 1997 Habitat: Fermented squid liver sauce Salt range for growth: optimum DNA G+C content: 36.5 mol% Type strain: JCM 10006

TAXONOMY OF HALOPHILES Phylum BXIV Actinobacteria, Class I Actinobacteria, Subclass V Actinobacteridae, Order I Actinomycetales, Family I Micrococcaceae Genus VII Nesterenkonia Nesterenkonia halobia Basonym: Micrococcus halobius References: Onishi and Kamekura, 1972; Stackebrandt et al., 1995; Mota et al., 1997 Habitat: Salterns and solar salt, Japan Salt range for growth: optimum DNA G+C content: 70-72 mol% Type strain: ATCC 21727 Suborder X Pseudonocardineae, Family I Pseudonocardiaceae Genus II Actinopolyspora Actlnopolyspora halophila Reference: Gochnauer et al., 1975 Habitat: Unknown; isolated as a culture contaminant Salt range for growth: optimum DNA G+C content: 64.2 mol% Type strain: ATCC 27976 Actinopolyspora mortivallis References: Yoshida, 1991; Yoshida et al., 1991 Habitat: Saline soil, Death Valley, California Salt range for growth: optimum DNA G+C content: 68 mol% Type strain: JCM 7550 Actinopolyspora iraqiensis Reference: Ruan et al., 1994 Habitat: Saline soil, Iraq Salt range for growth: optimum DNA G+C content: Not reported Type strain: A.S.4.1193 Suborder XII Streptosporangineae, Family II Nocardiopsaceae Genus I Nocardiopsis Nocardiopsis lucentensis Reference: Yassin et al., 1993 Habitat: Saline soil, Alicante, Spain Salt range for growth: DNA G+C content: 71 mol% Type strain: DSM 44048 Nocardiopsis halophila References: Al-Tai and Ruan, 1994 Habitat: Saline soil Salt range for growth: optimum DNA G+C content: Not reported Type strain: A.S.4.1195 Nocardiopsis kunsanesis Reference: Chun et al., 2000. Habitat: Saltern, Korea Salt range for growth: optimum DNA G+C content: 71 mol% Type strain: JCM 10721

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Phylum BXX Bacteroidetes, Class II "Flavobactcria", Family I Flavobacteriaceae Genus I Flavobacterium Flavobacterium salegens Reference: Dobson et al., 1993 Habitat: Hypersaline Antarctic lake Salt range for growth: optimum DNA G+C content: 39-41 mol% Type strain: DSM 5424 Genus of unknown affiliation: Genus ... Salinibacter Salinibacter ruber Reference: Antón et al., 2002 Habitat: Salterns, Spain Salt range for growth: optimum DNA G+C content: 66.5 mol% Type strain: DSM 13855

2.4. THE HALOPHILIC AND HALOTOLERANT EUCARYA The diversity of eukaryotic microorganisms able to grow at high salinities is relatively restricted. However, Eucarya do contribute significantly to the biota of hypersaline environments. Green algae of the genus Dunaliella are the main or sole primary producers in environments that arc too salty for even the best salt-adapted among the cyanobacteria. Dunaliella is a ubiquitous genus with species that are normal denizens of waters ranging in salinity from slightly brackish to saturated sodium chloride brines. Species found up to the highest salinity range are Dunaliella salina, Dunaliella bardawil (both often containing large amounts of and the smaller green Dunaliella viridis and Dunaliella parva. Little attention has been devoted to the taxonomic aspects of this group of algae, and their phylogenetic relationships are poorly known. Only recently have the first 18S rRNA genes of different Dunaliella species been compared (Olmos et al., 2000; Olmos-Soto et al., 2002). Two introns were found in the 18S rRNA gene of Dunaliella bardawil, the gene of Dunaliella salina contains a single intron, and no introns were found in other species of the genus (Olmos-Soto et al., 2002). The rDNA spacer region has also been used for molecular phylogenetic studies of the genus (González et al., 2001). Another interesting eukaryotic halophilic green alga that has only recently been discovered is Picocystis salinarum. This small (about organism was first isolated from saltern evaporation ponds salt) in San Francisco Bay (Lewin et al., 2000). A similar organism abounds in Mono Lake, CA (see also Section 16.2). The Mono Lake strain (designated strain ML) can grow over a very large range of salt concentrations, from (Roesler et al., 2002). Diatoms may be found up to salt concentrations of about A survey of the salinity tolerance of diatoms present in the salterns and hypersaline tidal channels of

TAXONOMY OF HALOPHILES

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Baja, California (Mexico) showed that most halophilic diatoms have their salinity optimum below 75 salt, while only a minority could grow at 125-150 and none grew at 175 salt. The most halotolerant strains found were two unidentified Amphora species, Amphora cf. subaculiuscula, Nitzschia fusiformis, and Entomoneis sp. (Clavero et al., 2000). Protozoa have often been reported from hypersaline lakes. The most comprehensive survey on the occurrence of protozoa at high salt concentrations was made in Hutt Lagoon, Western Australia (Post et al., 1983). This lagoon ranges in salinity from 180 in winter to NaCl saturation in summer, and is fed by a mixture of thalassohaline and athalassohaline water sources. Ten zooflagellates and four sarcodines were frequently observed in brines with above 150 salt. Ciliates found include the bacteriophagous Trachelocerca conifer, Metacystis truncata, Chilophrya utahensis, Rhodopalophrya salina, Uronema marinum, Condlylostoma sp., and Palmarella salina. Species eating both bacteria and algae were Nassula sp., Fabrea salina, Blepharisma halophila, Cladotrichia sigmoidea, and Euplotes sp. Ciliates feeding on other ciliates include Podophrya sp. and Trematosoma bocquetti. Among the zooflagellates recorded were several species of Monosiga, Rhynchomonas nasuta, Phyllomitus sp., Tetramitus salinus, Tetramitus cosmopolites, Bodo caudatus, Bodo edax, and three other distinctive Bodo species. The sarcodina included Heteramoeba sp. and Naegleria sp. Some protozoa evidently encyst in the salt crust and germinate on subsequent dissolution of the salt. Finally there are also fungi that have found a niche in hypersaline environments. The presence of fungi living at high salt concentrations has long been ignored. However, it is increasingly becoming clear that certain groups of fungi may significantly contribute to the biota of salterns and other highly saline waters. One of these isolates is the meristematic fungus Trimmatostroma salinum, first isolated from a saltern pond at the Adriatic coast (Zalar et al., 1999). Black yeasts such as Hortaea werneckii and others were found to be indigenous to such environments, and only now are we obtaining some insight in their importance in the ecosystem (Gunde-Cimerman et al., 2000).

2.5. REFERENCES Adkins, J.P., Madigan, M.T., Madelco, L., Woese, C.R., and Tanner, R.S. 1993. Arhodomonas aquaeolei gen. nov., sp. nov., an aerobic halophilic bacterium isolated from a subterranean brine. Int. J. Syst. Bacteriol. 43: 514-520. Al-Tai, A.M., and Ruan, J.-S. 1994. Nocardiopsis halophila sp. nov., a new halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44: 474-478. Amann, G., Stetter, K.O., Llobet-Brossa, E., Amann, R., and Antón, J. 2000. Direct proof for the presence and expression of two 5% different 16S rRNA genes in individual cells of Haloarcula marismortui, Extremophiles 4: 373-376. Antón, J., Llobet-Brossa, E., Rodríguez-Valera, F., and Amann, R. 1999. Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environ. Microbiol. 1: 517-523. Antón, J., Oren, A., Benlloch, S., Rodríguez-Valera, F., Amann, R., and Rosselló-Mora, R. 2002. Salinibacter ruber gen. nov., sp. nov., a novel extreme halophilic Bacterium from saltern crystallizer ponds. Int. J. Syst. Evol. Microbiol. 52: 485-491.

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Arahal, D.R., Márquez, M.C., Volcani, B.E., Schleifer, K.H., and Ventosa, A. 1999. Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int. J. Syst. Bacteriol. 49: 521-530. Arahal, D.R., Márquez, M.C., Volcani, B.E., Schleifer, K.H., and Ventosa, A. 2000. Reclassification of Bacillus marismortui as Salibacillus marismortui comb. nov. Int. J. Syst. Evol. Microbiol. 50: 1501-1503. Arahal, D.R., García, M.T., Ludwig, W., Schleifer, K.H., and Ventosa, A. 2001a. Transfer of Halomonas canadensis and Halomonas israelensis to the genus Chromohalobacter as Chromohalobacter canadensis comb. nov. and Chromohalobacter israelensis comb. nov. Int. J. Syst. Evol. Microbiol. 51: 1443-1448. Arahal, D.R., García, M.T., Vargas, C., Cánovas, D., Nieto, J.J., and Ventosa, A. 2001b. Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174. Int. J. Syst. Evol. Microbiol. 51: 1457-1462. Arahal, D.R., Ludwig, W., Schleifer, K.H., and Ventosa, A. 2002. Phylogeny of the family Halomonadaceae based on 23S and 16S rDNA sequence analyses. Int. J. Syst. Evol. Microbiol. 52: 241-249. Baumgarte, S., Moore, E.R.B., and Tindall, B.J. 2001. Re-examining the 16S rDNA sequence of Halomonas salina. Int. J. Syst. Evol. Microbiol. 51: 51-53. Baumgartner, J.G. 1937. The salt limits and thermal stability of a new species of anaerobic halophile. Food Res. 2: 321-329. Berendes, F., Gottschalk, G., Heine-Dobbernack, E., Moore, E.R.B., and Tindall, B.J. 1996. Halomonas desiderata sp. nov., a new alkaliphilic, halotolerant and denitrifying bacterium isolated from a municipal sewage works. Syst. Appl. Microbiol. 19: 158-167. Bhupathiraju, V.K., Oren, A., Sharma, P.K., Tanner, R.S., Woese, C.R., and McInerney, M.J. 1994. Haloanaerobium salsugo sp. nov., a moderately halophilic, anaerobic bacterium from a subterranean brine. Int. J. Syst. Bacteriol. 44: 565-572. Bhupathiraju, V.K., McInerney, M.J., Woese, C.R., and Tanner, R.S. 1999. Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine. Int. J. Syst. Bacteriol. 49: 953-960. Boone, D.R., and Baker, C.C. 2001. Genus VI. Methanosalsum gen. nov., pp. 287-288 In: Boone, D.R., and Castenholz, R.W. (Eds.), Bergey’s manual of systematic bacteriology, Ed., Vol. 1. Springer-Verlag, New York. Boone, D.R., and Castenholz, R.W. (Eds.), Garrity, G.M. (Editor-in-Chief). 2001. Bergey’s Manual of Systematic Bacteriology, Ed., Vol. 1. Springer-Verlag, New York. Boone, D.R., Mathrani, I.M., Liu, Y., Menaia, J.A.G.F., Mah, R.A., and Boone, J.E. 1993. Isolation and characterization of Methanohalophilus portucalensis sp. nov. and DNA reassociation study of the genus Methanohalophilus. Int. J. Syst. Bacteriol. 43: 430-437. Bouchotroch, S., Quesada, E., del Moral, A., Llamas, I., and Bejar, V. 2001. Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium. Int. J. Syst. Evol. Microbiol. 51: 1625-1632. Brandt, K.K., and Ingvorsen, K. 1997. Desulfobacter halotolerans sp. nov., a halotolerant acetate-oxidizing sulfate-reducing bacterium isolated from sediments of Great Salt Lake, Utah. Syst. Appl. Microbiol. 20: 366373. Brandt, K.K., Patel, B.K.C., and Ingvorsen, K. 1999. Desulfocella halophila gen. nov., sp. nov., a halophilic, fattyacid-oxidizing, sulfate-reducing bacterium isolated from sediments of the Great Salt Lake. Int. J. Syst. Bacteriol. 49: 193-200. Briones, C., and Amils, R. 2000. Nucleotide sequence of the 23S rRNA of Haloferax mediterranei and phylogenetic analysis of halophilic archaea based on LSU rRNA. Syst. Appl. Microbiol. 23: 124-131. Caumette, P., Baulaigue, R., and Matheron, R. 1988. Characterization of Chromatium salexigens sp. nov., a halophilic Chromatiaceae isolated from Mediterranean salinas. Syst. Appl. Microbiol. 10: 284-292. Caumette, R., Baulaigue, R., and Matheron, R. 1991a. Thiocapsa halophila sp. nov., a new halophilic phototrophic purple sulfur bacterium. Arch. Microbiol. 155: 170-176. Caumette, P., Cohen, Y., and Matheron, R. 1991b. Isolation and characterization of Desulfovibrio halophilus sp. nov., a halophilic sulfate-reducing bacterium isolated from Solar Lake (Sinai). Syst. Appl. Microbiol. 14: 3338. Caumette, P., Imhoff, J.F., Süling, J., and Matheron, R. 1997. Chromatium glycolicum sp. nov., a moderately halophilic purple sulfur bacterium that uses glycolate as substrate. Arch. Microbiol. 167: 11-18. Cayol, J-L., Ollivier, B., Lawson Anani Soh, A., Fardeau, M.-L., Ageron, E., Grimont, P.A.D., Prensier, G., Guezennec, J., Magot, M., and Garcia, J.-L. 1994a. Haloincola saccharolytica subsp. senegalensis subsp. nov., isolated from the sediments of a hypersaline lake, and emended description of Haloincola saccharolytica. Int. J. Syst. Bacteriol. 44: 805-811.

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Yakimov, M.M., Giuliano, L., Chemikova, T.N., Gentile, G., Abraham, W.R., Lunsdorf, H., Timmis, K.N., and Golyshin, P.N. 2001. Alcalilimnicola halodurans gen. nov., sp. nov., an alkaliphilic, moderately halophilic and extremely halotolerant bacterium, isolated from sediments of soda-depositing Lake Natron, East Africa Rift Valley. Int. J. Syst. Evol. Microbiol. 51: 2133-2143. Yassin, A.F., Galinski, E.A., Wohlfarth, A., Jalmke, K.-D., Schaal, K.-P., and Trüper, H.G. 1993. A new actinomycete species, Nocardiopsis lucentensis sp. nov. Int. J. Syst. Bacteriol. 43: 266-271. Yoon, J.-H., Lee, K.-C., Kho, Y.H., Kang, K.H., Kim, J.-W., and Park, Y.-H. 2002. Halomonas alimentaria sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int. J. Syst. Evol. Microbiol. 52: 123-130. Yoshida, M. 1991. A novel halophilic Actinopolyspora HS-1, pp. 84-96 In: Horikoshi, K., and Grant, W.D. (Eds.), Superbugs. Microorganisms in extreme environments. Japan Scientific Societies Press, Tokyo/Springer-Verlag, Berlin. Yoshida, M., Mastubara, K., Kudo, T., and Horikoshi, K. 1991. Actinopolyspora mortivallis sp. nov., a moderately halophilic actinomycete. Int. J. Syst. Bacteriol. 41: 15-20. Yu, I.K., and Kawamura, F. 1987. Halomethanococcus doii gen. nov., sp. nov.: an obligately halophilic methanogenic bacterium from solar salt ponds. J. Gen. Appl. Microbiol. 33: 303-310.

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Zalar, P., de Hoog, G.S., and Gunde-Cimerman, N. 1999. Trimmatostroma salinum, a new species from hypersaline water. Studies Mycol. 43: 57-62. Zavarzin, G.A., Zhilina, T.N., and Pusheva, M.A. 1994. Halophilic acetogenic bacteria, pp. 432-444 In: Drake, H.L. (Ed.), Acetogenesis. Kluwer Academic Publishers, Dordrecht. Zeikus, J.G., Hegge, P.W., Thompson, T.E., Phelps, T.J., and Langworthy, T.A. 1983. Isolation and description of Haloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophilc common to Great Salt Lake sediments. Curr. Microbiol. 9: 225-234. Zhilina, T.N. 1983. A new obligate halophilic, methane-producing bacterium. Mikrobiologiya 52: 375-382 (Microbiology 52: 290-297). Zhilina, T.N., and Zavarzin, G.A. 1987. Methanohalobium evestigatus, n.gen., n.sp., an extremely halophilic methanogenic archaebacterium. Dokl. Akad. Nauk. SSSR 293: 464-468 (in Russian). Zhilina, T.N., and Zavarzin, G.A. 1990. A new extremely halophilic homoacetogenic bacterium Acetohalobium arabaticum gen. nov., sp. nov. Dokl. Akad. Nauk. SSSR 311: 745-747 (in Russian). Zhilina, T.N., Zavarzin, G.A., Bulygina, E.S., Kevbrin, V.V., Osipov, G.A., and Chumakov, K.M. 1992a. Ecology, physiology and taxonomy studies on a new taxon of Haloanaerobiaceae, Haloincola saccharolytica gen. nov., sp. nov. Syst. Appl. Microbiol. 15: 275-284. Zhilina, T.N., Miroshnikova, L.V., Osipov, G.A., and Zavarzin, G.A. 1992b. Halobacteroides lacunaris sp. nov., new saccharolytic, anaerobic, extremely halophilic organism from the lagoon-like hypersaline lake Chokrak. Mikrobiologiya 60: 714-724 (Microbiology 60: 495-503). Zhilina, T.N., Zavarzin, G.A., Detkova, E.N., and Rainey, F.A. 1996. Natroniella acetigena gen. nov. sp. nov., an extremely haloalkaliphilic, homoacetogenic bacterium: a new member of Haloanaerobiales. Curr. Microbiol. 32: 320-326. Zhilina, T.N., Tourova, T.P., Lysenko, A.M., and Kevbrin, V.V. 1997a. Reclassification of Halobacteroides halobius Z-7287 on the basis of phylogenetic analysis as a new species Halobacteroides elegans sp. nov. Mikrobiologiya 66: 114-121 (Microbiology 66: 97-103). Zhilina, T.N., Zavarzin, G.A., Rainey, F.A., Pikuta, E.N., Osipov, G.A., and Kostrikina, N.A. 1997b. Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov., an alkaliphilic, sulfate-reducing bacterium. Int. J. Syst. Bacteriol. 47: 144-149. Zhilina, T.N., Tourova, T.P., Kuznetsov, B.B., Kostrikina, N.A., and Lysenko, A.M. 1999. Orenia sivashensis sp. nov., a new moderately halophilic anaerobic bacterium from Lake Sivash lagoons. Mikrobiologiya 68: 519527 (Microbiology 68: 452-459). Zhilina, T.N., Garnova, E.S., Tourova, T.P., Kostrikina, M.A., and Zavarzin, G.A. 2001. Halonatronum saccharophilum gen. nov., sp. nov.: a new haloalkaliphilic bacterium of the order Haloanaerobiales from Lake Magadi. Mikrobiologiya 70: 77-85 (Microbiology 70: 64-72). Zvyagintseva, I.S., and Tarasov, A.L. 1987. Extreme halophilic bacteria from saline soils. Mikrobiologiya 56: 839844 (Microbiology 56: 664-669). Zvyagintseva, I.S., Kudryashova, E.B., and Bulygina, E.S. 1996. Proposal of a new type strain of Halobacterium distributum. Mikrobiologiya 65: 399-402 (Microbiology 65: 352-354). Zvyagintseva, I.S., Bykova, S.A., and Gal'chenko. 1999. Taxonomic structure of haloarchaea based on the results of gel electrophoresis of cell proteins. Mikrobiologiya 68: 283-288 (Microbiology 68: 242-247).

CHAPTER 3 THE CELLULAR STRUCTURE OF HALOPHILIC MICROORGANISMS

The cell envelopes of Halobacterium halobium and Halobacterium salinarium actually dissolved in water or even a sufficiently diluted solution of NaCl. No covalent bonds were broken in the disaggregation, which yielded a purple-red 'solution' that, on high-speed centrifugation, yielded a purple sediment and a red supernatant solution of lipoproteins. I promptly threw the purple sediment down the sink and set out to try to find out something about the properties of the dissolved lipoproteins. Shortly after, Walter Stoeckenius became interested in this dissolving membrane. He too encountered the purple sediment, did not throw it down the sink and found the purple membrane. Since then the work on these organisms has exploded.

The above quotation from Duncan Brown's book on "Microbial Water Stress Physiology" (Brown, 1990) refers to some of the unique cellular structures of the archaeon Halobacterium: the dissolution of the cell, including its cell wall and membrane, when suspended in low salt media, and the presence of purple membrane with bacteriorhodopsin, which does not require salt for stabilization. The purple membrane and other pigments present in the different groups of halophilic microorganisms will be discussed in depth in Chapter 5. The present chapter provides an overview of the properties of different cell structures of halophilic Archaea, Bacteria, and Eucarya, structures that include cell walls, capsules, cytoplasmic and intracellular membranes, and intracellular organelles such as ribosomes, gas vesicles, and storage polymers.

3.1. CELLULAR STRUCTURES OF THE HALOPHILIC ARCHAEA 3.1.1. Cell walls of halophilic Archaea The shape of the cells of halophilic Archaea is determined, as in other microorganisms, by the properties of their cell wall. Species of the family Halobacteriaceae appear in a variety of shapes. In addition to rods (Halobacterium and others) and spheres (Halococcus, Natronococcus) there are flat pleomorphic species such as Haloferax, the shape of which has been compared with potato chips (Englert et al., 1992). A yet uncultured representative of the family appears as extremely thin perfect squares or rectangles, while others such as Haloarcula japonica may show triangular or rhomboid

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cells. Such unusual shapes are possible thanks to the fact that halophilic Archaea do not possess a significant turgor pressure (Walsby, 1971), so that shapes may occur within this group that may not be feasible for other groups of organisms. Triangular halophilic Archaea were first reported in 1986 by the group of Horikoshi in Japan. The isolate was described as Haloarcula japonica (Horikoshi et al., 1993; Otozai et al., 1991; Takashina et al., 1990). Cell division in this species was analyzed by time lapse microscopic cinematography. Cell plates are formed asymmetrically, generating triangular or rhombic daughter cells which then separate (Hamamoto et al., 1988) (Figure 3.1). The number of triangular cells observed in liquid culture may exceed 80 percent of the total population. Even more unusual than the triangular cells are the thin, flat square Archaea first observed by Walsby (1980) in a brine pool on the shore of the Sinai peninsula, Egypt (for a review see Oren, 1999). Square shapes occur very rarely in nature. The unusual square-shaped structures loaded with cylindrical gas vesicles have even erroneously been identified as square microcolonies of rod-shaped bacteria (Romanenko, 1981). The square cells grow to a rectangle, which then divides. Figure 3.2 shows electron micrographs of such square or rectangular cells, each cell containing many gas vesicles (see also Section 3.1.7). Daughter cells sometimes remain attached following cell division, and this may lead to the formation of sheets of flat cells resembling blocks of postage stamps (Figure 3.3). The square cells are extremely thin, from down to as little as as shown in transverse sections in the electron microscope (Figure 3.4). No cultures of this organism appear to be are extant, although there has been one report of the isolation of such gas vesicle-containing flat square Archaea (Torrella, 1986). Halococcus is often found as irregular clusters of cells. Wais (1985) described what was interpreted as Geodermatophilus-like clusters of halophilic red cells, which on basis of their sensitivity to antibiotics and the presence of ether lipids were identified as Archaea. The organism was stated to grow first as an amorphous cell mass of or more in diameter. This cell mass then underwent multiple internal cellular subdivisions to produce a multicellular structure consisting of cuboidal cells of submicron dimensions. These cells then disaggregated, elongated, became motile, and started to multiply by budding. Although a positive identification of the structures observed is not possible, the cells in cross-section greatly resemble those of Halococcus (see also Figure 3.9). Another species that displays a complex life cycle is Halorubrum distributum. As a result of irregular cell fissions in different planes, packages of various numbers of cells are formed, and these are surrounded by a common capsule. Resting forms ("halocysts") with multilayer walls were detected as well. Four morphological types were identified: 1, Single, rod-shaped cells with a thin (10 nm) wall; 2, Irregular clusters of tightly packed cells in a common fibrillar capsule, the thickness of which depended on culture conditions; 3, Single rods with a thickened (up to 50 nm) cell wall of fibrillar material, with nucleoids more compact than in normal cells; 4, Rounded cells with a three-layered capsule, consisting of an internal loose 50 nm thick fibrillar layer, an intermediate homogenous layer of 100 nm thickness, and an external 25 to 50 nm thick fibrillar layer (Kostrikina et al., 1991).

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Halophilic methanogens also come in a variety of shapes: irregular spheroid, flat or polygonal. No detailed studies have yet been made of the properties of their cell walls.

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While the coccoid genera Halococcus and Natronococcus possess thick rigid cell walls that do not depend on high salt for structural stability, the other members of the Halobacteriaceae have a cell wall (often referred to as an S-layer) that consists of subunits of a large glycoprotein. Absence of "normal" wall components such as Damino acids or teichoic acid in the Halobacterium cell wall had been noted long before the domain Archaea was recognized (Kushner and Onishi, 1968). No further wall layers are present outside the cytoplasmic membrane except for slime capsules found in some species (see below). The glycoprotein is essential for maintaining the rod shape of Halobacterium and the flat disk- or triangular shape of Haloferax and Haloarcula species. Considerable structural information on these wall glycoproteins has been obtained. The mature cell wall glycoproteins of Halobacterium salinarum, Haloferax volcanii, and Haloarcula japonica contain 818, 794, and 828 amino acids, respectively, with molecular masses of 86,538, 81,732 and 87,166 Da. SDS-PAA gel electrophoresis gives much higher apparent molecular masses (200, 170 and 170 kDa, respectively). Substitution of the proteins with sulfated sugar groups, as well as their modification with diphytanylglyceryl phosphate (Kikuchi et al., 1999) reduces their electrophoretic mobility. The carbohydrate content of the Halobacterium salinarum glycoprotein is about 10-12% (Mescher and Strominger, 1976a). The unusually high content of acidic residues in these proteins may lower their capacity to bind SDS, also contributing to the aberrant electrophoretic behavior. This Halobacterium glycoprotein requires high NaCl concentrations for structural stability (as do many other halophilic proteins, see Chapter 7). When suspended in low salt solutions, the wall protein denatures, and this leads to lysis and cell death (Kushner, 1964; Mohr and Larsen, 1963a; Soo-Hoo and Brown, 1967). Lysis of Halobacterium salinarum in hypotonic solutions is not due to the build-up of osmotic pressure (Mohr and Larsen, 1963b). Different enzyme inhibitors tested did not affect lysis either, thus excluding the possibility that enzymes are involved in the lysis phenomenon. It was concluded that: "The observations support the conclusion that the globular lipoprotein particles, which constitute the bulk of the material of the cell wall of these bacteria, are bound together mainly by electrostatic forces and secondary bonds. When the cells are exposed to hypotonic solutions, or to ions which bind strongly to proteins, or to chemicals which are believed to break secondary bonds between protein molecules, the linkages binding the lipoprotein particles together are weakened so that the wall structure disintegrates. Only in the presence of high concentrations of sodium and chloride ions, or other ions which bind loosely to proteins, is it possible for the proteinaceous particles of the cell wall to associate in an orderly array." (Mohr and Larsen, 1963b)

Measurements of intrinsic fluorescence and far-UV circular dichroism showed that the native conformation of the cell wall protein is lost at low salt concentrations (Hecht et al., 1986). Exposure to bacitracin, which interferes with the glycosylation of the protein, caused the formation of spherical cells, thus supplying further documentation for the structural, shape-maintaining role of the Halobacterium salinarum glycoprotein (Mescher and Strominger, 1976b). Bile acids in low concentrations have a destabilizing effect on the S-layer cell wall of many archaeal halophiles (Dussault, 1956a; Kamekura and Seno, 1991; Kamekura et al., 1988), thereby providing a simple diagnostic test to obtain information on the type of cell wall present (Dussault, 1956b). Loss of the native

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shape with the formation of spherical structures is also induced when the pH is lowered to values below 4 (Kushner and Bayley, 1963). In addition to high NaCl concentrations, relatively large amounts of magnesium or other divalent cations are required to maintain the structural stability of the glycoprotein cell wall. When suspended in media lacking magnesium, even in the presence of saturated NaCl concentrations, cells of many species become rounded and form spheroplasts. This phenomenon has been documented in Haloarcula marismortui, in Haloferax volcanii (Cohen et al., 1983) and in Haloarcula japonica (Horikoshi et al., 1993; Nakamura et al., 1992). Magnesium concentrations as high as 20-50 mM may be required in these species to protect the cell wall from structural damage. Spheroplast formation can often be induced by EDTA treatment, and this property has proven of great use in the design of genetic transformation protocols for halophilic Archaea (Dyall-Smith, 2001) (see also Section 10.2.3). It was reported already almost half a century ago that relatively high magnesium concentrations are needed for Halobacterium salinarum to grow as rods; at suboptimal magnesium growth as spheres was observed (Brown and Gibbons, 1955). In 4 M NaCl, 25 mM KCl and magnesium or calcium concentrations above 50 mM the protein molecules associated to well-defined assemblages of up to Da, but a wide sizedistribution of aggregates was observed at low magnesium concentrations (Hecht et al., 1986). Some magnesium may also be required by alkaliphilic rods to retain their native morphology: a not yet formally described rod-shaped species isolated from a soda lake in India becomes rounded at magnesium concentrations below 2 mM (Upasani and Desai, 1990). The structure and the biosynthesis of the Halobacterium salinarum cell wall glycoprotein have been investigated in great detail. The S-layer has been extensively characterized in the electron microscope. High quality electron micrographs showing a hexagonal pattern of protein subunits on the surface of Halobacterium cells were published already in 1956 (Houwink, 1956). Techniques such as negative staining, preparation of freeze-fracture replicas with rotary shadowing, and thin sectioning combined with freeze-substitution have been applied. Hexagonal patterns are observed with a center-to-center repeating distance between 15.5 and 17 nm (Blaurock et al., 1976; D'Aoust and Kushner, 1972; Kirk and Ginzburg, 1982; Steensland and Larsen, 1969; Stoeckenius and Rowen, 1967; Usukura et al., 1980). In Halobacterium salinarum the high molecular weight glycoprotein makes up between 40-50% of the wall protein, the remainder consisting of 15-20 proteins of smaller size (Mescher and Strominger, 1976a, 1976b; Mescher et al., 1974). Gene cloning and sequencing yielded detailed information on the structure of the S-layer glycoprotein and its biosynthesis (Lechner and Sumper, 1987; Lechner and Wieland, 1989; Lechner et al., 1985a; Mescher, 1981; Paul and Wieland, 1987; Paul et al., 1986; Sumper, 1987; Wieland, 1988; Wieland et al., 1980, 1981, 1982). The glycoprotein (molecular mass about 120 kDa) consists of a 87 kDa core protein rich in acidic amino acids, to which acidic and neutral saccharide chains are attached in a manner resembling animal proteoglycans (Kandler and König, 1993; Lechner and Sumper, 1987; Lechner and Wieland, 1989; Sumper, 1987). Figure 3.5 (part B) presents a model of the Halobacterium salinarum cell wall glycoprotein. Attached to the core protein are:

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1. A single large (around 10 kDa) acidic glycosaminoglycan composed of 10-15 repeats of a branched sulfated pentasaccharide which contains galacturonic acid (Figure 3.6). It is bound to the sub asparagine via a direct asparaginyl GalNAc N-linkage (Paul and Wieland, 1987; Paul et al., 1986). This saccharide complex is considered as the main shape-maintaining component of the Slayer. When its synthesis is inhibited by bacitracin, the rod-shaped cells are converted to spheres (Lechner and Wieland, 1989; Mescher and Strominger, 1976a, 1976b). 2. Low molecular weight tetrasaccharide units (probably about 12 copies per molecule), composed of 2-3 sulfated hexuronic acid residues (glucuronic acid, iduronic acid) attached to a glucose residue, bound by a direct N-linkage to asparagine residues distributed over the polypeptide chain (Lechner and Wieland, 1989).

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3. Collagen-like O-linked disaccharide units (probably 14 per molecule), bound via threonines clustered on the protein core domain 755-774 on top of the cell membrane, close to the postulated transmembranal terminal domain (Paul and Wieland, 1987; Wieland, 1988). The cell wall protein is synthesized with a 34-amino acids leader signal peptide. A hydrophobic stretch of 21 amino acids at the C-terminus probably serves as a transmembrane domain. The 14 disaccharide-bearing threonines are clustered adjacent to this membrane anchor (Lechner and Sumper, 1987). Lipid-bound intermediates (polyisoprenyl-phosphoglucose, polyisoprenyl-phosphomannose, polyisoprenyl pyrophospho-N-acetylglucosamine) were postulated as intermediates in the biosynthesis of the glycoprotein sugar moieties (Mescher et al., 1976). The biosynthesis of the sulfated saccharides linked to asparagine involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates. A 3-O-methylglucose containing intermediate was detected only at the lipid-linked level was but absent at the protein-linked level. Methylation appears to be an obligatory step in the synthesis of the glycoprotein (Lechner et al., 1985b). The high molecular weight saccharide is synthesized bound to a lipid anchor; only after completion is the fully sulfated saccharide transferred to the core protein (Wieland et al., 1982). Incorporation of the high molecular weight glycosaminoglycan is specifically inhibited by bacitracin (Wieland et al., 1980). The glycoprotein of Halobacterium salinarum also carries one or more diphytanylglyceryl groups covalently attached in thioester linkage (Kikuchi et al., 1999; see also Sagami et al., 1994, 1995). The pleomorphic Haloferax volcanii has also become a suitable model organism for the study of the properties of the archaeal cell wall glycoprotein. High-resolution electron micrographs of the periodic structure (lattice constant 16.8 nm) have been obtained (Figure 3.7). Optical diffraction analysis of the images enabled a detailed three-dimensional reconstruction of the wall structure at a resolution of 2 nm. The protein formed 3.4 nm high, dome-shaped complexes with a narrow pore at the apex, opening to a "funnel" towards the cell membrane. Six radial protrusions emanate from

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each morphological complex and join around the 3-fold axis to provide lateral connectivity (Kessel et al., 1988, 1988b) (Figure 3.8).

The structure of the Halobacterium salinarum S-layer shows much similarity to that of Haloferax volcanii. It was studied by electron microscopy of isolated negatively stained envelopes by cryo-fixation of intact cells in high salt growth medium, followed by freeze-substitution and tomography of thin sections. The two-dimensional projection map was highly similar to that of Haloferax volcanii with a hexagonal arrangement of morphological units with identical center-to-center spacing of 150 Å. The tomographic reconstruction of the Halobacterium salinarum S-layer shows a number of minor differences with that of Haloferax volcanii (Trachtenberg et al., 2000).

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NaCl alone cannot stabilize the Haloferax volcanii cell envelope. The cell wall is optimally stabilized by a combination of NaCl and divalent cations, which are required to ensure an equilibrium between the stabilization of the hydrophobic interactions and the charge shielding effects involved in the maintenance of the structure of cell envelope (Cohen et al., 1991). The primary structure of the protein backbone and the mode of its glycosylation vary among different species of halophilic Archaea. The glycosylation pattern of the Haloferax volcanii glycoprotein involves both N- and O-glycosidic bonds [glucosyldisaccharides O-linked to threonine residues] in a pattern that differs from that of Halobacterium salinarum (Sumper et al., 1990). The S-layer protein of Haloferax volcanii has 7 N-glycosylation sites, as compared to 12 in Halobacterium salinarum (including the sub negatively charged repeating unit saccharide which is absent in Haloferax volcanii). The N-linked oligosaccharides of Haloferax volcanii are repeating glucose residues attached via asparaginyl-glucose linkages (Mengele and Sumper, 1992) (see Figure 3.5, Part A). The replacement of charged sugar units (glucuronic acid) in Halobacterium salinarum by neutral sugars (glucose) in Haloferax volcanii results in a difference in charge density of the cell wall proteins of the two species (see Figure 3.5). This difference may be related to the different salt requirement of the two species (Mengele and Sumper, 1992); Haloferax volcanii was described as a moderate halophile with high magnesium tolerance (Mullakhanbhai and Larsen, 1975). The biosynthesis of the major Haloferax volcanii S-layer glycoprotein involves a maturation step following translocation of the protein across the membrane. Evidence for the processing step was obtained from an increase in its apparent molecular mass. This increase was unaffected by inhibition of protein synthesis and unrelated to glycosylation. Maturation also led to an increase in hydrophobicity (Eichler, 2001).

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This maturation process probably involves isoprenylation of the protein. A procedure for the preparation of inverted membrane vesicles of Haloferax volcanii was recently developed. It may prove useful in the study of the biosynthesis of the S-layer glycoprotein of Haloferax volcanii, its translocation and its glycosylation (Ring and Eichler, 2001). The cell envelope of Haloferax volcanii contains a number of additional glycoproteins. Using oligosaccharide staining and lectin binding, glycosylated membrane proteins of apparent molecular mass of 150, 98, 58 and 54 kDa were detected, proteins that are neither precursors nor breakdown products of the major Slayer glycoprotein (Eichler, 2000). In Haloarcula japonica, similar to in Haloferax volcanii, the glycoprotein envelope requires high magnesium concentrations for stabilization. The triangular shape is lost below 41 mM even in the presence of NaCl. Addition of EDTA also induces formation of spheroplasts (Nakamura et al., 1992). Part of the surface glycoprotein (apparent molecular mass 170 kDa) is released in low magnesium media (Nakamura et al., 1992). A three-dimensional reconstruction of the structure of the glycoprotein was achieved on the basis of electron microscopic images (Nishiyama et al., 1992). The gene coding for the glycoprotein has been cloned and sequenced. The deduced polypeptide has 828 amino acids with a 34 amino acids long signal peptide. The protein has 52.1% and 43.2% identity respectively with the Halobacterium salinarum and the Haloferax volcanii cell envelope glycoprotein. Five potential Nglycosylation sites were identified, sites that differ from those in the Halobacterium salinarum and the Haloferax volcanii glycoprotein (Wakai et al., 1997). The flat, square halophilic Archaea discovered by Walsby (1980) have not yet been cultured, but biomass collected from the environments in which these organisms thrive has supplied suitable material for electron microscopical analyses. Techniques such as thin sectioning, preparation of shadowed replicas, and negative staining have been successfully applied to these square cells (Kessel et al., 1985a). Double periodic components were often identified in the images, caused by superposition of the upper and the lower cell surface. These two components can be separated in the optical diffraction pattern, to resolve separate views of the upper and the lower cell wall (Kessel, 1983). The cell wall is comprised of regularly arranged subunits. Both hexagonal (lattice constants 16 or 22 nm) and tetragonal arrangements with a lattice constant of 14.6 nm were observed within the cell wall. Different lattice constants of the subunit arrangement were found in different cells examined, suggesting that the natural community may consist of more than one type (Kessel and Cohen, 1982). The coccoid Halococcus morrhuae and other Halococcus species do not have a glycoprotein subunit cell envelope but they are protected by a thick heteropolysaccharide cell wall that does not require high salt concentrations to maintain its rigidity. This cell wall does not dissolve even upon exposure to distilled water (Steensland and Larsen, 1971). Figure 3.9 shows the appearance of the Halococcus cell wall in the electron microscope.

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The earliest chemical analyses of the Halococcus cell wall identified glycine, glutamate, glucosamine and galactosamine among the components (Reistad, 1971). A more thorough analysis of the wall structure (Figure 3.10) has confirmed the presence of glucosamine and galactosamine derivatives in the sugar backbone. Sulfate groups are linked to hydroxyl groups in positions 2 and/or 3 of uronic acids, galactose and galactosamine residues. Glucose, galactose, galacturonic acid and all amino sugars are glycosidically linked to the cell wall polymer. Part of the glucose, the galactose, and to a lesser extent also the mannose residues possess more than two glycosidic linkages, and these residues represent possible branching points. Glycine residues may play a role in connecting glycan strands through peptide linkages between the amine groups of glucosamine and the carboxyl group of an uronic acid or gulosaminuronic acid(Schleifer et al., 1982; Steber and Schleifer, 1975, 1979).

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The alkaliphilic coccoid Natronococcus occultus also has a thick cell wall that retains its shape in the absence of salt. Its structure differs greatly from that of the cell wall polymer of Halococcus. It consists of repeating units of a poly(L-glutamine) glycoconjugate (Niemetz et al., 1997). Figure 3.11 presents a model of this structure.

3.1.2. Extracellular capsules of halophilic Archaea Several species of halophilic Archaea deposit polysaccharide capsules outside the cell wall. Massive amounts of exopolysaccharides are excreted by members of the genus Haloferax (Paramonov et al., 1998; Parolis et al., 1996; Severina et al., 1989, 1990). An early analysis of the Haloferax mediterranei extracellular slime showed it to be a heteropolysaccharide that contains mannose as a major component. Glucose, galactose, and another unidentified sugar, as well as amino sugars and uronic acids were also detected, as was a considerable amount of bound sulfate (Antón et al., 1988). The

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structure of this exopolysaccharide has now been resolved (Parolis et al., 1996). It consists of repeating units of the following sulfated trisaccharide:

The polysaccharide excreted by Haloferax gibbonsii has a different structure (Paramonov et al., 1998):

Haloferax denitrificans produces yet another type of exopolysaccharide, with a repeating tetrasaccharide of the following structure (Parolis et al., 1999):

in which acid.

is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic

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An exopolysaccharide of Haloferax volcanii has been characterized in part. It contained mannose as major component together with small amounts of hexuronic acids. Sulfate groups were also present (Severina et al., 1990). An acidic exopolysaccharide is also formed by some Haloarcula isolates obtained from a marine saltern in Tunisia (Nicolaus et al., 1999). A completely different type of extracellular polymer was recently identified in Natrialba aegyptiaca (aegyptia). acid) is the principal component of this polymer, which consists of about 85% glutamic acid, 12.5% carbohydrates, and 2.5% unidentified compounds (Hezayen et al., 2000, 2001). At least some of the extracellular polymers excreted by halophilic Archaea (and halophilic Bacteria as well, see Section 3.2.2) may have considerable biotechnological interest. More information on their potential applications is given in Section 11.2.5. In accordance with the variations observed in the surface layer structure, the halophilic Archaea display a considerable antigenic diversity with limited cross-reaction between species. Antigenic fingerprinting may thus be useful for rapid identification. Immunological techniques may also be used to study the species composition of natural communities (Conway de Macario et al., 1986). Classic immunological techniques may be hampered by the fact that antibodies may react poorly or not at all in the presence of salt concentrations approaching saturation. However, antibodies against halobacterial flagella raised in chickens were shown to interact with the antigen also in NaCl (Alam and Oesterhelt, 1984). Typing of the surface layers with different lectins that binding to specific sugar residues has also been employed in the characterization of halophilic Archaea (Gilboa-Garber et al., 1998).

3.1.2.

Flagella of halophilic Archaea

Many members of the Halobacteriales are motile by means of flagella. Halobacterial flagella have a right-handed helix, this in contrast to flagella of Bacteria that are lefthanded (Alam and Oesterhelt, 1984; Alam et al., 1984; Houwink, 1956) (Figures 3.12 and 3.13). Motility has been extensively studied in Halobacterium salinarum. This organism produces bundles of flagellar filaments (Figure 3.12). In the exponential growth phase cells are predominantly monopolarly, in the stationary phase bipolarly flagellated (Alam et al., 1984). Using atomic force microscopy, the individual flagellar motors could be observed as knobs at the end of single flagella (Jaschke et al., 1994). Cell envelopes with the flagella intact were isolated by treatment with taurodeoxycholate. After solubilization of these envelopes with Triton X-100 at a high ionic strength, round patches of envelope material were isolated from which numerous flagella emerged, thus showing that the flagellar bundle is inserted into a differentiated polar cap (Kupper et al., 1994) (Figure 3.14). SDS-polyacrylamide electrophoresis of isolated flagella resolved three proteins with apparent molecular masses of 23.5, 26.5, and 31 kDa (Alam and Oesterhelt, 1987; Alain et al., 1984). These three flagellins are related glycoproteins that carry sulfated oligosaccharides. The flagellins show a high degree of similarity with the sulfated glycoproteins found in the Halobacterium salinarum cell envelope (see Section 3.1.1),

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including presence of the unusual asparaginylglucose moiety as linkage unit (Lechner and Wieland, 1989; Sumper, 1987; Wieland et al., 1985).

The flagellin proteins are encoded by the flgB genes. Additional genes are involved in the biogenesis of flagella in Halobacterium salinarum. A detailed analysis has been

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made of the fla gene cluster, located upstream of the flagellin genes. This cluster encodes for nine open reading frames. It was concluded that the flaI gene product is involved in the biosynthesis, transport or assembly of the flagella (Patenge et al., 2001). Characterization of the flagellin genes of Natrialba magadii suggest that the genes may have been acquired from the bacterial domain by lateral transfer exchange (Serganova et al., 2002).

Clockwise rotation of the flagella of halophilic Archaea results in forward movement of the cells, counterclockwise rotation in backward movement. Flagellar bundles do not fly apart during the change from clockwise to counterclockwise rotation or vice versa when the direction of movement changes (Alain and Oesterhelt, 1984; Marwan et al., 1987, 1991). Considerable understanding has been achieved of the signals that govern flagellar motion and directed movement of motile halophilic archaeal cells. A complex array of chemo- and photosensors and transducers is present in Halobacterium salinarum. There are at least 17 homologous methyl-accepting proteins, 6 response regulator genes, and 14 histidine kinase-encoding genes in the genome of Halobacterium strain NRC-1 (Ng et al., 2000). More information on phototaxis and chemotaxis is given in Section 5.4.3.

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3.1.4. The cytoplasmic membrane and its lipids The cytoplasmic membrane is composed of lipids and proteins. It contains all the functions needed for respiratory electron transport, inward and outward transport of ions, nutrients, and other compounds, sensors that provide information about the extracellular environment and their transducers, and many other components. In addition, the retinal ion pumps bacteriorhodopsin and halorhodopsin are present in the membranes of many halophilic Archaea. The bacteriorhodopsin proton pump is sometimes organized in differentiated patches within the membrane – the purple membrane. The properties of many of the proteins present in the cytoplasmic membrane and their functions will be discussed in later chapters (see e.g. Section 4.1.3 for information on the components of the respiratory chain), and the retinal pigments will be described in depth in Section 5.4. The members of the Halobacteriaceae have archaeal-type lipids based on branched 20-carbon (phytanyl) and sometimes also 25-carbon (sesterterpanyl) chains, bound to glycerol by ether bonds. Considerable effort has been devoted to the structural elucidation of the polar and neutral lipids found in the different representatives of the family. Analytical methods such as mass spectrometry (fast atom bombardment mass spectrometry, HPLC combined with electrospray mass spectrometry), and NMR spectroscopical techniques are useful in the structural characterization of lipid mixtures, isolated lipid components, or chemical degradation products of these lipids (Fredrickson et al., 1989a, 1989b; Klöppel and Fredrickson, 1991; Qiu et al., 2000). One- or twodimensional thin-layer chromatography is a useful technique for the rapid characterization of the lipids present in halophilic archaeal isolates (Norton et al., 1993; Oren et al., 1996; Torreblanca et al., 1986). Details on the lipid composition of halophilic Archaea, the biosynthetic pathways involved in their synthesis, and the analytical methods used in their characterization are found in several reviews (Kamekura, 1993, 1998; Kamekura and Kates, 1988, 1999; Kates, 1993, 1996; Kates and Kushwaha, 1995; Kates and Moldoveanu, 1991). The diether core lipid that forms the basis for most polar lipid structures present in the family Halobacteriaceae is 2,3-di-O-phytanyl-sn-glycerol Certain species, however, contain in addition the asymmetric 2-O-sesterterpanyl-3-O-phytanylsn-glycerol in different amounts. A thin layer chromatographic procedure has been developed to separate the and lipid species (Ross et al., 1981). The core lipid is found in many of the alkaliphilic types, as well as in the neutrophilic Natrialba asiatica (Kamekura and Dyall-Smith, 1995; Matsubara et al., 1994), and in the genera Natrinema (McGenity et al., 1998) and Halococcus. core lipids were also detected in "Halobacterium halobium IAM13167", which may be a Halobacterium salinarum strain (Morita et al., 1998). In some cases 2,3-di-Osesterterpanyl-sn-glycerol is encountered as well as a minor component (De Rosa et al., 1982, 1983). Membranes containing a large percentage of lipids may be intermediate in stability between the rigid tetraether monolayer of the extremely thermophilic Archaea and the regular bilayer of lipids present in most neutrophilic halophiles. The lipids are expected to form 'zip' type bilayer membranes as exemplified in Figure 3.15 (De Rosa et al., 1982, 1983). A mono-

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isoprenyl analog of the methyl ester of phosphatidylglycerophosphate (Me-PGP) has been detected in Halococcus saccharolyticus (Moldoveanu et al., 1990).

The proportion of and core lipids differs among the species. In the haloalkaliphilic strain SP8 9% of the lipids were based on 85% on and 6% on (De Rosa et al., 1983). In a study of the haloalkaliphilic species Natronomonas pharaonis, Natrialba magadii, Natronobacterium gregoryi, and Natronococcus occultus, together with a number of additional unidentified isolates, strains were found with as much as 89% or as little as 0.1% of the lipids based on the core structure (Tindall, 1985). The content of lipids never exceeded 1% of the total. The ratio between the and lipids also depends on the growth conditions. In Natronobacterium gregoryi and Natrialba magadii, growth at increasing medium salinity led to an increased content of in the lipids (Morth and Tindall, 1985a, 1985b; Tindall et al., 1991). The distribution of the and core

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structures may vary among the different polar lipids: in Natronococcus occultus an increase in Me-PGP (the methyl ester of phosphatidylglycerophosphate) containing and a decrease in PG (phosphatidylglycerol) containing was observed with increasing medium salinity. The content of and was independent of the salt concentration at which the cells had been grown (Nicolaus et al., 1989). The hydrophobic isoprenoid chains are in most cases fully saturated. However, unsaturated phytanyl ("phytenyl") side chains occur in Halorubrum lacusprofundi from Deep Lake, Antarctica, a species that grows at temperatures down to 4 °C (Franzmann et al., 1988). Introduction of double bonds in the carbon chains is probably important to control membrane fluidity at low temperatures. In Halobacterium salinarum double bonds are transiently formed during the biosynthesis of the phytanyl chains (Moldoveanu and Kates, 1988). A haloalkaliphilic isolate from India contains phytanyl and sesterterpanyl chains that may be both hydroxylated and unsaturated (Upasani et al., 1994). A great variety of polar lipids, including phospholipids, sulfolipids, and glycolipids, is encountered in the different representatives of the Halobacteriaceae. All known species contain the diether derivatives of PG and Me-PGP (Figure 3.16 a and b). The presence of the methyl ester group in the Me-PGP structure was recognized only relatively recently (Kates, 1996; Kates et al., 1993), although its presence was already suggested from fast bombardment mass spectrometry data published in 1989 (Fredrickson et al., 1989a; see also Tsujimoto et al., 1989). Phosphatidylglycerosulfate (PGS, Figure 3.16 c) is present in many neutrophilic species (Hancock and Kates, 1973). Its absence in certain genera (Haloferax, Natrialba, Halobaculum, Halococcus, Halogeometricum) is a useful diagnostic feature in the classification and identification of the Halobacteriaceae. The alkaliphilic members characterized thus far all lack PGS. PG, Me-PGP, and PGS are the only phospholipids in most neutrophilic representatives of the Halobacteriales. Additional unidentified phospholipids have been detected in the genus Natrinema (McGenity et al., 1998). In Natronococcus occultus a phospholipid with a cyclic phosphate group has been identified: 2,3-di-O-phytanyl-sn-glycero-lphosphoryl-3'-sn-glycerol-1,2-cyclic phosphate (Figure 3.16 d). A 2-O-sesterterpanyl3-O-phytanyl glycerol diether form of the novel phospholipid is also present (Lanzotti et al., 1989). Yet unidentified novel phospholipids designated PL1, PL2, and PL3 are present, in addition to PG and Me-PGP, in certain haloalkaliphiles (De Rosa et al., 1988; Morth and Tindall, 1985a; Tindall, 1985). Table 3.1 gives their distribution. The glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-Ophytanyl-1-phosphoglycerol-3-phospho-sn-2,3-di-O-phytanylglycerol, has been identified in Halobacterium salinarum as a lipid specifically associated with bacteriorhodopsin in the purple membrane (Corcelli et al., 2000). A variety of glycolipids has been identified in different members of the Halobacteriales. These include di-, tri-, and tetraglycosyl diether lipids. Part of these glycolipids carry sulfate groups on the sugar moieties. Some of the following structures are given in Figure 3.17. DGD-1 found in minor amounts in Haloferax species (Kushwaha et al., 1982a, 1982b).

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DGD-2, a minor diglyceride lipid of unknown structure, containing mannose and glucose. It is found as a minor component in Haloarcula species (Evans, 1980). DGD-4, documented from the haloalkaliphilic strain SSL1, isolated from an Indian soda lake (Upasani et al., 1994). Two possible structures have been proposed for this glycolipid: and

S-DGD-1 sn-glycerol) (Figure 3.17 a), the major glycolipid in the genus Haloferax (Kushwaha et al., 1982a, 1982b). It is also found as sole or minor glycolipid in the genera Halobaculum (Oren et al., 1996) and Halococcus, and was also reported from Halorubrum saccharovorum (Lanzotti et al., 1988). S-DGD-1 was found as the major or sole glycolipid in the biomass of the Dead Sea during an archaeal bloom (Oren and Gurevich, 1993) and in a saltern crystallizer ponds in Eilat, Israel (Oren, 1994). Lipid

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analysis of field samples also suggested that S-DGD-1 may be the main glycolipid of the flat gas vesicles containing square Archaea (Oren et al., 1996).

S-DGD-3 glycerol), the glycolipid of several Halorubrum species, including Halorubrum sodomense (Trincone et al., 1990) and Halorubrum lacusprofundi (Tindall, 1990a). S-DGD-5 glycerol), found in Halorubrum trapanicum (Trincone et al., 1993).

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• a bis-sulfated glycolipid -glucose]-2,3-di-O-phytanyl- or phytanyl sesterterpenyl-sn-glycerol) (Figure 3.17 b), found in Natrialba asiatica (Kates, 1996; Matsubara et al., 1994; Onishi et al., 1985). • TGD-1 phytanyl-sn-glycerol), a minor glycolipid of Halobacterium salinarum (Smallbone and Kates, 1981). • TGD-2 phytanyl-sn-glycerol) (Figure 3.17 c), the sole or major glycolipid of most Haloarcula species (Evans et al., 1980). • S-TGD-1 glucose]-2,3-di-O-phytanyl-sn-glycerol) (Figure 3.17 d), found in the genus Halobacterium (Kates, 1978; Kates and Deroo, 1973). • TeGD a minor glycolipid of Halobacterium salinarum (Smallbone and Kates, 1981). • S-TeGD (Figure 3.17 e), found as well in Halobacterium (Smallbone and Kates, 1981). • A novel complex phosphosulfoglycolipid, being a derivative of bisphosphatidylglycerol, has been identified in Halobacterium salinarum, associated with the purple membrane: phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycerol] (Corcelli et al., 2000). Some confusion still exists on the correct structures of the glycolipids that occur in different members of the genus Halorubrum. Lanzotti et al. (1988) reported that the Halorubrum saccharovorum glycolipid is identical to S-DGD-1 from Haloferax. However, Kamekura (1998) stated that S-DGD-3 is the main glycolipid in the genus Halorubrum. In addition, different isolates designated as Halorubrum saccharovorum appeared to contain different glycolipids (Tindall, 1990b). A recent report that claims that a sulfated diglycosyl-2,3-di-O-phytanyl-sn-glycerol may be the major glycolipid of Halobacterium salinarum strain R1 (rather that S-TGD-1 and S-TeGD; Qiu et al., 2000) also does not agree with other published data. The ratios in which the different polar lipids occur in the cytoplasmic membrane of halophilic Archaea may vary according to the growth conditions. When grown at very high salt concentrations, Haloferax mediterranei shows an increase in S-DGD-1 with a concomitant decrease in PG (Kamekura and Kates, 1988; Kushwaha et al., 1982b). Glycolipids have become useful taxonomic markers in the classification of halophilic Archaea. Polar lipid composition analysis has successfully been used to obtain information on the types of Archaea that dominate in the biomass collected from environments such as the Dead Sea or saltern crystallizer ponds (Oren and Gurevich, 1993; Oren et al., 1996). However, the increased emphasis on the use of 16S rDNA sequence comparisons in of halophilic Archaea has led to many taxonomic rearrangements (see also Chapter 2). In the current classification there are a number of cases in which members of one genus contain greatly different glycolipids, and the presence of certain glycolipids is shared by representatives of more than one genus.

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Table 3.2 presents a – not necessarily complete – overview of the distribution of different glycolipids among the genera of the Halobacteriaceae.

Neutral lipids may represent about 10% of the total lipid content of the halophilic Archaea of the family Halobacteriaceae (Kamekura and Kates, 1988). The following types have been reported: Carotenoids (further discussed in Section 5.3). Quinones isoprenoid lipids: geranylgeraniol. Neutral glycerol phytanyl ethers: DL-O-phytanyl-sn-glycerol and 2,3-di-O-phytanylsn-glycerol (Kushwaha and Kates, 1978). compounds, including squalene, dihydrosqualene, tetrahydrosqualene, and dehydrosqualene (Kushwaha et al., 1972; Mullakhanbhai and Francis, 1972). Indole may occur in the membrane of Halobacterium salinarum and Halococcus sp. (Kushwaha et al., 1977). The major respiratory quinones in the Halobacteriaceae are MK-8 and two menaquinones with eight isoprenoid units (Collins et al., 1981; Tindall and Collins, 1986). Quinones may amount to about 9% of the total neutral lipid content of the cells (Kamekura and Kates, 1988). In in Halococcus morrhuae the terminal double bond has become saturated (Tindall and Collins, 1986). The relative amounts of MK-8 and depend on the growth conditions (Tindall et al., 1991). Novel types of methylated menaquinones were detected in Natronobacterium gregoryi, which contains a range of octaprenyl menaquinones as well as monomethylated and dimethylated menaquinones (Collins and Tindall, 1987).

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Information on the biosynthetic pathways of different lipids mentioned above has been presented by Kamekura and Kates (1988). Activities of three key enzymes involved in the production of the isoprenoid chains, isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and 3-O-geranylgeranylglycerol phosphate synthase, have been detected in the cytoplasm of Halobacterium salinarum (Zhang and Poulter, 1993). Although isoprenoid hydrophobic chains are present in the major lipids in the family Halobacteriaceae, straight-chain fatty acids are present in these Archaea as well. The presence of a fatty acid synthetase complex in Halobacterium salinarum has been ascertained more than thirty years ago. Activities detected were low, and the enzyme is inhibited by salt at such concentrations reported to occur intracellularly (Pugh et al., 1971). The fatty acid biosynthesis inhibitor cerulenin inhibits growth of Halobacterium salinarum, and this inhibition can be relieved by the addition of stearate or oleate (but not by palmitate) to the medium (Dees and Oliver, 1977). The straight-chain fatty acids produced are not incorporated into the membrane lipids but they serve to acylate membrane proteins. Both palmitate and stearate are bound to proteins, and small amounts of myristic acid and 18:1 acids can also be found. These fatty acids are bound to protein through both ester and amide linkages. Acylation of proteins increases their hydrophobicity, and was postulated to stabilize the membrane through increased protein-lipid interaction (Pugh and Kates, 1994). Free palmitate is associated with halorhodopsin in Halobacterium salinarum at a ratio of 1-2 fatty acid molecules per molecule of halorhodopsin (Colella et al., 1998; Corcelli et al., 1996).

3.1.5. Ribosomes The ribosomes of Haloarcula marismortui have become a paradigm for the understanding of the structure of prokaryote ribosomes in general (Yonath, 2002). Since the first attempts were made toward crystallization of the large subunit of the Haloarcula ribosome (Shevack et al., 1985) to the full structural elucidation using a combination of X-ray crystallography and cryo-electron microscopy, much information has been gained about the organization and functioning of the halophile ribosome and ribosomes in general (Francheschi et al., 1994; Penczek et al., 1999). The complete atomic structure of the large ribosomal subunit has been resolved at 2.4 Å resolution by X-ray diffraction studies of crystals that include 2,833 of the subunit's 3,045 nucleotides and 27 of its 31 proteins (Ban et al., 2000). Halobacterium salinarum contains, in addition to the 5S, 16S and 23S ribosomal RNAs, another species of stable RNA (7S, consisting of 304 nucleotides), not associated with the ribosome (Moritz and Goebel, 1985). The molecule is now known to be part of the signal recognition particle that directs proteins to be secreted to the cytoplasmic membrane (Zwieb and Eichler, 2002).

3.1.6. Fibrocrystalline bodies An unusual intracellular organelle has occasionally been observed inside the cytoplasm of Halobacterium salinarum. This organelle, referred to as a fibrocrystalline body,

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appears in thin sections in the electron microscopy as a group of wavy alternating electron-dense and electron-light bands (Cho et al., 1967). The organelle is frequently found in close association with the cell membrane. At higher magnification the dense bands are shown to be divided into smaller units by transverse strands. These smaller units are 120 to 130 by 75 to 100 Å in size, separated by a 45 to 50 Å electron-light gap (Cho et al., 1967). Robertson et al. (1982) described these fibrocrystalline bodies as up to long and thick, and consisting of a bundle of hollow tubes, 12 nm in diameter, each with a wall of about 4 nm thick, and interconnected by cross bridges of 15 nm. The tubules are arranged in a hexagonal array with a lattice constant of 15 nm. In longitudinal section the body displays regular dense and light cross striations spaced at a period of about 22 nm. Isolation of these fibrocrystalline bodies from Halobacterium salinarum has recently been reported, and similar structures were detected also in Halorubrum saccharovorum, Haloarcula hispanica, Haloferax volcanii, and Natronomonas pharaonis (Alba et al., 2001). Vincristine, an antitumor drug that targets tubulin, affects the structure of the organelle. The drug causes fragmentation of the fibrocrystalline body, causes changes in cell shape, and leads to growth inhibition (Alba et al., 2001). Sensitivity of Halobacterium salinarum to certain antitubulin drugs has been noted earlier (Sioud et al., 1987). Indications for the presence of tubulin-like proteins and the possible existence of a cytoskeleton in halophilic Archaea were also obtained from the characterization of a gene in Halobacterium salinarum homologous to the cell division gene ftsZ. Expression of the ftsZ gene protein (39 kDa; 32% identity to the corresponding protein of Escherichia coli) in Halobacterium salinarum induced morphological changes, leading to the loss of the rod shape. The protein is related more to tubulins than are the FtsZ proteins from the domain Bacteria, In the Bacteria this protein polymerizes to form a circumferential ring at the division site, constricting at the leading edge of the invaginating septum that will eventually separate the two daughter cells. Altered FtsZ levels, and hence altered interactions between the FtsZ division polymer and the cell wall, could perturb the shape-determining features of the S-layer without causing lysis. This idea is consistent with the presence of a tubulin-like FtsZ cytoskeleton in these cells, the structure of which is changed when the FtsZ levels are altered (Margolin et al., 1996). Another protein that may be involved in the formation of a cytoskeleton in Halobacterium is a cytoplasmic 71-kDa protein belonging to a ubiquitous P-loop ATPase superfamily with head-rod-tail structure (Hp71), a protein that has similarities with eukaryotic cytoskeleton proteins. Heterologous production of Hp71 in Escherichia coli allowed the isolation of antibodies against the protein, and these were then used to determine the location of the protein in Halobacterium by immune electron microscopy. Homologous overproduction of Hp71 in Halobacterium salinarum and heterologous production in Haloferax volcanii resulted in modifications of cell morphology from rods to extended rods and from pleomorphic cells to rods, respectively. The protein is supposedly involved in cytoskeleton formation and/or chromosome partitioning (Ruepp et al., 1998).

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3.1.7. Gas vesicles The occurrence of gas vesicles within cells of Halobacterium and a number of additional genera of halophilic Archaea has drawn the attention of the investigators since early times. Drawings by Helena Petter (1931) of Halobacterium cells containing gas vesicles were reproduced in Figure 1.2. The early electron micrographs of Halobacterium salinarum by Houwink (1956) provided pictures of surprisingly high quality, and these even included stereopairs, allowing a three-dimensional visualization of the gas vesicles. Gas vesicles have been reported in Halobacterium salinarum, Haloferax mediterranei, Halorubrum vacuolatum (Mwatha and Grant, 1993) and Halogeometricum borinquense (Montalvo-Rodriguex et al., 1998). In addition, they abound within the flat square, not yet cultivated cells discovered by Walsby (1980) (Figure 3.2). Gas vesicles are hollow cylindrical or spindle-shaped structures, built of protein subunits. An old report that the gas vesicles of Halobacterium also contain a considerable amount (about 1% by weight) of bound galactose (Krantz, and Ballou, 1973) has not been confirmed since. The morphology of the vesicles is genetically determined: a gas vesicle-defective mutant of Halobacterium salinarum strain 5 showed mostly cylindrical vesicles, while the vesicles of the wild-type are spindle-shaped (Simon, 1981). The vesicles are not solubilized by extremes of pH or by presence of urea, detergents, or organic solvents. The gas vesicle protein resists digestion by pronase, trypsin, thermolysin, and papain. Gas vesicles can easily be isolated in pure form from lysates in distilled water; the gas vesicles remain intact, and they can then be collected by flotation. The gas vesicle protein is one of the few proteins in halophilic Archaea that do not require salt for stabilization. The gas vesicles of the Halobacteriaceae are sensitive to pressure, similar to gas vesicles from other prokaryotes, and they collapse already at relatively low pressures. A pressure of 90 kN (0.9 atmospheres) causes collapse of half of the gas vesicles in Halobacterium salinarum, while the weakest gas vesicles within the cells are already destroyed by a pressure of (Walsby, 1971). Halobacterium cells prepared for electron microscopy after a centrifugation step show large amounts of "intracytoplasmic membranes" of unusual structural characteristics, being the remnants of gas vesicles broken by the force exerted by high-speed centrifugation (Larsen et al., 1967; Stoeckenius and Kunau, 1968; Stoeckenius and Rowen, 1967). The presence of gas vesicles and the ability to float to the brine surface is advantageous for an aerobic halophilic microorganism, as the solubility of oxygen and other gases in salt-saturated brines is low. The advantage of possessing gas vesicles was proven in competition experiments between a Halobacterium salinarum mutant with a low amount of deficient gas vesicles and the wild type. In shaken culture both strains grew equally well. However, in static cultures, where steep vertical oxygen concentration gradients were established, cells of the wild type floated and became dominant, probably due to their successful competition for oxygen which was in short supply. In shallow static cultures the mutant won the competition. This was explained by its smaller protein burden, i.e., the wild type wasted much energy to produce gas vesicles that were unnecessary under the conditions employed (Beard et al., 1997). The

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idea that gas vesicles may also be effective as a shield against harmful UV radiation was not confirmed in laboratory experiments (Simon, 1980). The major structural component of the gas vesicles is a 8 kDa protein (the product of the gvpA gene, see below) with a very high content of hydrophobic amino acids. The GvpA protein spontaneously assembles in a helix of low pitch. The GvpA protein is highly conserved in all prokaryotes that produce gas vesicles, the protein from Halobacterium or Haloferax being very similar to that of cyanobacteria (domain Bacteria) (Walker et al., 1984). A second structural component is GvpC, a 42 kDa protein containing internal repeats. This protein strengthens the structure of the vesicles, assists in their assembly, and to a large extent determines the shape of the gas vesicles. The genetic control of gas vesicle biosynthesis is quite complex. It has been observed long ago that Halobacterium salinarum spontaneously loses its ability to produce gas vesicles with a high frequency (DasSarma et al., 1987; Larsen et al., 1967). In many cases is the property of gas vesicles production plasmid-coded (DasSarma, 1993; Simon, 1978; Weidinger et al., 1979). In Halobacterium strain NRC-1, operons coding for the production of gas vesicles are located both on the chromosome (the c-vac region) and on the large plasmid pNRC100 (the p-vac region). Under normal circumstances the gas vesicles are spindle-shaped, and are formed using the plasmidlinked genes, which are expressed constitutively. Predominantly cylindrical gas vesicles are synthesized by the chromosomal c-vac region, which is closely controlled (Offner et al., 1998). The formation of gas vesicles is governed by clusters of 13 or 14 genes. Their tentative functions are as follows (DasSarma and Arora, 1997; Pfeifer and Englert, 1992; Pfeifer et al., 2002): GvpA (8 kDa) is the major structural gas vesicle protein; it has 76 (p-vac) or 79 (cvac) amino acids (Horne and Pfeifer, 1989). GvpB (8 kDa) is a structural protein associated with the cylindrical, chromosomal encoded vesicles. GvpC (42 kDa) is a structural protein involved in strengthening of the vesicles, and contains internal repeats. GvpD (59 kDa) is probably a negative regulator. It contains a nucleotide-binding motif. gvpD deletion mutants overproduce gas vesicles (Englert et al., 1992). Sitedirected mutation is now being used to identify the functionally relevant parts of the protein responsible for its repression activity (Pfeifer et al., 2001). GvpE (21 kDa) is a possible regulator (activator) of transcription of gvpA. GvpF, G, H, K, and L are possibly minor structural proteins or assembly proteins. Their sizes are 24, 10, 20, 13, and 32 kDa, respectively. GvpI (16 kDa) is possibly a minor structural protein or an assembly protein. GvpJ (12 kDa) and GvpM (9 kDa) are likely minor structural proteins. GvpN (39 kDa) is possibly a minor structural protein or an assembly protein that assists in growth of the vesicle. It contains a nucleotide binding motif. GvpO (12-15 kDa) is possibly a regulator. In the Halobacterium salinarum plasmid the p-vac genes are organized in the sequence gvpMLKJIHGFEDACN(O). A rightward operon ACN(O) codes for genes for proteins with either a structural or an assembly role, while the leftward operon

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gvpDEFGHIJKLM is composed of genes for putative regulatory proteins (DasSarma and Arora, 1997; Offner et al., 1998; Pfeifer et al., 2002). Antibody probes have been used to identify the gvpA and gvpC gene products (Halladay et al., 1993). Genetic transformation of a gas vesicle-less mutants of Halobacterium salinarum has been achieved with a gvp-cluster cloned on a Halobacterium salinarum - Escherichia coli shuttle plasmid (Halladay et al., 1992). Heterologous complementation of gvp clusters derived from different vac regions demonstrated that the formation of chimeric gas vesicles is possible. Analysis of such chimeric types showed that the shape of the vesicles is not exclusively determined by GvpA (Offner et al., 1998). Halobacterium NRC-1 mutants carrying deletions in the p-vac region, as well as Halobacterium strains GN101 and YC819-9, synthesize gas vesicles already in the early stationary phase, and contain the maximal amount of c-vac transcript mRNA in the stationary phase. The wild type synthesizes gas vesicles exclusively by expression of the p-vac genes, the maximal mRNA level being observed during exponential growth; transcripts of the c-vac genes were not detectable. It was therefore suggested that p-vac gene expression might directly or indirectly repress transcription of the c-vac genes (Home and Pfeifer, 1989). Haloferax mediterranei has a single, chromosomal mc-vac gene cluster that codes for gas vesicle production. Organization of the genes within the operons is similar to that in Halobacterium salinarum. Gas vesicles are only produced when the salt concentration in the medium exceeds (Englert et al., 1990; Pfeifer et al. 1997; Röder and Pfeifer, 1996). The relative abundance of mc-vac mRNA in cells grown at salt was sevenfold higher than in cells grown in (Englert et al., 1992). Also in this species is the property of gas vesicle production easily lost by mutation. The structural and genetic aspects of the Haloferax gas vesicles and the mechanisms involved in the regulation of their formation have been described in depth (Offner et al., 1998; Pfeifer and Englert, 1992; Pfeifer et al., 1997). The arrangement of the genes within the nv-vac cluster of Halorubrum vacuolatum differs from that in Halobacterium and in Haloferax (Pfeifer et al., 1997). Little research has been devoted as yet to the properties of the gas vesicles of Halorubrum vacuolatum and to the regulation of their production.

3.1.8. Storage materials Some members of the Halobacteriaceae accumulate massive amounts of the storage polymer (PHA). When Haloferax mediterranei is grown in medium containing acetate under conditions in which phosphate is in limited supply, PHA granules occupy a large part of the cell volume (Fernandez-Castillo et al., 1986; Lillo and Rodriguez-Valera, 1990; Rodriguez-Valera and Lillo, 1992) (Figure 3.18). The potential biotechnological application of Haloferax mediterranei for PHA production is discussed in further detail in Chapter 11. Also Haloarcula marismortui accumulates PHA granules under suitable conditions (Kirk and Ginzburg, 1972). The accumulation of inorganic polyphosphate as phosphorus storage has been documented in Halobacterium salinarum, but probably more phosphate may be present intracellularly in the form of magnesium phosphate (Smirnov et al., 2002).

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3.1.9. Cysts and endospores Halornbrum distributum produces thick-walled cyst-like structures, which probably represent resting stages (Kostrikina et al., 1991). No information is available on the cell wall structure of these cysts as compared to that of cells in growing cultures. Formation of true endospores has never yet been described in the archaeal domain. A report on the alleged occurrence of heat-resistant endospores in a putative archaeon isolated from "bagoong" (a Japanese fish paste) (Fujii, 1980) still awaits confirmation, The organism grew optimally at and no growth was observed below The endospores formed resisted heating to 80 °C for 30 minutes.

3.2. CELLULAR STRUCTURES OF THE HALOPHILIC BACTERIA The properties of the cellular structures of halophilic and halotolerant Bacteria have been subject of much less research than the components of the halophilic Archaea. This is understandable as the halophilic Bacteria have much in common with the nonhalophilic representatives of the domain, while the Archaea tend to have special and

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unusual properties. Still, as shown below, there are quite a number of special features in the cellular structures of the halophilic and halotolerant members of the Bacteria.

3.2.1. Cell walls of halophilic Bacteria The cell envelope of Halomonas elongata shows marked changes in its hydrophobicity as a function of the salt concentration at which the cells were grown. Cells grown in NaCl are far more hydrophilic than cells grown in salt, as shown by the behavior of the cells when shaken in a water-hexadecane mixture (Hart and Vreeland, 1988). Surface hydrophobicity tends to increase as cultures approach the stationary phase. Surface hydrophobicity is probably a function both of the structure of the cell wall and of the cytoplasmic membrane. The increase in charged phospholipids at higher NaCl concentrations (see below) may in part explain the enhanced hydrophilicity. A hydrophilic cell surface makes the cell more attractive to water molecules in the waterpoor environment of high-salinity media, thus preventing desiccation (Hart and Vreeland, 1988). An interesting feature of the peptidoglycan of Halomonas is the presence of the hydrophobic amino acid leucine, which is expected to add to the overall hydrophobicity of the cell wall (Vreeland et al., 1984). The hydrophobicity of the cells is not influenced by trypsin treatment, in contrast to many other microorganisms in which trypsin treatment converts hydrophobic cells to hydrophilic ones. To study the saltinduced changes in cell wall composition, rabbits were immunized with Halomonas elongata cells grown at different NaCl concentrations. Antiserum prepared against lowsalt grown cells reacted with cells grown in all salt concentrations, but antiserum against high-salt grown cells proved specific for this type of cells. Different possible explanations for the findings were suggested: either the low salt cells possessed a unique antigen, or some type of conformational change in the surface antigens had caused the rabbits to respond to a determinant that was not exposed in low-salt cells (Vreeland et al., 1991). The cell wall of the photosynthetic purple nonsulfur bacterium Rhodothalassium salexigens is covered by an S-layer consisting of subunits with a periodic distance (center to center) of 10.5 nm (Golecki and Drews, 1980). No lipopolysaccharide was delected in the cell wall, which thus appears to consist only of a thin peptidoglycan layer and a protein S-layer (Tadros et al., 1982). The cell wall of Actinopolyspora halophila has a conventional peptidoglycan cell wall (Johnson et al., 1986). Similar to other chemotype IV actinomycetes such as Micropolyspora and Saccharomonospora, its cell wall does not contain mycolic acids (Kates et al., 1987). Interesting wall structures can be seen in the budding prosthecate Dichotomicrobium thermohalophilum isolated from Solar Lake (Sinai) and in similar strains obtained from a shallow coastal saline lake in Brazil (Hirsch and Hoffmann, 1989). The nearly tetrahedral mother cells produce up to four hyphae, at the tips of which non-motile buds are formed. Dichotomous branching of the hyphae result in the formation of chains and nets (Figure 3.19).

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Direct electron microscopical examination of cell material collected from hypersaline environments has yielded information on the fine structure of cell walls of halophilic microorganisms of unknown phylogenetic affiliation. The surface layer of the stalk of a yet unidentified prosthecate microorganism found in the Gavish sabkha (Sinai, Egypt) at salt concentrations reaching saturation, showed a periodic array of electron dense trimers with a spacing of 9 nm, a structure reminiscent of that of porins of Escherichia coli (Kessel et al., 1985b).

3.2.2. Extracellular capsules of halophilic Bacteria Halomonas eurihalina produces an exopolysaccharide of interesting properties. It contains 42% carbohydrates (mostly hexoses) and 15% protein; the nature of the remainder is unknown. The polymer is highly viscous, especially at acid pH, and is thermostable (Quesada et al., 1993). More information on halophilic bacterial exopolysaccharides, including on their possible biotechnological uses, is given in Section 11.3.4. Another recently described exopolysaccharide producer is Halomonas maura (Bouchotroch et al., 2001). Polysaccharide slimes are also produced by halophilic unicellular cyanobacteria of the Aphanothece type (Sudo et al., 1995).

3.2.3.

Flagella of halophilic Bacteria

Motility by means of flagella is widespread among the different groups of halophilic Bacteria. Especially thick, sheathed polar flagella were observed in the haloalkaliphilic

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photosynthetic sulfur bacterium Halorhodospira abdelmalekii (Imhoff and Trüper, 1981).

3.2.4.

The cytoplasmic membrane and its lipids

The properties of the cytoplasmic membrane of halophilic Bacteria are to a large extent regulated by the outside salt concentration to adjust such functions as ion permeability and the activity of integral membrane proteins (Russell, 1993). Haloadaptation is therefore at least in part a response of the cell envelope to osmotic stress (Russell and Kogut, 1985). Salt-dependent changes in the properties of the cell membrane have been identified both on the level of the types of phospholipids that dominate in the membrane structure and in the types of fatty acid chains in the lipids.

3.2.4.1. Polar lipid metabolism of halophilic Bacteria. The major polar lipids present in most species are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Additional types of lipids may occur such as diphosphatidylglycerol (cardiolipin, CL) and glycolipids. Generally the content of negatively charged phospholipids (PC, CL) increases at the expense of neutral phospholipids (PE) as salinity increases (Russell, 1993, Vreeland, 1987, Vreeland et al., 1984). Table 3.1 shows examples of the phospholipid composition of halophilic Bacteria grown at different salt concentrations.

To explain the shift toward a higher negative charge density on the membrane at increasing salt concentration it was first postulated that the increase in anionic lipids is necessary to allow charge balance at the membrane surface exposed to high concentrations (Hiramatsu et al., 1980a, 1980b). However, this explanation alone is insufficient to account for the high salt requirement (Russell and Kogut, 1985). Another idea proposed is that the high content of negatively charged phospholipids contributes to

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the regulation of the selective permeability to cations (Ohno et al., 1979). Nowadays it is generally agreed that the change in polar lipid composition provides a mechanism for preserving the membrane bilayer structure. PE containing unsaturated fatty acids tends to form nonbilayer phases, while PG forms stable bilayers. A functional membrane requires a correct proportion of bilayer-forming and non-bilayer forming lipids. Adjustment of the phospholipid ratio in the membrane is required to preserve the integrity of the lipid bilayer in the face of an increased tendency of PE to form nonbilayer phases as a consequence of the raised external salinity. The tendency to form nonbilayer phases may activate specific phospholipid-synthesizing enzymes in the membrane, resulting in a rise in the proportion of lipids such as PG that prefer the formation of a lamellar phase, counteracting the disruptive forces of the nonbilayerphase-forming lipids (Russell, 1989, 1993; Sutton et al., 1991). Halomonas elongata cells grown at NaCl contain four times as much CL as cells in salt. The PE content of the membrane decreases accordingly, while the PG content does not change greatly (Vreeland et al., 1984). The major lipid components of Chromohalobacter israelensis are PG and PE. Additional components present are CL, (Peleg and Tietz, 1973), and an acidic glucuronic acid-containing glycolipid. The content of CL increases in the stationary phase cell at the expense of PG (Stern and Tietz, 1973a). The biosynthesis of glucosylphosphatidylglycerol proceeds from UDP-glucose and PG. and ions are required for activation of the biosynthetic system, while KCl and NaCl are inhibitory, even at the low concentration of 0.1 M (Stern and Tietz, 1978). Incubation of a cell-free preparation with acid and diglycerides led to the formation of labeled glucuronosyldiglyceride. The reaction was inhibited by more than 80% by 0.5 M KCl or NaCl (Stern and Tietz, 1973b). "Pseudomonas halosaccharolytica" contains PE, PG, and CL as major phospholipids. An unidentified phosphoglycolipid is present as well (a glucosyl derivative of PG containing phosphate, glycerol, fatty acids and glucose in the ratio 1:2:2:1). This phosphoglycolipid may be identical to the glycerol identified in Chromohalobacter israelensis (Ohno et al., 1976). Also here UDP-glucose serves as the glucosyl donor for its biosynthesis (Ohno et al., 1980). When grown in the lower salinity range PE was twice as abundant as PG, but above NaCl the amounts of PE and PG were approximately equal (Hiramatsu et al., 1980a, 1980b; Ohno et al., 1976, 1979). At a combination of high salt and high temperature, acidic phospholipids were very abundant (73.6 mol%), as a result from an increase in CL and glucosylphosphatidylglycerol and a decrease in PE (Hara et al., 1980). Pulse-labeling experiments with radiolabeled presursors such as serine, acetate and glycerol showed a decrease in PE biosynthesis with increasing NaCl, while incorporation in PG remained constant (Hara and Masui, 1985; Hiramatsu et al., 1980a, 1980b; Ohno et al., 1979; for a critical assessment of these experiments see also Russell, 1993). Upon entering the stationary growth phase the content of CL increased at the expense of PG. When 1% glucose was added to the growth medium, the phosphoglycolipid content rose to 25% of the total lipid. The glucuronosyldiglyceride of Chromohalobacter israelensis was not detected in "Pseudomonas halosaccharolytica" (Ohno et al., 1976). The outer membrane proteins

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of "Pseudomonas halosaccharolytica" show a 20-26 mol% excess of acidic amino acids over positively charged ones, a level similar to that found in the halophilic Archaea. In a yet unidentified halophilic Vibrio-like organism strain HX, PE and PG together formed more than 90% of the membrane lipids. The PE/PG ratio decreased from 1.6 to 1.0 when the NaCl concentration in the medium was raised from 60 to (Adams et al., 1990). The major lipids of Salinivibrio costicota are PG and PE, with lesser amounts of CL, lysophosphatidylethanolamine, a glycolipid, and two other phospholipids which were tentatively identified as lysocardiolipin and lysophosphatidylglycerol. These two minor lipid components may play an important role in determining the phase behavior of the membranes, as binary mixtures of PG and PE showed a different behavior as a function of salinity than the native membranes (Sutton et al., 1991). Also here an increase in medium salinity induces an increase in PG and a decrease in PE content of the membrane. The growth phase of the culture appears to have little effect on the lipid composition (Hanna et al., 1984; Sutton et al., 1991). Salt-sensitive mutants have been isolated that have a phospholipid composition similar to that of the wild type when grown in NaCl, but show only a partial change in phospholipid composition upon transfer to higher salt concentrations. A correct phospholipid composition may thus be essential for haloadaptation (Kogut et al., 1992). The lipid profile of the membrane depends not only on the salt concentration, but on temperature as well (Adams and Russell, 1992). When the salt concentration is increased, growth and phospholipid synthesis by Salinivibrio costicola are initially inhibited. Growth resumes within a few hours, as does the biosynthesis of PG. The synthesis of PE, however, remains at a low level, and this results in a decrease in the PE/PG ratio. Synthesis of new proteins is not required during the this phase of the adaptation. Only when the correct membrane composition has been achieved does the organism resume growth (Kogut and Russell, 1984; Russell et al., 1985). These changes in biosynthetic rates are reversed when the external NaCl concentration is restored to the previous level (Adams et al., 1987; Russell et al., 1985). The phospholipid biosynthetic enzymes are located in the cytoplasmic membrane, and they probably respond directly to alterations in membrane fluidity and/or the lipid phase when the external solute concentration is suddenly changed (Russell et al., 1985). The salt upshock is probably sensed via osmotic pressure effects, as non-ionic solutes such as sucrose can cause similar effects on growth and phospholipid synthesis (Adams et al., 1987; Russell et al., 1985, 1986). The major polar lipids of the alkaliphilic phototrophic sulfur bacterium Halorhodospira halophila are PG, CL, PC and PE. When grown in increasingly saline media the content of PC was strongly enhanced. PG increased to a lesser extent. However, upon a salinity upshock, biosynthesis of PG was stimulated, while dilution stress led to increased synthesis of PE (Thiemann and Imhoff, 1991). In Gram-positive halophilic Bacteria the increase in the anionic lipid fraction with salinity is generally due to an increase in CL rather that PG. The effect may be unspecific, as an increase in CL at the expense of PG is often associated with slowly growing cells, and at high salt concentrations growth rates are often reduced (Russell, 1989, 1993; Russell and Kogut, 1985). Glyco(phospho)lipids are also commonly found (Russell, 1993). PE was reported to be absent in Bacillus haloalkaliphilus (Tindall,

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1988). A Planococcus sp. showed an increase in anionic phospholipids and a decrease in PE with increasing salt (Miller, 1985). When grown at high monovalent cations concentrations, the ratio CL/PG was increased, while this ratio remained unchanged when or were used to increase medium salinity (Miller, 1986). Elevated temperatures and salinity had an antagonistic effect on phospholipid composition: a rise in temperature caused an increase in PE and a decrease in CL content (Miller, 1985; Russell, 1993).

3.2.4.2. Fatty acid metabolism of halophilic Bacteria. The rod-shaped Gram-negative halophilic Bacteria (Halomonas, Chromohalobacter, and related organisms) mainly contain common straight-chain saturated and monounsaturated fatty acids (C 16:0, 16:1, and especially 18:1) in their membrane lipids. Cyclopropane fatty acids were detected as well in many isolates (Franzmann and Tindall, 1990; Monteoliva-Sanchez and Ramos-Cormenzana, 1987a; Monteoliva- Sanchez et al., 1988; Peleg and Tietz, 1971; Skerratt et al., 1991). The content of cyclopropane fatty acids and unsaturated fatty acids generally increases with salt concentration, while the abundance of branched fatty acids decreases. The effect has been reported in Halomonas halophila (MonteolivaSanchez et al., 1988), in Halomonas halmephila (Monteoliva-Sanchez and RamosConnenzana, 1986), in "Pseudomonas halosaccharolytica" (Ohno et al., 1979), and in Vibrio HX (Adams et al., 1990). Cyclopropane fatty acids are synthesized by addition of a methyl group (derived from S-adenosylmethionine) across the double bond of a monounsaturated fatty acid. Such modification rigidifies the central part of the acyl chain. An increase in unsaturated fatty acids is accompanied by an increase in membrane fluidity. The effect of cyclopropane fatty acids on the membrane fluidity is less clear. The physical properties of cyclopropane fatty acids (e.g. cyc17:0) are intermediate between the corresponding saturated fatty acid (17:0) and the unsaturated fatty acid from which it was derived (16:1), so an increase in cyclopropane fatty acids at the expense of unsaturated fatty acids would tend to decrease membrane fluidity (Russell, 1989, 1993). Halomonas halophila shows an increasing content of cyclopropane fatty acids with increasing salt with a concomitant decrease in monounsaturated fatty acids. It was suggested that the cyclopropane fatty acid synthetase is activated or induced by high salt levels (Monteoliva-Sanchez et al., 1988). When the salt concentration was raised from 50 to cyc17:0 increased from 0 to 8.5%, and cyc19:0 from 4.2 to 14.9%, while the content of the saturated branched-chain 15:0 decreased from 42.9% to 31.9% (Monteoliva-Sanchez and Ramos-Cormenzana, 1986). The metabolism of cyclopropane fatty acids (cyc17:0 and cyc19:0) has been further studied in "Pseudomonas halosaccharolytica". The content of cyclopropane fatty acids increased with salinity, with a decrease in the corresponding monounsaturated species. It was suggested that cyclopropane fatty acid synthetase was induced by high NaCl levels. The proportion of cyclopropane fatty acids also increased with temperature and also when the cultures entered the stationary phase. The cyclopropane fatty acid synthetase is either an integral membrane protein or is associated with the membrane. Its activity is inhibited by NaCl or KCl, but stimulated up to 12-fold by 3 M glycine betaine (Hara et al., 1980; Hiramatsu et al., 1978, 1980a, 1980b; Hyono et al., 1979,

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1980;Kuriyama et al., 1982; Monteoliva-Sanchez et al., 1993; Ohno et al., 1976, 1979). Electron paramagnetic resonance studies of rotational movements of lipids in the membranes in intact "Pseudomonas halosaccharolytica" cells showed the presence of an unusually thick (up to a depth of 12 carbons from the hydrophobic surface) viscous surface region in the membrane bilayer. At the 12th carbon from the polar surface the viscoelastic properties change very drastically. The membrane has thus two viscous outer regions and one fluid central region, the border of the viscous region being located near the double bonds or the cyclopropane rings located at carbon no. 11-12 of the acyl chains. Increasing salinity caused an increase of the rigidity, i.e. of the micro-viscosity of the polar surface in the lipid bilayer. The presence of thick viscous regions may be related to the need to withstand high osmotic pressure or to the requirement for a low permeability to (Hiramatsu et al., 1980a, 1980b; Hyono et al., 1979, 1980; Kuriyama et al., 1982). Vibrio HX contained twice as much cyclopropane fatty acids when grown at 230 g salt than at (Adams et al., 1990). When the medium NaCl concentration was raised from 60 to the endogenous activity of cyclopropane fatty acid synthetase measured in cell lysates was doubled. NaCl was found inhibitory to both fatty acid synthetase (82% inhibition by NaCl) and cyclopropane fatty acid synthetase (97% inhibition by NaCl), but both activities were up to 100-fold stimulated by 2-3 M glycine betaine. Enzyme induction is not required to achieve the salt-dependent alterations in cyclopropane fatty acid content observed in vivo. Sucrose and trehalose were less effective than glycine betaine (Kuchta and Russell, 1994). The psychrophilic halophile Flavobacterium salegens from Organic Lake, Antarctica has a high abundance of i15:0, 15:0, a15:0, and i16:0. The structure of the branched C15 monounsaturated fatty acids is unusual. This fatty acid can therefore be used as a taxonomic marker for this type of organism in ecological studies of Antarctic lakes (Skerratt et al., 1991). Salinivibrio costicola does not produce cyclopropane fatty acids. Its major fatty acids are 16:0 and with minor amounts of 14:0, 17:1, and 18:0. Unsaturated fatty acids are synthesized using the anaerobic pathway, common to most prokaryotes. The content of 16:0 was lowest at the optimal salinity and increased at the low and high salt extremes of growth (30 and NaCl); the content of 18:1 was highest at the optimum salinity (Hanna et al., 1984; Russell, 1993; Sutton et al., 1990a, 1991). Differences in the position of the double bond in the unsaturated fatty acids were found in the fatty acid composition of PG and PE: and were present in phospholipids of cells grown in but not in NaCl, indicating that salinity may affect the specificity of the fatty acid synthetase. Salinity had a greater influence on the fatty acid composition of PG than of PE, which suggests that PG and PE biosynthesis are controlled separately by a selective effect of NaCl which occurs after the cytosine diphosphate-diacylglycerol branch point (Sutton et al., 1990b). In the Gram-positive genera Marinococcus, Nesterenkonia, and Halobacillus, branched fatty acids such as 15:0 and 17:0 are dominant (Monteoliva-Sanchez et al., 1989). The content of these branched fatty acids changes with salinity (Russell, 1993). In Marinococcus halophilus an increase in salinity gave a response similar to that found when increasing the temperature: the content of saturated fatty acids (mainly 18:0) increased, while the branched anteiso-15:0 decreased. The unsaturated 16:1 increased

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from 40.9 to 48.8% when salt concentration was raised from 50 to 150 g Cyclopropane fatty acids were not detected (Monteoliva-Sanchez et al., 1987b). In Planococcus sp. strain A4a (ATCC 35671), anteiso-15:0 is the major fatty acid, and branched fatty acids make up more than 85% of the fatty acid content at all salinities (Miller, 1985). The phototrophic alkaliphile Halorhodospira halophila contains straight saturated and monounsaturated C16 and C18 as major fatty acids. With increasing temperature the content of 18:0 and unsaturated fatty acids decreased with a rise in 16:0 and saturated fatty acids (Imhoff and Thiemann, 1991).

3.2.4.3. Respiratory Quinones in Halophilic Bacteria. Both ubiquinone and menaquinone type isoprenoid quinones have been detected in halophilic Bacteria. Ubiquinones (Q-7, Q-8, and Q-9) are found in halophilic Proteobacteria in different amounts (Table 3.4).

Salinivibrio costicola contains both ubiquinones (Q-8) and menaquinones (MK-8). In Actinopolyspora halophila a complex mixture of partially hydrogenated menaquinones was found, dominated by tetrahydrogeated menaquinones with nine isoprene units (Collins et al., 1981).

3.2.4. Photosynthetic membranes Photosynthetic prokaryotes possess elaborate intracellular membrane systems on which the photosynthetic apparatus is located. Cyanobacteria, including the halophilic representatives within the group, contain intracellular thylacoids. A great variety of different types of intracellular photosynthetic membranes is found among halophilic anoxygenic photosynthetic bacteria. Halochromatium and Thiohalocapsa have intracellular membranes of the vesicular type. Vesicular-type photosynthetic membrane are also present in Rhodovibrio salinarum and Rhodovibrio sodomensis (Mack et al., 1993; Nissen and Dundas, 1984). Rhodothalassium salexigens has photosynthetic membranes located parallel to the cytoplasmic membrane (Drews, 1981). Stacks of thylacoid-like membranes are characteristic for the moderately halophilic genus Ectothiorhodospira and the truly halophilic genus Halorhodospira (Imhoff and Trüper, 1981; Imhoff et al., 1991; Oren et al., 1989; Ventura et al., 1988). Figure 3.20 shows an example.

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Based on analysis of serial thin sections and other electron microscopic techniques, a three-dimensional model has been made of the photosynthetic membrane system of Halorhodospira halochloris (Wanner et al., 1986). The disc-shaped thylacoidal sacs were found to be connected both to each other and to the cytoplasmic membrane by small membranous "bridges". The lumina of all thylacoids are therefore continuous with the periplasmic space (Figure 3.21).

3.2.6. Gas vesicles Gas vesicles, so prominent in many halophilic Archaea (see Section 3.1.7), are rarely found in halophilic Bacteria. The truly halophilic representatives of the cyanobacteria, a group in which gas vesicle production is frequently encountered, do not contain gas vesicles. Ectothiorhodospira vacuolata is characterized by gas vesicle production, but this species is only slightly halophilic, as it only grows between 10 and NaCl (Imhoff et al., 1991). There are two interesting reports of the synthesis of gas vesicles in endosporeproducing anaerobic members of the Halobacteroidaceae. Growing cells of

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Sporohalobacter lortetii and Orenia sivashensis do not contain gas vesicles. However, when endospore formation is initiated, gas vesicles are synthesized, and these remain attached to the mature endospores, possibly aiding in their dispersion (Oren, 1983; Zhilina et al., 1999).

3.2.7. Endospores Heat-tolerant endospores are produced by several groups of halophilic Bacteria. These include the representatives of the families Clostridiaceae such as Clostridium halophilum (Fendrich et al., 1990) and Desulfotomaculum halophilum (Tardy-Jaquenod et al., 1998), the Halanaerobiaceae (Natroniella acetigena; Zhilina et al., 1996), the Halobacteroidaceae with Sporohalobacter lortetii (Oren, 1983), Orenia species (Mouné et al., 2000; Oren et al., 1987; Zhilina et al., 1999), and Halonatronum saccharophilum (Zhilina et al., 2001), and the Bacillaceae with the genera Bacillus, Gracilibacillus, Halobacillus, and Salibacillus (Arahal et al., 1999; Chaiyanan et al., 1999; Garabito et al., 1997; Spring et al., 1996). Pasteurization of hypersaline sediment samples is a reliable tool for the selective isolation of anaerobes of the order Halanaerobiales (Oren, 1987). No in-depth studies have yet been performed on the physiology and genetics of endospore formation in halophiles.

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3.3. CELLULAR STRUCTURES OF THE HALOPHILIC EUCARYA The cellular organization of the Eucarya is much more complex than that of the Archaea and the Bacteria. This is true to the same extent for the halophilic representatives within the eukaryotic domain. Species of the halophilic or halotolerant algal genus Dunaliella do not possess a rigid cell wall. The cells can therefore swell and shrink as an immediate reaction to changes in the salinity of their environment. The cytoplasmic membrane that surrounds the cell enables rapid endocytotic uptake of large molecules such as dextrans and other large molecules with molecular masses of up to The molecules taken up become located in intracellular membrane vesicles. Rapid efflux of compounds contained in these vesicles is possible as well. It was suggested that these vesicles may be a source of membrane material, to be used when the cell has to expand upon decrease in salt concentration (Ginzburg et al., 1999). Additional evidence for the presence of such a supply of available membrane to be used when the cells swell during a hypoosmotic shock is provided by electron micrographs which document the event of the fusion of such membrane vesicles with the plasmalemma (Maeda and Thompson, 1986) (see also Section 8.4).

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Dunaliella cells contain additional cell inclusions of interest. Besides the globules accumulated in the interthylacoid space within the (single) chloroplast of some species (to be discussed in Section 5.1) and the starch-containing pyrenoid, there have been reports on the presence of other inclusions of unknown nature in Dunaliella salina cells. Laser microprobe analysis showed these inclusions to be rich in silica, phosphorus, sulfur, and calcium (Bochem and Sprey, 1979). Heavy metals such as uranium may accumulate in these inclusions (Sprey and Bochem, 1981) (Figure 3.22). Sterols are a major component of the lipids of halophilic black yeasts. Ergosterol and abound in all species of halophilic fungi examined (Méjanelle et al., 2000). Other sterols and are specifically found in Cladosporium, Alternaria alternata, and Hortaea werneckii. These sterols can be used as specific biomarkers for such fungi (Méjanelle et al., 2000; see also Section 14.3). The biosynthetic pathways leading to ergosterol formation have been elucidated in different halophilic melanized fungi (Méjanelle et al., 2001). 3.4. REFERENCES Adams, R.L., and Russell, N.J. 1992. Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola. Can. J. Microbiol. 38: 823827. Adams, R., Bygraves, J., Kogut, M., and Russell, N.J. 1987. The role of osmotic effects in haloadaptation of Vibrio costicola. J. Gen. Microbiol. 133: 1861-1870. Adams, R.L., Kogut, M., and Russell, N.J. 1990. The effect of salinity on growth and lipid composition of a moderately halophilic Gram-negative bacterium HX. Biochem. Cell Biol. 68: 249-254. Alam, M., and Oesterhelt, D. 1984. Morphology, function and isolation of halobacterial flagella. J. Mol. Biol. 176: 459-475. Alam, M., and Oesterhelt, D. 1987. Purification, reconstitution and polymorphic transition of halophilic flagella. J. Mol. Biol. 194: 495-499. Alam, M., Claviez, M., Oesterhelt, D., and Kessel, M. 1984. Flagella and motility behaviour of square bacteria. EMBO J. 3: 2899-2903. Alba, I., Torreblanca, M., Sánchez, M., Colom, M.F., and Meseguer, I. 2001. Isolation of the fibrocrystalline body, a structure present in haloarchaeal species, from Halobacterium salinarum. Extremophiles 5: 169175. Antón, J., Meseguer, I., and Rodríguez-Valera, F. 1988. Production of an extracellular polysaccharide by Haloferax mediterranei. Appl. Environ. Microbiol. 54: 2381-2386. Arahal, D.R., Márquez, M.C., Volcani, B.E., Schleifer, K.H., and Ventosa, A. 1999. Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int. J. Syst. Bacteriol. 49: 521-530. Ban, N., Nissen, P., Hansen, J., Moore, P., and Steitz, T.A. 2000. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905-934. Beard, S.J., Hayes, P.K., and Walsby, A.E. 1997. Growth competition between Halobacterium salinarium strain PHH1 and mutants affected in gas vesicle synthesis. Microbiology UK 143: 467-473. Blaurock, A.E., Stoeckenius, W., Oesterhelt, D., and Scherphof, G.L. 1976. Structure of the cell envelope of Halobacterium halobium. J. Cell Biol. 71: 1-22. Bochem, H.-P., and Sprey, B. 1979. Laser microprobe analysis of inclusions in Dunaliella salina. Z. Pflanzenphysiol. 95: 179-182. Bouchotroch, S., Quesada, E., del Moral, A., Llamas, I., and Béjar, V. 2001. Halomonas maura sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium. Int. J. Syst. Evol. Microbiol. 51: 1625-1632. Brown, A.D. 1990. Microbial water stress physiology. Principles and perspectives. John Wiley & Sons, Chichester

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Soo-Hoo, T.S., and Brown, A.D. 1967. A basis for the specific sodium requirement for morphological integrity of Halobacterium halobium. Biochim. Biophys. Acta 135: 164-166. Sprey, B., and Bochem, P.-P. 1981. Uptake of uranium into the alga Dunaliella detected by EDAX and LAMMA. Fresenius Z. Anal. Chem. 308: 239-245. Spring, S., Ludwig, W., Marquez, M.C., Ventosa, A., and Schleifer, K.-H. 1996. Halobacillus gen. nov., with descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov. Int. J. Syst. Bacteriol. 46: 492-496. Steber, J., and Schleifer, K.H. 1975. Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall. Arch. Microbiol. 105: 173-177. Steber, J., and Schleifer, K.H. 1979. N-glycyl-glucosamine: a novel constituent in the cell wall of Halococcus morrhuae. Arch. Microbiol. 123: 209-212. Steensland, H., and Larsen, H. 1969. A study of the cell envelope of the halobacteria. J. Gen. Microbiol. 55: 325-336. Steensland, H., and Larsen, H. 1971. The fine structure of the extremely halophilic cocci. Kong. Norske Vidensk. Selsk. Skr. 8: 1-5. Stern, N., and Tietz, A. 1973a. Glycolipids of a halotolerant, moderately halophilic bacterium. I. The effect of growth medium and age of culture on lipid composition. Biochim. Biophys. Acta 296: 130-135. Stern, N., and Tietz, A. 1973b. Glycolipids of a halotolerant, moderately halophilic bacterium. II. Biosynthesis of glucuronosyldiglyceride by cell-free particles. Biochim. Biophys. Acta 296: 136-144, Stern, N., ad Tietz, A. 1978. Glycolipids of a halotolerant, moderately halophilic bacterium. Biosynthesis of glucosylphosphatidylglycerol by cell-free particles. Biochim. Biophys. Acta 530: 357-366. Stoeckenius, W. 1981. Walsby's square bacterium: fine structure of an orthogonal procaryote. J. Bacteriol. 148: 352-360. Stoeckenius, W., and Kunau, W.H. 1968. Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes. J. Cell Biol. 38: 337-357. Stoeckenius, W., and Rowen, R. 1967. A morphological study of Halobacterium halobium and its lysis in media of low salt concentration. J. Cell Biol. 34: 365-393. Sudo, H., Burgess, J.G., Takemasa, H., Nakamura, N., and Matsunaga, T. 1995. Sulfated exopolysaccaride production by the halophilic cyanobacterium Aphanothece halophytica. Curr. Microbiol. 30: 219-222. Sumper, M. 1987. Halobacterial glycoprotein biosynthesis. Biochim. Biophys. Acta 906: 69-79. Sumper, M., Berg, E., Mengele, R., and Strobel, I. 1990. Primary structure and glycosylation of the S-layer protein of Haloferax volcanii. J. Bacteriol. 172: 7111-7118. Sutton, G.C., Quinn, P.J., and Russell, N.J. 1990a. The effect of salinity on the composition of fatty acid double bond isomers and sn-1/sn-2 positional distribution in membrane phospholipids of a moderately halophilic eubacterium. Curr. Microbiol. 20: 43-46. Sutton, G.C., Russell, N.J., and Quinn, P.J. 1990b. The effect of salinity on the phase behaviour of purified phosphatidylethanolamine and phosphatidylglycerol isolated from a moderately halophilic eubacterium. Chem. Phys. Lipids 56: 135-147. Sutton, G.C., Russell, N.J., and Quinn, P.J. 1991. The effect of salinity on the phase behaviour of total lipid extracts and binary mixtures of the major phospholipids isolated from a moderately halophilic eubacterium. Biochim. Biophys. Acta 1061: 235-246. Tadros, M.H., Drews, G., and Evers, D. 1982. Peptidoglycan and protein, the major cell wall constituents of the obligate halophilic bacterium Rhodospirillum salexigens. Z. Naturforsch. 37c: 210-212. Takashina, T., Hamamoto, T., Otozai, K., Grant, W.D., and Horikoshi, K. 1990. Haloarcula japonica sp. nov., a new triangular halophilic archaebacterium. Syst. Appl. Microbiol. 13: 177-181. Tardy-Jaquenod, C., Magot, M., Patel, B.K.C., Matheron, R., and Caumette, P. 1998. Desulfotomaculum halophilum sp. nov., a halophilic sulfate-reducing bacterium isolated from oil-producing facilities. Int. J. Syst. Bacteriol. 48: 333-338. Thiemann, B., and Imhoff, J.F. 1991. The effect of salt on the lipid composition of Ectothiorhodospira. Arch. Microbiol. 156: 376-384. Tindall, BJ. 1985. Qualitative and quantitative distribution of diether lipids in haloalkaliphilic archaebacteria. Syst. Appl. Microbiol. 6: 243-246. Tindall, B.J. 1988. Prokaryotic life in the alkaline, saline, athalassic environment, pp. 31-67 In: RodriguezValera, F. (Ed.), Halophilic bacteria, Vol. I. CRC Press, Boca Raton. Tindall, B.J. 1990a. Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol. Lett. 66: 199-202. Tindall, B.J. 1990b. A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst. Appl. Microbiol. 13: 128-130.

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CHAPTER 4 CELLULAR METABOLISM AND PHYSIOLOGY OF HALOPHILIC MICROORGANISMS

Even with the recent advances in our understanding of the physiology of these micro-organisms the basis for extreme obligate halophilism is far from clearly understood and remains an exciting scientific challenge. In addition to this, many scientists not primarily interested in extreme halophilism find that halophilic bacteria, their enzymes and organelles, are uniquely well suited to a study of general biological phenomena such as electron transport or transport of molecules across membranes. (Dundas, 1977)

This chapter presents a general overview of the physiology of different groups of halophilic microorganisms - Archaea, Bacteria, and Eucarya, with special emphasis on those aspects of their dissimilatory and assimilatory metabolism that are unique. The ion metabolism and the production of organic osmotic solutes by different types of halophiles are discussed separately in Chapter 6 and in Chapter 8, respectively. No extensive discussions will be devoted here to the biochemistry and the physiology of oxygenic photosynthesis in halophilic cyanobacteria and eukaryotic algae, as the process does not greatly differ from photosynthesis in their non-halophilic relatives.

4.1. PHYSIOLOGY OF THE HALOPHILIC ARCHAEA Of all Archaea, the halophiles are the easiest to handle in the laboratory. Organisms such as Halobacterium salinarum and Haloferax volcanii have therefore become convenient laboratory models to study archaeal metabolism. Since the earlier reviews on metabolic aspects of the Halobacteriaceae by Dundas (1977) and by Hochstein (1988), much new information has become available. Little effort has been devoted thus far to the study of halophilic methanogenic Archaea beyond their taxonomic characterization (see Section 2.2). Section 4.1.10 reviews the limited information available on the metabolism of these organisms.

4.1.1. Nutritional demands of aerobic halophilic Archaea Members of the Halobacteriaceae differ greatly in their nutritional demands. Some have complex requirements that can only be met in culture by including high concentrations of yeast extract or other rich sources of nutrients in their medium.

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Others grow well on single carbon sources while using ammonia as nitrogen source. In addition to simple substrates such as amino acids, sugars, and organic acids, certain polymeric substances can be degraded by some halophilic Archaea. Many species of the Halobacteriaceae produce exoenzymes such as proteases, lipases, DNAses, and amylases. The nutritional demands of Halobacterium salinarum, the best known archaeal halophile species, are complex. Defined media designed for the growth of different isolates may contain between 10 and 21 amino acids, in some cases supplemented with vitamins, up to 5 different nucleosides, and glycerol (Dundas et al., 1963; Grey and Fitt, 1976; Onishi et al., 1965; Shand and Perez, 1999). When Halobacterium salinarum was grown in a defined medium containing inorganic salts, five nucleosides, 21 amino acids, glycerol, and the vitamins folic acid, thiamine, and biotin, a complex growth curve was obtained in which a number of phases could be discerned within the "exponential" growth phase, each with a different growth rate (Shand and Perez, 1999). There are some indications that even rich media based on yeast extract, peptones, etc. may not provide all the compounds required by some fastidious members of the group. When attempting to enumerate halophilic Archaea present in saltern evaporation and crystallizer ponds, Wais (1988) reported that colony recovery rates greatly improved when the medium was amended with a lysate of Halobacterium salinarum cells as a source of additional growth factors. The concensus, expressed in older studies, that the nutritional requirements of all Halobacteriaceae are complex, had to be changed when a strain was isolated, later described as Haloferax mediterranei, that grows on simple compounds such as succinate, acetate, and others as single carbon and energy source. Inorganic salts may supply the need for nitrogen, sulfur and other essential elements (Rodriguez-Valera et al., 1980, 1983). Such simple growth requirements are commonly found in the genera Haloferax and Haloarcula. Sometimes vitamins may be stimulatory: a synthetic medium described as satisfactory for the growth of Haloferax volcanii contains glycerol and succinate as carbon and energy sources, thiamine and biotin as stimulatory vitamins, and inorganic salts, including ammonium ions as nitrogen source (Kauri et al., 1990). Field studies have shown that in environments such as the Dead Sea or saltern crystallizer ponds acetate is utilized very poorly by the halophilic archaeal community: acetate turnover times in the order of weeks to months were measured (Oren, 1995b). A study of a hypersaline cyanobacterial mat that contained halophilic Archaea (including Haloarcula japonica and Halorubrum distributum) suggested that tricarboxylic acid cycle intermediates excreted by Microcoleus chthonoplastes, the dominant cyanobacterium present, may be the major carbon and energy source on which the Archaea thrive in situ (Zvyagintseva et al., 1995b). Simple sugars such as glucose and sucrose are not readily used by all members of the Halobacteriaceae, but species of Haloferax, Haloarcula and some additional genera do grow on sugars. More information on the carbohydrate metabolism of the Halobacteriaceae is presented in Section 4.1.4. While amino acids are the preferred nitrogen source for most species, some can use ammonia or nitrate. A nitrite reductase required during the assimilatory use of nitrate or nitrite has recently been purified from Haloferax mediterranei (Martinez-Espinosa et al., 2001).

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4.1.2. Membrane transport systems for nutrients Most halophilic Archaea preferentially use amino acids as carbon and energy source. Different amino acid transport systems in the cytoplasmic membrane of Halobacterium salinarum have been characterized, using membrane vesicles as model systems. In many of these studies membranes containing bacteriorhodopsin were used, so that the transport processes could be energized by light. Many of the amino acid transport systems specifically depend on ions. Thus, leucine transport was facilitated by symport with ions (MacDonald and Lanyi, 1975). A mechanism also drives uptake of glutamate (Birkeland and Ratkje, 1985; Lanyi et al., 1976; Stevenson, 1966). Tyrosine can be transported by two specific transporters as well as by a common transport system for aromatic amino acids (Lobyreva et al., 1994). Measurements of the size of the intracellular pools of amino acids in Halobacterium salinarum showed glutamate and aspartate as the most abundant compounds; pool sizes of the different amino acids were correlated with their rate of transport (Lobyreva et al., 1991). Transport systems for acetate and propionate have been studied in the alkaliphilic Natronococcus occultus. Two systems were detected. One of these is a high affinity system, driven by the gradient over the membrane (Kevbrina et al., 1989). Sodiumdriven transport systems are also responsible for the transport of glucose and fructose in Haloferax volcanii (Takano et al., 1995; Tawara and Kamo, 1991). Active transport of phosphate into Halobacterium salinarum cells depends on the cellular ATP level. or gradients alone do not drive the process. It was suggested that ATP acts as a regulator for phosphate transport which is supposedly driven (Zoratti and Lanyi, 1987). Sulfate, used as an electron acceptor by Desulfonatronovibrio hydrogenovorans, enters the cell via an electroneutral symport with ions (Sydow et al., 2002). Multi-drug efflux transporters have been detected in the membrane of halophilic Archaea. A mutant of Haloferax volcanii was found that had acquired resistance to doxorubicin, a compound that otherwise inhibits growth. ATP-driven extrusion of the drug was demonstrated (Miyauchi et al., 1992). When grown in the presence of glucose or other sugars, wild type Haloferax volcanii activity of the multi-drug efflux transporter is increased (Miyauchi et al., 1997). It was suggested that metabolism of glucose or fructose produces some substances that are cytotoxic, upset cell homeostasis, and prevent growth. The cells then activate the efflux transporter to remove these toxic substances (Kaidoh et al., 1996).

4.1.3. Chemotaxis and transducer proteins Halobacterium salinarum shows a strong chemotactic behavior toward leucine, isoleucine, valine, methionine, cysteine, arginine and several peptides. Deletion analysis of potential transducers has led to the identification of the Car protein, a cytoplasmic sensor for arginine chemotaxis. The signaling pathway from extracellular arginine to the flagellar motor has been elucidated; it involves an arginine:ornithine antiporter system (Storch et al., 1999). The membrane-bound BasT, another transducer protein for amino acid detection in Halobacterium salinarum, mediates chemotaxis to

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the other five attractants, leucine, isoleucine, valine, methionine, and cysteine, as appeared in a study of strains in which this transducer was mutated. The Car and the BasT transducers together thus cover the whole spectrum of amino acids that serve as attractants (Kokoeva and Oesterhelt, 2000). A myoglobin-like, heme-containing protein (HemAT-Hs) has been identified that functions as an oxygen-sensing transducer for the aerotaxis reaction in Halobacterium salinarum (Hou et al., 2000). The polyhistidine-tagged protein has been cloned in Escherichia coli, and could be purified by affinity chromatography (Piatibratov et al., 2000). A series of methyl-accepting taxis proteins have been identified in Halobacterium salinarum, ranging in molecular mass from 90 to 135 kDa. Positive chemotaxis increased their methylation. During negative chemotaxis they became demethylated. An increase in demethylation was noted both during positive and negative photostimulation (Alam et al., 1989). Using a probe designed after the signaling domain of HtrI, a halobacterial transducer for phototaxis (see also Section 5.4.3), a series of four new related transducer proteins were discovered (Rudolph et al., 1996). Thirteen soluble and membrane-bound transducer proteins were identified in Halobacterium salinarum, belonging to at least three subfamilies: 1, a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; 2, a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains, and 3, a cytoplasmic domain without hydrophobic transmembrane segments (Zhang et al., 1996). The soluble transducer protein HtrXI has been studied in further detail. It is a 49.5 kDa soluble protein, involved in chemotactic responses to histidine, aspartate, and glutamate. Addition of histidine increases the degree of methylation of the protein (Brooun et al., 1997).

4.1.4. Dissimilatory biochemical pathways The Halobacteriaceae are basically aerobic heterotrophs. Aerobic degradation of carbon sources is based on the tricarboxylic acid cycle and a respiratory electron transport involving a chain of cytochromes, if necessary in combination with the glyoxylate cycle, and reactions of the Embden-Meyerhof pathway and of a modified Entner-Doudoroff pathway. All enzymes of the tricarboxylic acid cycle are present in Halobacterium salinarum (Aitken and Brown, 1969). A study by NMR spectroscopy following addition of glucose or alanine and subsequent monitoring of the appearance of the label in different atoms of glutamate suggested rapid cycling of carbon through the pathway (Ghosh and Sonawat, 1998). Of all pyruvate thus entering the tricarboxylic acid cycle, 90% was oxidized through the pyruvate:ferredoxin oxidoreductase, and 10% was carboxylated to oxaloacetate by pyruvate carboxylase (Ghosh and Sonawat, 1998). By following the labeling pattern of glutamate after pyruvate addition it was concluded that extensive randomizing of the label occurs in the tricarboxylic acid cycle by scrambling of the label at the fumarate-succinate stage and operation of malic enzyme (Bhaumik and Sonawat, 1994; Majumdar and Sonawat, 1998). Although most pyruvate oxidation proceeds via a ferredoxin-linked oxidoreductase, an operon coding for a pyruvate dehydrogenase multienzyme complex,

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involving dihydrolipoyl acyltransferase and dihydrolipoamide dehydrogenase, has been found both in the genome of Halobacterium salinarum NRC-1 and in the genome of Haloferax volcanii (Jolley et al., 2000). There is, however, no evidence as yet that this operon is transcribed and used; its function is unknown. Dihydrolipoamide dehydrogenase activity is present in the halophilic Archaea, as is lipoic acid. Dihydrolipoamide dehydrogenase-negative mutants have growth characteristics identical to those of the wild type, showing that lipoate is not necessary for any essential cellular function. Metabolism of acetate in the Halobacteriaceae has a number of unusual features. Organisms such Halococcus saccharolyticus, Haloferax volcanii and Halorubrum saccharovorum excrete acetate when grown on glucose, and they are able to use acetate as growth substrate as well. Halococcus saccharolyticus grown on glucose contained activity of ADP-forming acetyl-CoA synthase (ADP-ACS; E.C. 6.2.1.13) rather than the conventional acetate kinase and phosphate acetyltransferase or the AMP-forming acetyl-CoA synthase. In stationary phase the excreted acetate was consumed again, and the cells then contained AMP-forming acetyl-CoA synthetase (AMP-ACS, EC 6.2.1.1.), while the activity of ADP-ACS was significantly reduced. When grown on acetate as carbon and energy source, the cells contained AMP-ACS rather than ADP-ACS or acetate kinase. Metabolism of acetate depended strictly on the presence of AMS-ACS. Suspensions of cells grown in glucose did not consume acetate. These cells contained ADP-ACS but had no AMP-ACS. Formation of acetate is thus catalyzed by ADP-ACS (catalyzing the reaction while activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS (acetate + + AMP + pyrophosphate) (Bräsen and Schönheit, 2001). In Natronococcus occultus acetate is activated via acetylphosphate via acetate kinase and acetyl-CoA synthetase (Kevbrina and Plakunov, 1992). Aitken and Brown (1969) first reported the presence in Halobacterium salinarum of both key enzymes of the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2). These enzymes were detected only when acetate had been included in the growth medium. Interestingly, genes for isocitrate lyase and malate synthase could not be identified in the Halobacterium strain NRC1 genome (Ng et al., 2000). Acetate-grown Haloferax mediterranei and Haloferax volcanii had high activities of both enzymes (Oren and Gurevich, 1995a; Serrano et al., 1998), but low activity was found in representatives of the genera Halobacterium and Haloarcula (Oren and Gurevich, 1995a). The glyoxylate bypass operon (ace) of Haloferax volcanii was recently sequenced and subjected to phylogenetic and transcriptional analysis. The isocitrate lyase is similar to the eukaryotic and the bacterial enzyme, but the malate synthase is very different from earlier described counterparts. Both genes cotranscribed in a single 2.7 kb mRNA (Serrano et al., 2001). A functional glyoxylate cycle has also been demonstrated in Natronococcus occultus (Kevbrina and Plakunov, 1992). In addition to the enzymes of the modified Entner-Doudoroff pathway (see below) and gluconeogenesis, the methylglyoxal bypass may also be operative in the halophilic Archaea. In this pathway, dihydroxyacetone phosphate is converted to methylglyoxal by methylglyoxal synthase (EC 4.2.99.11). Methylglyoxal is then transformed to D-

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lactate by glyoxalase I (S-lactoylglutathione methylglyoxal lyase; EC 4.4.1.5) which forms S-lactoylglutathione, and by glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) (Oren and Gurevich, 1995b). In the halophilic Archaea, is used instead of glutathione (Newton and Javor, 1985). Methylglyoxal synthase has been detected in all strains tested except in Halobacterium salinarum, while glyoxalase I was present in all species examined (Oren and Gurevich, 1995b). Glycerol is used by many species as a carbon source. Glycerol is produced as an osmotic solute by the halophilic green alga Dunaliella (see Section 8.4), and this widely available compound may therefore be one of the main substrates that support growth of halophilic Archaea in salt lakes (Borowitzka, 1981; Javor, 1984; Oren, 1993, 1995a; Oren and Shilo, 1985). There are two possible pathways of glycerol activation: phosphorylation by glycerol kinase (EC 2.7.1.30) followed by dehydrogenation of the glycerol-3-phosphate formed, or first oxidation to dihydroxyacetone by glycerol dehydrogenase (EC 1.1.1.6) followed by a phosphorylation step. A constitutive NADdependent glycerol dehydrogenase was found in Halobacterium salinarum. No such activity was found in Halorubrum saccharovorum and in Halorubrum sodomense, nor in Haloferax or Haloarcula species, not even when grown in the presence of glycerol. Glycerol kinase is present constitutively in all species examined (Oren, 1994b; Oren and Gurevich, 1994a). Different enzymatic pathways were found for the use of lactate: at least three types of lactate dehydrogenase were detected in the Halobacteriaceae: Haloarcula had high levels of an NAD-linked enzyme, but only low activities were detected in Haloferax mediterranei; NAD-independent enzymes (assayed by the reduction of dichlorophenol indophenol) that oxidize L- or D-lactate were found in all species tested, but activities in Haloarcula were very low. Lactate racemases were found in Haloarcula species (constitutive) and in Haloferax volcanii (inducible) (Oren, 1994b; Oren and Gurevich, 1994b). Halobacterium does not grow on sugars, although addition of carbohydrates to the growth medium may stimulate growth to some extent (Gochnauer and Kushner, 1969). However, different carbohydrates can be used by a variety of other genera of the Halobacteriaceae. Carbohydrate utilization was first demonstrated in Halorubrum saccharovorum (Tomlinson and Hochstein, 1972a, 1976). Breakdown of glucose by Halorubrum saccharovorum and other members of the group follows a modified Entner-Doudoroff pathway in which the phosphorylation step is postponed. Glucose is oxidized via gluconate to 2-keto-3-deoxygluconate, followed by phosphorylation to 2keto-3-deoxy-6-phosphogluconate, which is then split into pyruvate and glyceraldehyde-3-phosphate (Tomlinson et al., 1974) (Figure 4.1, left part). Genes coding for glucose dehydrogenase and for 2-keto-3-deoxygluconate kinase, both enzymes of the semiphosphorylated Entner-Doudoroff pathway, have been identified in the genome of Halobacterium NRC-1 (Ng et al., 2000), in spite of the fact that Halobacterium reportedly does not metabolize sugars. However, NMR studies showed that added glucose was transformed to gluconate by Halobacterium salinarum (Sonawat et al., 1990). The gene for 2-keto-3-deoxy-6-phosphogluconate aldolase remains to be assigned in the genome of Halobacterium NRC-1 (Ng et al., 2000). A study of Haloferax mediterranei and Haloarcula vallismortis suggested that the glycolytic Embden-Meyerhof pathway may not be functional in the

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Halobacteriaceae as hexokinase and phosphofructokinase activities are low, and moreover, these enzymes are strongly salt-inhibited (Rawal et al., 1988a).

A functional modified Embden-Meyerhof pathway is, however, present in some members of the Halobacteriaceae. While glucose degradation follows the abovedescribed modification of the Entner-Doudoroff pathway, fructose breakdown in Haloarcula vallismortis proceeds via an initial phosphorylation step to yield fructose-1phosphate, catalyzed by an ATP-dependent fructose-1-phosphotransferase (ketohexokinase), rather than by the well-known phosphoenolpyruvate-dependent fructose phosphotransferase system. Fructose-1-phosphate is then converted by 1phosphofructokinase to fructose-1,6-bisphosphate (Altekar and Rangaswamy, 1990, 1991). The degradation of fructose originating from catabolism of sucrose and mannitol has been studied in Haloarcula vallismortis and in Haloferax mediterranei. Endogenously arising fructose was metabolized in a pathway similar to that used for exogenously supplied fructose in a modified Embden-Meyerhof pathway, initiated by ketohexokinase. The enzymes of this pathway were present under all conditions, but elevated levels were detected in the presence of fructose (Altekar and Rangaswamy, 1992) (Figure 4.1, right part). Existence of activities related to the Embden-Meyerhof pathway in Halobacterium salinarum was also shown in NMR studies with glucose; glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate were detected in addition to labeled gluconate (see above). Some of the enzymes of the

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Embden-Meyerhof pathway also function in gluconeogenesis in halophilic Archaea (Sonawat et al., 1990). The fructose- 1,6-bisphosphate aldolase involved in the pathway has been characterized (Krishnan and Altekar, 1991). Two classes of fructose-1,6bisphosphate aldolase are present in the halophilic Archaea. The Class-I (Schiff-base) type was found in the genera Haloarcula and Halorubrum, Class-II enzymes (metalloenzymes, inhibited by EDTA) are present in Halobacterium and Haloferax (Dhar and Altekar 1986a, 1986b). A recent study of sugar metabolism in Halococcus saccharolyticus in which labeled substrates and NMR techniques were used to identify the intermediates and products formed, confirmed the use of different dissimilatory pathways for glucose and fructose. Glucose is metabolized via the modified Entner-Doudoroff pathway, while fructose utilization almost entirely follows the Embden-Meyerhof pathway, and only a small fraction (4%) is degraded via the reactions of the Entner-Doudoroff pathway (Figure 4.1). Glucose-grown cells showed increased activities of gluconatc dehydratasc and 2-keto-3-deoxygluconate kinase. Fructose-grown cells contained higher activities of ketohexokinase and fructose-1-phosphate kinase, the key enzymes of the modified Embden-Meyerhof pathway. When both glucose and fructose were offered to the cells, diauxic growth occurred in which glucose was degraded first, followed by fructose (Johnsen et al., 2001). Use of carbohydrates by halophilic Archaea is often associated with the production of acids, as oxidation of such substrates is incomplete (Hochstein, 1978, 1988). When grown on glucose, galactose, lactose, fructose or sucrose, Halorubrum saccharovorum excretes acetate and pyruvate (Tomlinson and Hochstein, 1972b; Tomlinson et al., 1978). Aldonic acids may be formed from other sugars. Galactonic acid is produced by Halorubrum saccharovorum in the presence of galactose (Hochstein et al., 1976). Catabolism of lactose yields lactobionic acid which is not metabolized further. Lactose metabolism is not coupled to growth (Tomlinson et al., 1978). Arabinose, ribose, and xylose are also oxidized to the corresponding aldonic acids (Hochstein, 1978, 1988). Acetate, pyruvate, and D-lactate were identified in cultures of a variety of Haloferax and Haloarcula species grown in the presence of glycerol (Oren and Gurevich, 1994b). Halobacterium salinarum produces considerable amounts of acids in media containing glycerol (Gochnauer and Kushner, 1969). Production of D-lactate, acetate and pyruvate from glycerol could also be demonstrated in natural communities of halophilic Archaea from the Dead Sea and from saltern crystallization ponds amended with micromolar concentrations of glycerol (Oren and Gurevich, 1994b). Although there are no reports of growth of Halobacterium on fatty acids, the genes for the fatty acid pathway are present in the Halobacterium NRC-1 genome (Ng et al., 2000). Some other halophilic Archaea have been shown to degrade aliphatic hydrocarbons and derivatives. Red halophilic Archaea were isolated from a salt marsh in the south of France from an enrichment culture with the hydrocarbon eicosane (Bertrand et al., 1990). One of the isolates degraded a wide variety of compounds, including even- and odd carbon number saturated hydrocarbons (tetradecane, hexadecane, eicosane, heneicosane), saturated isoprenoid alkanes (pristane), and also long-chain fatty acids such as palmitic acid. No taxonomic description of the isolate has been published as yet. Halophilic Archaea are sometimes found in association with oilfield brines (Zvyagintseva et al., 1995a), and hydrocarbon-degrading isolates

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taxonomically close to Halobacterium salinarum, Haloferax volcanii and Halorubrum distributum were isolated from hypersaline brines from an oil deposit in Tatarstan (Kulichevskaya et al., 1991). Degradation of aromatic compounds has also been documented in certain strains. The above-mentioned hydrocarbon degraders isolated from a salt marsh in the south of France (Bertrand et al., 1990) also break down aromatic molecules such as acenaphtene, phenanthrene, anthracene, and 9-methylanthracene. A Haloferax strain (designated strain D1227), obtained from soil contaminated with oil brine grows on different aromatic compounds, including benzoate, cinnamate, and 3-phenylpropionate, as sole carbon and energy sources (Emerson et al., 1994). The metabolism of 3phenylpropionate has been characterized in further depth (Fu and Oriel, 1998, 1999; Oriel et al., 1997). Degradation is initiated by a two-carbon shortening of the side chain to yield benzoyl-CoA via a mechanism similar to of fatty acids. Subsequently, the aromatic ring is attacked using the gentisate pathway, involving gentisate 1,2-dioxygenase (Fu and Oriel, 1999) (Figure 4.2). All members of the Halobacteriaceae are chemoheterotrophs, while some have the additional ability of photoheterotrophic growth (see Section 5.4.1). Autotrophic growth has never been demonstrated. Therefore the finding of ribulose-1,5-bisphosphate carboxylase (RuBisCo) activity in certain Haloferax and Haloarcula species came as a surprise (Altekar and Rajagopalan, 1990; Rawal et al., 1988b). When cell extracts were incubated with in the presence of ribulose-1,5-bisphosphate, labeled 3phosphoglycerate was formed. The RuBisCo of Haloferax mediterranei is a hexadecamer of 500 kDa apparent molecular mass, and is composed of 54 and 14 kDa subunits (Rajagopalan and Altekar, 1994). It cross-reacts with anti-spinach RuBisCo antibodies (Altekar and Rajagopalan, 1990). In contrast to most other proteins of halophilic Archaea (see Section 7.2), the Haloferax RuBisCo does not possess an excess of acidic amino acid residues (Rajagopalan and Altekar, 1994). Additional enzymes of the Calvin cycle, including phosphoribulokinase, a key enzyme of the pathway, have been identified in RuBisCo-containing halophilic Archaea (Rawal et al., 1988a). A correlation has been observed between the presence of RuBisCo activity and the ability to accumulate as storage polymer, shown in Haloferax mediterranei, Haloferax volcanii, and Haloarcula marismortui (see also Section 3.1.8). However, the nature of the connection between the two phenomena remains unclear. No information is available on the contribution of fixed via the RuBisCo to the total carbon assimilated during growth. Additional modes of fixation, not involving RuBisCo, have been indicated in different halophilic Archaea. Halobacterium salinarum cells containing bacteriorhodopsin showed a light-induced stimulation of uptake. The process was suggested to involve a reductive carboxylation of propionate (Danon and Caplan, 1977). Alternative mechanisms have been proposed such as the operation of a novel pathway of non-reductive fixation involving a glycine synthase reaction with and a methyl carbon derived from the cleavage of propionate, released as methylenetetrahydrofolate (Javor, 1988), or exchange of with the carboxylic group of pyruvate (Rajagopalan and Altekar, 1991). Light-stimulated incorporation by members of the Halobacteriaceae was also observed in a bacteriorhodopsin-containing

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community of halophilic Archaea (probably including Halorubrum sodomense) in the Dead Sea (Oren, 1983a).

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The respiratory electron transport chain has been analyzed in Halobacterium salinarum, in Haloferax volcanii, and in Natronomonas pharaonis (Schäfer et al., 1999). Halobacterium salinarum membranes contain NADH-oxidizing activity that can be attributed to a type-II NADH oxidase not involved in energy conservation (Sreeramulu et al., 1998). In Halobacterium salinarum, b-type cytochromes are present in the largest quantities; four different b-type cytochromes have been identified (Hallberg and Baltscheffsky, 1979, 1981; Hallberg and Hederstedt, 1981; HallbergGradin and Colmsjö, 1989). Low levels of a c-type cytochrome and cytochrome o and are present as well (Cheah, 1970; Hallberg and Baltscheffsky, 1979, 1981). An type cytochrome oxidase of 40 kDa molecular mass has been isolated (Fujiwara et al., 1987, 1989), and its amino acid sequence has been analyzed (Denda et al., 1991). Cytochrome b was suggested as the likely electron donor for the reduction of cytochrome (Fujiwara et al., 1993). There is also spectral evidence for the presence of a cytochrome bd analog (Sreeramulu et al., 1998). In cells grown under low oxygen tension a d-type cytochrome was detected, and a complex of a Rieske iron-sulfur cluster with b- and ctype cytochromes has been partially purified (Sreeramulu et al., 1998). Presence of cytochrome o (a b-type cytochrome) was demonstrated in the brown membrane of Halobacterium salinarum, which is a developmental precursor of purple membrane. This cytochrome was postulated to act more as a "bacterial hemoglobin" than as a terminal oxidase, and may not have an electron accepting function (Hartsel et al., 1988). A cytochrome has been purified from Haloferax volcanii. The protein consists of subunits of 44 and 35 kDa. Reduced cytochrome c purified from the membrane of Haloferax volcanii is oxidized by cytochrome No evidence was found for the involvement of cytochrome b in the reduction of cytochrome in Haloferax volcanii (Tanaka et al., 2002). The oxidase complex of Natronomonas pharaonis was spectroscopically characterized as a oxidase (Mattar and Engelhard, 1997). It consists of two major polypeptides of 36 and 4 kDa (Schäfer et al., 1999). Subunit I contains six heme and five Cu-A ligands. The midpoint redox potentials of the heme centers in the membranes were determined as + 268 mV (cytochrome b) and + 358 mV (cytochrome Subunit II provides all the ligands to form the mixed-valence center (Mattar and Engelhard, 1997). It may function physiologically either as a cytochrome c oxidase or as the oxidase for halocyanin (see below). Information on a complex III equivalent is rudimentary, although a cytochrome bc fraction could be isolated from solubilized membrane. In addition, a ferredoxin has been purified from Natronomonas pharaonis (Scharf et al., 1997). Halocyanin is a small (15.5 kDa) blue copper protein that may functionally substitute for cytochrome c in membranes of Natronomonas (Scharf and Engelhard, 1993). The soluble protein contains one atom of copper per mol and has a broad absorption band around 600 nm. EPR spectra revealed a close resemblance to plastocyanin, suggesting a type I copper ligation with two histidine residues, one methionine, and one cysteine. This is in line with primary sequence data, which also predict the presence of an N-terminal membrane anchor (Hildebrandt et al., 1994; Mattar et al., 1994). The open reading frame codes for a 163 amino acid protein of

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17,223 Da. The copper-free native protein is only 15,356 Da in size, suggesting posttranslational processing of the gene product. The copper binding site, located at the Cterminal end, has a high degree of sequence identity with other small copper proteins. The respiratory electron transport chain of Natronomonas thus consists of a cytoplasmic cytochrome c (-142 mV), a membrane-bound cytochrome bc (midpoint redox potentials of the heme B and heme C groups -117 and -44 mV, respectively), the copper heme B protein halocyanin (+ 128 mV), and the membrane-bound cytochrome with a heme group (+ 358 mV) (Scharf et al., 1997). Due to the low solubility of oxygen in salt-saturated brines, oxygen may easily become a limiting factor for development of members of the Halobacteriaceae. A few representatives of the group may overcome oxygen limitation by producing gas vesicles that enable them to float toward the air-water interface (see Section 3.1.7). An alternative strategy to cope with lack of molecular oxygen is the use of alternative electron acceptors in respiration. Anaerobic growth with nitrate as electron acceptor has been demonstrated in Haloferax denitrificans, in several members of the genus Haloarcula, and in Halogeometricum borinquense. Nitrate is reduced to gaseous products. Molecular nitrogen is generally the main product, but formation has also been observed in several species (Hochstein, 1988; Hochstein and Tomlinson, 1985; Mancinelli and Hochstein, 1986; Tomlinson et al., 1986). The dissimilatory nitrate reductases of Haloferax denitrificans and Haloferax mediterranei have been characterized (Alvarez-Ossorio et al., 1992; Hochstein, 1991; Hochstein and Lang, 1991). Analysis of nitrate respiration-deficient mutants of Haloferax volcanii has enabled the genetic identification of three ABC transporters as essential elements of nitrate respiration. One of these is a glucose transporter that solely mediates glucose transport under anaerobic conditions. A second one is proposed to be molybdatespecific, which explains its essential role in nitrate respiration: nitrate reductases all contain molybdenum. The third is proposed to be anion-specific (Wanner and Soppa, 1999). The ecological relevance of anaerobic growth on nitrate has never been confirmed. In view of the low concentrations of nitrate generally encountered in hypersaline brines and the apparent lack of regeneration of nitrate by nitrification at high salt concentrations, the process can be expected to occur only to a limited extent in nature (Oren, 1994a) (see also Section 4.4). Other alternative electron acceptors for respiration in many species are dimethylsulfoxide (DMSO), trimethylamine N-oxide (TMAO), and fumarate. Reduction of DMSO and TMAO was found to be coupled with growth in several species. Halobacterium salinarum, Haloarcula marismortui, Haloarcula vallismortis, and Haloferax mediterranei grew anaerobically in the presence of DMSO or TMAO; in Haloferax volcanii DMSO supported anaerobic growth, while TMAO did not (Oren and Trüper, 1990). The ecological importance of DMSO reduction is not clear. However, TMAO may be available as an electron acceptor in salted fish, being often present in high concentrations within the fish tissues as an osmotic solute. Fumarate-driven anaerobic growth was reported in certain Halobacterium salinarum strains, in Haloferax volcanii, and in Haloferax denitrificans (Oren, 1991). Elemental sulfur and thiosulfate have also been suggested to serve as potential electron acceptors (Tindall and Trüper, 1986), but little information is available on the nature of the process.

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Halobacterium salinarum can grow anaerobically by fermentation of L-arginine (Bickel-Sandkotter et al., 1996; Hartmann et al., 1980; Oestcrhelt, 1982). Earlier experiments had already shown that in a defined medium containing a mixture of amino acids, arginine rapidly disappeared already in the beginning of the exponential growth phase without any relation to its requirement for assimilatory purposes. Citrulline accumulated in the medium concomitant with the disappearance of arginine (Ducharme et al., 1972). Arginine is fermented via citrulline to ornithine and carbamoylphosphate, which is then split into carbon dioxide and ammonia with the gain of ATP (Dundas and Halvorson, 1966). The arginine deiminase (EC 3.5.3.6) of Halobacterium salinarum has been purified and its properties studied (Monstadt and Holldorf, 1991). A kinetic analysis of the ornithine carbamoyltransferase (EC 2.1.3.3) has been performed as well (Ruepp et al., 1995), and the genes involved in the arginine fermentation pathway have been studied in-depth (Ruepp and Soppa, 1996). The genes for arginine deiminase (arcA), carbamate kinase (arcC), and catabolic ornithine carbamoyltransferase (arcB) are organized together with the gene for a putative regulatory protein in an operon (arcRACB). Anaerobic growth on arginine is not widespread among the halophilic Archaea: of all isolates tested only strains belonging to the genus Halobacterium showed fermentative growth on arginine (Oren, 1994b; Oren and Litchfield, 1999). A specific enrichment procedure for members of the genus Halobacterium could therefore be developed on the basis of this finding (Oren and Litchfield, 1999). Light can drive anaerobic growth in Halobacterium salinarum, provided the cells contain the light-driven proton pump bacteriorhodopsin (Hartmann et al., 1980; Oesterhelt, 1982; Oesterhelt and Krippahl, 1983). Section 5.4.1 provides a more detailed description of the bacteriorhodopsin system. However, the biosynthesis of the retinal moiety of bacteriorhodopsin from is oxygen-dependent, and therefore either trace concentrations of oxygen should be present to enable sustained light-driven anaerobic growth, or retinal or its derivatives should be supplied to the medium (Oesterhelt and Krippahl, 1983). ATP synthesis in cells or in membrane vesicles of halophilic Archaea can be driven by respiration, by light (in case bacteriorhodopsin is present, see Section 5.4.1), or by artificially induced pH gradients (Michel and Oesterhelt, 1980; Mukohata and Yoshida, 1987a, 1987b; Mukohata et al., 1986; Stan-Lotter et al., 1993). ATP synthesis and ATP hydrolysis by isolated membranes are sensitive to dicyclohexylcarbodiimide (DCCD) (Kristjansson and Hochstein, 1985), which suggests that they are mediated by a protontranslocating ATPase (archaeal ATP-synthase or A-ATP synthase). This ATPase differs in many aspects from F-ATPase that had been considered ubiquitous in aerobic organisms as the central enzyme in ATP synthesis. Inhibitor studies also suggested a high degree of resemblance to V-ATPases (Hochstein and Lawson, 19931 Ihara et al., 1992). The existence of a DCCD-binding proteolipid has been demonstrated, but there have been contradictory reports on its apparent molecular mass: 9.7 kDa (Steinert et al., 1997), 14 kDa (Dane et al., 1992), or 42 kDa (Konishi and Murakami, 1984). The Halobacterium salinarum genome contains a sequence for a putative 8 kDa proteolipid DCCD-binding protein with two transmembrane helices (Schäfer et al., 1999). No complete ATP synthase complex has yet been isolated from any of the members of the Halobacteriaceae. The ATPase of Halorubrum saccharovorum, solubilized by octylglucoside as a 350-kDa complex, is composed of polypeptides with apparent

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molecular masses of 87, 60, 29 and 20 kDa (Hochstein et al., 1987; Stan-Lotter and Hochstein, 1989; Stan-Lotter et al., 1993). A cryptic, detergent-inducible ATPase from membranes of Halorubrum saccharovorum that can be activated by dithiothreitol contains polypeptides with molecular masses of 98, 71, 31, and 22 kDa (Bonet and Schobert, 1992; Schobert and Lanyi, 1989). Subunit A (98 kDa) is the catalytic subunit, as shown by the presence of a high-affinity ADP binding site. The Halorubrum saccharovorum ATPase has a high degree of resemblance to the type ATPase according to its reaction kinetics and by its inhibition by ADP and in the absence of inorganic phosphate, as well as by its inhibition by N-ethylmaleimide and by NBD-Cl (7-chloro-4-nitrobenz-2-oxa-l,3-diazole), an inhibitor of ATPases (Bonet and Schobert, 1992; Hochstein, 1992; Schobert, 1991, 1992; Sulzner et al., 1992). A reaction mechanism analogous to that of ATPase was inferred from the nonlinear kinetics of ATP hydrolysis and from the inhibition by tight binding of ADP (Schobert, 1991). Like F-type ATPases, this enzyme is inhibited by azide. A common feature of the enzymes from Halobacterium salinarum and Haloferax volcanii is the preference for over as an activating ion (Bickel-Sandköttcr et al., 1998; Dane et al., 1992; Nanba and Mukohata, 1987). All require high salt concentrations for optimal activity. The Halobacterium salinarum ATPase in its membrane-bound state is inhibited by N-ethylmaleimide, by NBD-Cl and by nitrate, but not by azide (Mukohata and Yoshida, 1987b). It differs in its properties and inhibition patterns from (Mukohata and Yoshida, 1987b). Treatment with alkaline EDTA releases a 320 kDa complex from the membrane, consisting of two polypeptides (86 and 64 kDa) (Nanba and Mukohata, 1987). In addition to ATP, the enzyme hydrolyzes ITP, GTP, and CTP. In contrast, the ATPase from Haloferax volcanii is specific for ATP. The soluble enzyme is sensitive to N-ethylmaleimide but not to azide, bafilomycin, NBD-Cl, or nitrate (Steinert et al., 1995). Apparent molecular masses of the subunits were 63, 51, 37 and 12 kDa. The sizes of the subunits, as deduced from the gene sequences, are 64.5, 52, 22 and 11.6 kDa (Steinert and Bickel-Sandkötter, 1996). A three-dimensional model of the Haloferax volcanii ATPase showed most of the negatively charged amino acids (representing about 20% of the amino acids in the proteins) on the outer surface (Bickel-Sandkötter et al., 1998), as is typical for halophilic proteins (see Chapter 7). Haloferax mediterranei ATPase is inhibited by azide, nitrate, and NBD-Cl, but only weakly by N-ethylmaleimide. DCCD did not inhibit the membrane-bound ATPase, but it did bind to a 14-kDa polypeptide of the detergent-solubilized enzyme. Apparent molecular masses of the putative subunits in this preparation were 53.5, 49, 42, 22, 21, 14, 12, and 7.5 kDa (Dane et al., 1992). A membrane-associated ATPase purified from Natronococcus occultus had an apparent molecular mass of 130 kDa with 74 and 61 kDa subunits. Activity was sensitive to nitrate, vanadate, DCCD, and bafilomycin A, a behavior characteristic of Atype ATPases (Eddy and Jablonski, 2000).

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4.1.5. Assimilatory biochemical pathways The biosynthetic pathways for the production of amino acids in Haloarcula hispanica have been studied by feeding the cells with glycerol followed by examination of the products by NMR techniques. Evidence was obtained for a split pathway for isoleucine biosynthesis. Little more than half the isoleucine was produced from threonine and pyruvate via the threonine pathway, and the remainder was synthesized from pyruvate and acetyl-CoA via the pyruvate pathway (Figure 4.3). No evidence was obtained for the presence of the glutamate pathway, a third biosynthetic pathway that leads to the production of isoleucine (Hochuli et al., 1999). Lysine biosynthesis proceeds via the diaminopimelate pathway involving diaminopimelate dehydrogenase (Figure 4.4, part B, route 2); operation of the pathway could not be demonstrated. Comparison of the data thus obtained with the existing information on the pathways of amino acid metabolism in the Bacteria and the Eucarya supports the theory that the pathways for amino acid biosynthesis had been established before the diversion between the three domains occurred. Analysis of the labeling pattern of the tyrosine formed indicated the existence of a novel, yet to be characterized pathway, as significant deviations were observed from the expected labeling when tyrosine would have been synthesized from erythrose-4-phosphate and phosphoenolpyruvate via the shikimate pathway (Hochuli et al., 1999). A central point in the biosynthesis of phenylalanine is a dehydratase reaction. In different organisms this reaction may be performed by prephenate dehydratase, arogenate dehydratase, or by cyclohexadienyl dehydratase. Haloarcula vallismortis was shown to possess an interlock-type prephenate dehydratase of a type characteristic of Gram-positive Bacteria. The enzyme is subject to feedback inhibition by Lphenylalanine and to allosteric effects by a number of effectors not directly involved in the biosynthetic pathway of phenylalanine (Jensen et al., 1988). In Halobacterium salinarum the biosynthesis of 5-aminolevulinic acid, a precursor for the formation of tetrapyrrole compounds, proceeds by the pathway used also in the cyanobacteria, and in the and In this pathway, the carbon skeleton of glutamate is converted to 5-aminolevulinic acid in three enzymatic steps: an ATP-dependent glutamyl-tRNA synthase, an NADPH-dependent dehydrogenase, and a pyridoxal phosphate-dependent amidotransferase. The alternative pathway found in the involving 5-aminolevulinic acid synthase that synthesizes 5aminolevulinic acid by condensation of glycine and succinyl-CoA, is not found (OhHama et al., 1991). A recent study showed that Halobacterium salinarum has a surprisingly high content of D-amino acids, both in its proteins and in the free intracellular amino acid pool (Nagata et al., 1999) (Table 4.1). D-enantiomers of serine, alanine, proline, glutamate/glutamine, and aspartate/asparagine were detected. D-serine constituted about 12% of the total free serine pool, and also D-aspartate and D-proline were abundant in the free pool (4-7% and 3-4%, respectively). No D-amino acid oxidase activity was detected in Halobacterium salinarum cell extracts. The function of the Damino acids in halophilic Archaea is yet unknown.

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The members of the Halobacteriaceae differ in the extent to which the synthesis of stable RNA and protein are controlled by the availability of amino acids. Haloferax volcanii and Halococcus morrhuae are under stringent control, which means that amino acid starvation arrests not only protein synthesis but also the production of stable RNA. In the process guanosine tetra- and pentaphosphate (ppGpp and pppGpp) accumulate, the synthesis of which is triggered by binding of uncharged tRNAs to the acceptor site of the ribosomes. ppGpp acts as a negative effector in the synthesis of stable RNA. Control in Halobacterium salinarum strains was found to be partially or fully relaxed, as RNA biosynthesis proceeded upon amino acid starvation. Stringency in halophilic Archaea is dependent on the deaminoacylation of tRNA, as in the Bacteria. ppGpp is

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not an effector of stringent control over stable RNA synthesis in halophilic Archaea (Cimmino et al., 1993).

4.1.6. Life at temperature extremes Some members of the Halobacteriaceae tolerate quite high temperatures. Haloterrigena thermotolerans grows optimally at 50 °C, and its maximum temperature for growth is 60 °C (Montalvo-Rodriguez et al., 2000). Haloferax mediterranei can grow up to 54-55 °C. A detailed analysis of the dependence of its growth rate on temperature showed a complex relationship that could not be described by a simple Arrhenius curve (Shand and Perez, 1999). Halorubrum lacusprofundi from Deep Lake, Antarctica, is relatively tolerant to low temperatures. At 4 °C the species grows at about 10% the optimal rate, which is achieved at 30-35 °C (Franzmann et al., 1988). A mathematical analysis of the temperature dependence of its growth rate, based on plots of the square root of the reciprocal of the growth rate versus temperature, yielded theoretical minimum temperatures of 2.4 and 1.1 °C for strains ACAM 32 and 34 (the type strain of the species) and optima of 31.2 and 36.4 ºC, respectively (McMeekin and Franzmann, 1988). 4.1.7. Reactions to the presence of reactive oxygen species As all other microorganisms adapted to an aerobic life style, the Halobacteriaceae have to cope with the presence of reactive oxygen species such as peroxides and superoxide radicals. Both catalase and superoxide dismutase are present in the aerobic halophilic Archaea and take part in the defense against oxidative damage. When Halobacterium salinarum was suspended in low salt concentrations (1 or 1.25 M NaCl) for 12 hours, a nearly 100-fold increase in catalase activity was observed compared with cells kept at high NaCl concentrations, and peroxidase activity increased 4- to 5-fold. It was noted that the solubility of oxygen increases with decreasing salinity, thereby increasing the potential for production of active oxy-intermediates. The newly induced catalase activity was based on the induction of a mesohalic form of catalase, distinct from the catalase-peroxidase that is present constitutively (BrownPeterson and Salin, 1994; Brown-Peterson et al., 1994, 1995). The catalase-peroxidase gene of Halobacterium salinarum has been subcloned in shuttle vectors and expressed under different archaeal promoters. No induction was observed by a variety of

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environmental stress factors such as exposure to intense light, darkness, high temperatures, redox inhibitors, or heavy metals (Long and Salin, 2000). Halobacterium salinarum also contains a separate peroxidase with a lower molecular mass than the combined catalase-peroxidase (Fukumori et al., 1985). Halophilic Archaea also display superoxide dismutase activity (May and Dennis, 1987). The genes for the manganese-containing superoxide dismutase of Halobacterium salinarum and Haloferax volcanii show a high degree of sequence similarity with the bacterial gene, suggesting that they may possibly have been acquired by lateral gene transmission (May and Dennis, 1987; May et al., 1989). Exposure of Haloferax volcanii to paraquat, which generates superoxide radicals intracellularly, slowed down the growth rate and induced a large increase in superoxide dismutase activity. Paraquat treatment ofHalobacterium salinarum also caused an initial increase in superoxide dismutase levels, but the activity decreased again following prolonged exposure (May et al., 1989; Salin and Brown-Peterson, 1993). The gene for superoxide dismutase in Halobacterium salinarum is arranged adjacent to that of photolyase, an enzyme that repairs pyrimidine dimers (Takao et al., 1989). Addition of inhibitors of aerobic respiration such as cyanide or azide to Halobacterium salinarum at concentrations sufficiently low not to affect the oxidation rate of NADH resulted in a two-fold increase in superoxide dismutase activity (BrownPeterson et al., 1995). When cells were subjected to hypo-saline stress (a treatment that also induced a large increase in catalase activity, as described above), the superoxide dismutase activity increased up to ten-fold (Brown-Peterson and Salin, 1994). Halobacterium salinarum membrane vesicles exposed to a hypo-saline environment showed enhanced production of superoxide radicals. Vesicles prepared from cells grown in NaCl oxidized more NADH, consumed more oxygen, and generated more superoxide than vesicles prepared from cells grown in NaCl (BrownPeterson et al., 1994). Exposure of Halobacterium salinarum to moderate heat shock (50 °C for 2.5 hours) resulted in a more than two-fold enhancement in superoxide dismutase activity (Begonia and Salin, 1991), and a five-fold increase was observed upon exposure of the cells to 60 °C for up to eight hours (Salin and Brown-Peterson, 1993). The enzyme is thus induced also by stressful conditions not directly related to increased oxygen levels. Glutathione, the tripeptide known to protect against oxidative and free radical damage in most types of cells, was not detected in Halobacterium salinarum, Haloarcula marismortui, Haloferax volcanii, and Halorubrum saccharovorum. Instead, is present in millimolar levels (Newton and Javor, 1985; Sundquist and Fahey, 1989). Activity of an NADPHdependent reductase, analogous to glutathione reductase (EC 1.6.4.2), was detected in Halobacterium salinarum (Sundquist and Fahey, 1988, 1989).

4.1.8. Pterins and polyamines Two unusual pterins have been reported from Haloarcula marismortui: sulfohalopterin2 and phosphohalopterin-1 (Lin and White, 1987, 1988) (Figure 4.5). No information is available as yet on their function in the cell.

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Polyamines, which are useful chemotaxonomic markers in many groups of prokaryotes, are of little or no taxonomic value in the halophilic Archaea. Concentrations of polyamines encountered are very low, and sometimes they are altogether undetectable (Chen and Martynowicz, 1984). Agmatine, putrescine, spermidine, and spermine have been detected in different representatives (CarteníFarina et al., 1985; Hamana et al., 1985, 1995; Kamekura et al., 1986, Kneifel et al., 1986). The polyamines may be derived from the growth medium, and the cellular polyamine composition is to a large extent influenced by the type of medium employed.

4.1.9. Sensitivity to antibiotics and other antibacterial substances Members of the Halobacteriaceae are typically resistant to such Bacteria-specific antibiotics as penicillin, ampicillin, cycloserine, kanamycin, neomycin, polymyxin, and streptomycin (Bonelo et al., 1984; Hilpert et al., 1981; Pecher and Böck, 1981). Most are sensitive to novobiocin and bacitracin. Novobiocin is a DNA gyrase inhibitor (Holmes and Dyall Smith, 1991; Sioud et al., 1988), and acts on the same target in the Archaea as in sensitive Bacteria. Halococcus saccharolyticus was reported to be resistant to novobiocin inhibition (Montero et al., 1989), but Halococcus morrhuae is sensitive (Hunter and Millar, 1980). Bacitracin inhibits incorporation of the highmolecular-weight saccharide into the cell wall glycoprotein of non-coccoid halophilic Archaea (Wieland et al., 1980; Wieland et al., 1982) (see Section 3.1.1); it also inhibits lipid biosynthesis in these organisms (Basinger and Oliver, 1979 Moldoveanu and Kates, 1989). Anisomycin, a protein synthesis inhibitor of eukaryotic ribosomes, inhibits protein synthesis of all members of the Halobacteriales tested (Pecher and Böck, 1981). Halobacterium salinarum is further sensitive to haloquinone, an antibiotic produced by Streptomyces venezuelae subsp. xanthophaeus that affects DNA synthesis also in some Bacteria (Arthrobacter spp., Mycobacterium, and others) (Ewersmeyer-Wenk et al., 1981; Krone et al., 1981). Conflicting information exists with regard to the sensitivity to chloramphenicol of halophilic Archaea. Most reports state resistance to

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chloramphenicol (see e.g. Hilpert et al., 1981). However, Pecher and Böck (1981) found that Halobacterium salinarum was sensitive to chloramphenicol. They also reported some sensitivity to tetracycline and aureomycin. Mankin and Garrett (1991) also claimed a high sensitivity to chloramphenicol in Halobacterium strain Rl. A resistant mutant was found to carry a point mutation in the 23S rRNA. In another study Halobacterium salinarum was inhibited only by very high concentrations of chloramphenicol and susceptible to tetracycline at concentrations above (Chow and Mark, 1980). However, Bonelo et al. (1984) reported a very low sensitivity to chloramphenicol (minimal inhibitory concentrations up to 750in most species tested; in Haloferax mediterranei). Some species are inhibited by high concentrations of erythromycin (Bonelo et al., 1984; Chow and Mark, 1980; Pecher and Böck, 1981). The Halobacteriaceae are sensitive to aphidicolin, a DNA polymerase inhibitor that prevents cell division and often causes the formation of elongated cells (Forterre et al., 1984; Schinzel and Burger, 1984). Inhibition of halophilic Archaea by coumarin and quinolone antibacterial compounds provided evidence for the presence of DNA gyraselike enzymes (Sioud et al., 1988). Ciprofloxacin inhibits most Haloferax and Haloarcula species at concentrations between but Halobacterium cells proved less sensitive. At sub-lethal concentrations, swelling and elongation of the cells were observed. Sensitivity to ciprofloxacin and other quinolone derivatives (norfloxacin, perfloxacin) was decreased at increased magnesium concentrations (Oren, 1996; Sioud et al., 1988). Haloferax volcanii became more resistant when the magnesium concentration in the growth medium was increased. Alkaliphiles such as Natronomonas pharaonis are very sensitive these quinolones (Oren, 1996). Certain antitumor drugs that act on topoisomerase II (adriamycin, daunorubicin, etoposide) and drugs that act on actomyosin (cytochalasin B and D) or on tubulin (vincristine, podophyllotoxin, nocodazole) also inhibit Halobacterium salinarum (Sioud et al., 1987; see also Section 3.1.6). The sensitivity of Halobacterium salinarum to cerulenin, a potent inhibitor of the synthesis of the straight-chain fatty acids by the fatty acid synthetase complex (Dees and Oliver, 1977) was described in Section 3.1.4. At least certain species of halophilic Archaea are sensitive to gardimycin (inhibiting cell wall biosynthesis), virginiamycin (acting on the protein synthesis machinery), to monensin and lasalocid (which target the cell membranes) (Hilpert et al., 1981), and to amphotericin B, vibriostat O/129, sulfafurazole, and josamycin (Tindall, 1992). A special case is the sensitivity of many members of the Halobacteriaceae to rifampicin (Bonelo et al., 1984; Hilpert et al., 1981; Pecher and Böck, 1981). The target of rifampicin action in the halophilic Archaea is probably not the DNA-dependent RNA polymerase as in the Bacteria, but the inhibition is probably due to its detergent effect on the cell membrane, causing cell lysis (Pfeifer, 1988).

4.1.10. Physiology of the halophilic methanogenic Archaea Few studies have been made of the physiology of the halophilic species among the methanogens beyond the characterization of basic properties necessary for taxonomic

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descriptions. All halophilic species described thus far obtain their energy from disproportionate reactions using methylated amines or methanol as substrates. No halophilic methanogens are known that use acetate as an energy source, or that live chemoautotrophically on hydrogen + carbon dioxide (with a single possible exception, see Pérez-Fillol et al., 1985; see also Section 4.4). The pathways leading to the biosynthesis of the aromatic amino acids Lphenylalanine and L-tyrosine were examined in Methanohalophilus mahii. These amino acids are made via phenylpyruvate and 4-hydroxyphenylpyruvate. L-arogenate is not an intermediate, as arogenate dehydrogenase and arogenate dehydratase activities were not detected. Prephenate dehydrogenase is the key enzyme in the pathway. This enzyme preferentially uses as cofactor, but can also function with The enzyme is highly sensitive to feedback inhibition by L-tyrosine. The prephenate dehydratase is also subject to multimetabolite control by feedback inhibition by phenylalanine and by a series of allosteric activators (Fischer et al., 1993). This "interlock" type of prephenate dehydratase is broadly distributed among the Grampositive lineage of the Bacteria, and also exists in the halophilic Archaeon Haloarcula vallismortis (see Section 4.1.5).

4.2. PHYSIOLOGY OF HALOPHILIC BACTERIA The metabolic diversity of the halophilic Bacteria is much greater than that of the halophilic Archaea. Among the halophilic Bacteria we find aerobic and anaerobic heterotrophic modes of life, as well as oxygenic and anoxygenic phototrophy with autotrophic growth, photoheterotrophy, and chemolithotrophy. In the sections below only those aspects of the metabolism are discussed that are unique to the halophiles. Special emphasis will be on those halophilic genera or families of halophilic Bacteria that have been extensively studied, such as the family Halomonadaceae and the genus Salinivibrio (aerobic or facultatively anaerobic, heterotrophic, see also Ventosa et al., 1998) and the order Halanaerobiales with the families Halanaerobiaceae and Halobacteroidaceae (anaerobic, fermentative). Aspects related to the ion metabolism and the accumulation of organic osmotic solutes of the halophilic Bacteria are presented in Chapter 6 and 8, respectively. 4.2.1. Nutritional demands of the aerobic heterotrophic Bacteria Halomonas elongata, Chromohalobacter salexigens, and other members of the family Halomonadaceae have been extensively used as model organisms to study halophilic behavior in the bacterial domain. The nutritional requirements of the Halomonadaceae are generally simple, and most species can grow in mineral media supplemented with single carbon sources (sugars, amino acids, organic acids, etc.) (Kushner, 1993). Vitamins need to be supplied in some cases. A defined medium enabling sustained growth of Halomonas halodenitrificans should contain thiamine. For anaerobic growth with nitrate or other oxidized nitrogen compounds, methionine addition is required. Methionine can be replaced by vitamin suggesting that under anaerobic conditions the cobalamin-dependent pathway for methionine biosynthesis is impaired (Hochstein

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and Tomlinson, 1984). Salinivibrio costicola, another popular model organism in physiological studies, has somewhat more complex needs. Defined media developed for this bacterium contain glucose supplemented with a number of amino acids (cysteine, glutamate, arginine, valine and isoleucine) (Flannery and Kennedy, 1962) or glucose, glutamate, choline, thiamine, and biotin (Kamekura et al., 1985), in addition to inorganic salts. Assimilation of inorganic nutrients in this group of organisms has been little studied. Nitrate assimilation activity in Chromohalobacter israelensis is associated with the respiratory electron transport in the membrane, the electrons for assimilatory nitrate reduction being derived from NADH, succinate, or malate. A soluble assimilatory nitrite reductase that accepts electrons from ferredoxin was found in this organism. It is induced by the presence of nitrate and repressed by ammonium ions (Hochman et al., 1988). A specific demand for chloride has been demonstrated in Halobacillus halophilus (Roeßler and Müller, 1998). 4.2.2. Membrane transport systems for nutrient accumulation Bioenergetic processes involving the cell membrane have been extensively studied in Salinivibrio costicola. The amino acid analog has often been used as a model substrate in membrane transport experiments. Transport of aminoisobutyrate is driven by the membrane potential, but for optimal rates a gradient is specifically required. Transport is competitively inhibited by glycine, alanine, and to some extent by methionine (Hamaide et al., 1984a, 1984b; Kushner, 1993; MacLeod, 1986). Dependence of transport on a sodium gradient has also been documented in Halomonas elongata (Martin et al., 1983). Transport of different amino acids (aspartate, arginine, alanine) through the membranes of Gram-negative (Salinivibrio costicola, Halomonas halodenitrificans) and Grampositive aerobic halophiles (Nesterenkonia halobia, Planococcus citreus) is sodiumdependent as well (Matveeva et al., 1990; Nikolayev and Matveyeva, 1990, Nikolayev et al., 1990).

4.2.3. Degradation of unusual organic compounds by aerobic halophilic Bacteria In addition to substrates such as simple sugars, organic acids and amino acids, certain more difficult to degrade compounds can be used by some halophilic Bacteria. Hydrocarbons support growth of Marinobacter hydrocarbonoclasticus (Gauthier et al., 1992). This organism attaches to the hydrocarbon and produces an extracellular emulsifying agent. It grows on eicosane at NaCl as profusely as at (Fernandez-Linares et al., 1996). An incompletely characterized haloalkaliphilic member of the Halomonadaceae can grow on 2,4-dichlorophenoxyacetic acid (2,4-D). Optimal growth on 2,4-D occurred at pH 8.4-9.4 in the presence of NaCl. One isolate showed reasonable growth (25% of the rate under optimal conditions) at NaCl (Maltseva et al., 1996; Oriel et al., 1997). Chromohalobacter marismortui is able to break the C-S bond in

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aminomethanesulfonate, and it can use this compound both as sulfur and as nitrogen source (Ternan and McMullan, 2002). Certain halophilic Bacteria produce the enzyme organophosphorus acid anhydrase that enables them to use organophosphorus compounds such as phosphonoacetate, 2aminoethyl-, 3-aminopropyl-, 4-aminobutyl- methyl- and ethyl-phosphonate as sources of phosphate for growth (DeFrank and Cheng; 1991; DeFrank et al., 1993; Hayes et al., 2000). This property and its biotechnological potential is discussed further in Section 11.3.2.

4.2.4. Denitrification in members of the Halomonadaceae Some members of the Halomonadaceae can grow anaerobically using nitrate as electron acceptor. Well-known examples are Halomonas halodenitrificans and Halomonas desiderata. The dissimilatory nitrite reductase of Hatomonas halodenitrificans has been characterized. The membranes reduce nitrite to nitric oxide and/or to nitrous oxide. The nitrite reductase spectrally resembles a cd-type cytochrome (Grant and Hochstein, 1984; Grant et al., 1984). When cells were grown anaerobically in the presence of nitrite, both membrane-bound and cytoplasmic nitrite reductase activity was detected. When assayed with phenazine methosulfate and ascorbate as electron donors, the membranebound enzyme produced nitrous oxide whereas the cytoplasmic enzyme yielded nitric oxide. However, when the cytoplasmic enzyme was assayed with methylviologen and dithionite as electron donors, ammonia was produced. The two enzymes showed an identical electrophoretic behavior. They may be identical proteins, whose properties depend on their location within the cell (Mancinelli et al., 1986).

4.2.5. Life at temperature and pressure extremes The Antarctic isolate Halomonas subglaciescola is relatively tolerant to low temperatures. A mathematical analysis of the temperature dependence of the growth rate, based on plots of the square root of the reciprocal of the growth rate versus temperature, yielded a theoretical minimum temperature of-3.3 °C, an optimum of 23.4 °C, and a maximum of 32.3 °C, respectively, for the type strain of the species (strain ACAM 12). For other isolates theoretical minimum temperatures of -9.2 and -7.9 °C were calculated (McMeekin and Franzmann, 1988). Formation of heat shock proteins was examined in Chromohalobacter marismortui. When grown in salt, transfer to 42 °C resulted in complete inhibition of the synthesis of the normal proteins, and induced the formation of heat shock proteins. In cells grown in salt, a heat shock at 42 °C still allowed the synthesis of part of the normal proteins. The higher the salt concentration in which the cells were grown, the higher the upper temperature limit at which heat shock proteins could be synthesized (Katinakis, 1989). A similar phenomenon was observed in Halomonas halophila (Karamanou and Katinakis, 1988). A marine isolate of Nesterenkonia rosea (Micrococcus roseus) that grows up to 150 salt was found to be highly pressure-tolerant. The survival rate at a pressure of 139

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MPa (equivalent to 1,372 atmospheres) increased with the salinity at which the cells were grown (Tanaka et al., 2001).

4.2.6. Reactions to the presence of reactive oxygen species The factors influencing sensitivity to hydrogen peroxide have been examined in Halomonas halophila. Pre-exposure of the cells to a low concentration of protected against the otherwise lethal effect of a subsequent exposure to concentrations of 1-2 mM. The pretreatment induced the formation of "hydrogen peroxide-inducible proteins", at least some of which have apparent molecular masses identical to those of known heat-shock proteins. The extent of their induction was salt concentration-dependent: the response was fastest at the lower salinities, and the level of resistance achieved was higher in NaCl than in No difference in catalase activity was observed in cells grown at different salt concentrations (Mylona and Katinakis, 1992).

4.2.7. Polyamines of halophilic Bacteria Some moderately halophilic Bacteria may lack polyamines altogether. Thus, no polyamines were detected in Salinivibrio costicola (Hamana, 1997; Kamekura et al., 1986). However, when grown in a complex medium, this organism incorporates polyamines such as putrescine, cadaverine, spermidine, and spermine from the medium. The presence of these polyamines did not increase the osmotic stability of the cells upon salt downshock (Kamekura et al., 1986). Halomonas and Chromohalobacter species contain spermidine at a concentration of 0.3 to per g of wet cells, which is in the concentration range generally found in nonhalophilic bacteria (Hamana, 1997). The function of the polyamines in cell metabolism of the halophiles, if present at all, is as yet unknown. 4.2.8.

deposition by aerobic halophilic Bacteria

Several strains of halophilic Bacteria have been reported to mediate the precipitation of (calcite, aragonite) and other minerals (Del Moral et al., 1987). Isolates that were in the past assigned to the genus Flavobacterium produce calcite and magnesium calcite with 0.04 to 0.32 mol% magnesium. Acinetobacter strains make both calcite, aragonite, and magnesium calcite; up to 14% aragonite was found at the highest salinities. High temperatures and low ionic strength favor crystal formation (Ferrer et al., 1988b). Similarly, 63 halophilic Vibrio isolates (possibly belonging to the genus Salinivibrio) isolated from an inland saltern in Spain stimulate crystal formation (Rivadeneyra et al., 1994). In a test of 27 isolates of Halomonas halophila, all caused the formation of crystals as long as conditions allowed bacterial growth. Most crystals were spherical and consisted of calcite and magnesium calcite containing from 75 to 85% and 25 to 15% The ratio between these minerals depended on the salinity and the composition of the medium. High concentrations inhibited

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precipitation by Halomonas halophila. Growth at low salinity favored crystal formation, while at low temperature and/or at high salinity crystal formation was repressed. The bacteria influenced the type of crystal formed in vitro in a species-specific way (Del Moral et al., 1987; Ferrer et al., 1988a; Rivadeneyra et al., 1989, 1991). Calcification commences with the formation of a nucleus by aggregation of a few calcified bacterial cells and subsequent accumulation of more calcified cells and carbonate. This results in the formation of spherical bioliths of about in diameter (Rivadeneyra et al., 1996). Halomonas eurihalina catalyzes the formation of magnesium calcite, aragonite, and/or monohydrocalcite in proportions that depend on the medium salinity and composition (Rivadeneyra et al., 1998). To what extent a local increase in pH triggers precipitation or whether the bacteria served only as crystallization nuclei has never been ascertained. Bioliths can also be produced by Gram-positive halophiles: spherical 10-100 large carbonate bioliths were precipitated by Marinococcus albus and Marinococcus halophilus isolates obtained from the Salar de Atacama (Chile). The bioliths consisted of magnesium calcite, the content increasing with increasing salinity and Mg/Ca ratio of the medium. Marinococcus halophilus formed crystals at all salt concentrations from 30 and with an optimum at No bioliths were formed by Marinococcus albus at 200 g salt. (Rivadeneyra et al., 1999).

4.2.9. Physiology of the Halanaerobiales The order Halanaerobiales, families Halanaerobiaceae and Halobacteroidaceae, forms a phylogenetically coherent group that consists entirely of halophiles. All members of the order are strict anaerobes. Most species of the order grow fermentatively on sugars with the production of acetate, ethanol, hydrogen, and carbon dioxide. Some also produce butyrate, lactate, propionate, and/or formate. Halanaerobium congolense does not form ethanol (Ravot et al., 1997). The glucose transport system of a number of Halobacteroides strains has been characterized in part (Senyushkin et al., 1992; Severina et al., 1992). Different polysaccharides can be degraded as well, such as chitin (by Halanaerobacter chitinivorans) (Liaw and Mah, 1992) and cellulose (by Halocella cellulosilytica (Bolobova et al., 1992; Simankova et al., 1993). Glycerol is fermented at a low rate by Halanaerobium saccharolyticum and by Halanaerobium strain FR1H isolated from an oil field reservoir. Products were acetate, hydrogen, and carbon dioxide. Glycerol dissimlation was significantly increased in co-culture with Desulfohalobium retbaense which served as hydrogen scavenger in a mechanism based on interspecics hydrogen transfer (Cayol et al., 2002). Amino acid fermentation has been documented in Sporohalobacter lortetii (Oren, 1983b). Halanaerobacter salinarius can grow on a mixed amino acid fermentation (Stickland reaction) in which serine (or hydrogen) is oxidized while glycine betaine is reduced with formation of trimethylamine (Mouné et al., 1999). Glycine betaine is fermented by Haloanaerobium alcaliphilum with production of acetate and trimethylamine (Tsai et al., 1995).

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Some representatives of the Halobacteroidaceae have a homoacetogenic metabolism. Acetohalobium arabaticum and Natroniella acetigena produce acetate as the main end product of their energy metabolism. Acetohalobium arabaticum can grow as a lithoautotroph on hydrogen + carbon dioxide or on carbon monoxide, as a methylotroph on trimethylamine, and as an organotroph on other substrates (formate, trimethylamine, glycine betaine, lactate, pyruvate, histidine, aspartate, glutamate, and asparagine) (Kevbrin et al., 1995; Zhilina and Zavarzin, 1990a, 1990b). Electron transport from hydrogen during autotrophic growth is mediated by a flavoprotein; NAD, cytochromes or quinones are not involved (Pusheva and Detkova, 1996; Pusheva et al., 1992). Monensin, an inhibitor of antiport and ATP synthesis, inhibited growth. It was therefore postulated that the potential generated via antiport is essential for the functioning of the cell (Pusheva and Detkova, 1996). The corrinoid metabolism of this organism has been studied as well (Bykhovsky et al., 1994). Natroniella acetigena cannot grow chemolithotrophically; it produces acetate from lactate, ethanol, pyruvate, glutamate, and propanol (Zhilina et al., 1996). Acetate is formed by the acetyl-CoA pathway involving CO dehydrogenase. Also in this organism does the energy metabolism rely on the generation of a transmembrane electrochemical gradient of via antiporter activity (Pusheva et al., 1999, 1999b). None of the species mentioned above possess cytochromes. However, a cytochrome-containing member of Halobacteroidaceae was recently described: Selenihalanaerobacter shriftii. This obligate anaerobe oxidizes glycerol or glucose to acetate and carbon dioxide by anaerobic respiration, using selenate as a terminal electron acceptor. Selenate is reduced to a mixture of selenite and elemental selenium. Growth on glycerol with nitrate or trimethylamine-N-oxide as an electron acceptor is possible as well (Switzer-Blum et al., 2001). The order Halanaerobiales contains thermophiles(Halothermothrix orenii grows up to 68 °C with an optimum at 60 °C) and alkaliphiles (Natroniella acetigena, Haloanaerobium alcaliphilum). Little is known about the special adaptations that enable these bacteria to withstand both the salt stress and the additional stress caused by the high temperature or the high pH, respectively. Several fermentative members of the order (Halanaerobium saccharolyticum, Halanaerobacter lacunarum, Halobacteroides halobius, Halobacteroides elegans) can use methanethiol as the sole source of assimilatory sulfur for growth (Kevbrin and Zavarzin, 1992a; Zhilina et al., 1997). Many representatives of the Halanaerobiales may use oxidized sulfur compounds as electron acceptors or electron sinks. Thus, addition of thiosulfate or sulfur to Halanaerobium congolense increased the growth yield 6-fold and 3-fold, respectively. In the presence of thiosulfate, carbohydrate utilization was improved and growth rates were enhanced. Addition of thiosulfate also alleviated growth inhibition by accumulating hydrogen (Ravot et al., 1997). Reduction of elemental sulfur to sulfide was also reported in Halanaerobium saccharolyticum (Zhilina et al., 1992b), in Halanaerobacter lacunarum (Zhilina et al., 1992a), and in Halobacteroides elegans (Kevbrin and Zavarzin, 1992a; Zhilina et al., 1997). Thiosulfate reduction was also observed in Orenia marismortui (Oren et al., 1987). The homoacetogen Acetohalobium arabaticum slowly reduces sulfur to sulfide, but growth rates were not enhanced by elemental sulfur (Kevbrin and Zavarzin, 1992b; Zavarzin et

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al., 1994). Halanaerobium praevalens and Orenia marismortui reduce nitrosubstituted aromatic compounds such as nitrobenzene, nitrophenols, 2,4-dinitrophenol and 2,4dinitroaniline to the corresponding amino derivatives (Oren et al., 1991, 1992).

4.2.10. Physiology of anoxygenic phototrophic Bacteria The electron transport chain of the anoxygenic photosynthetic sulfur bacteria of the genus Halorhodospira has been studied in some detail. Halorhodospira halophila contains cytochrome c-551 and a pair of high redox-potential ferredoxins as the major soluble electron transport proteins (Meyer, 1985). In addition, a small photosensitive yellow protein was detected, which has since become the subject of intensive studies. More information about this yellow protein is given in Section 5.5. The properties of cytochrome c-551 were investigated in further depth in Halorhodospira abdelmalekii. This cytochrome functions as a sulfide:acceptor oxidoreductase. Electrons from sulfide are transferred to the cytochrome during its oxidation to elemental sulfur; polysulfides are transiently accumulated during the process (Then and Trüper, 1983). The pathway of electron flow was also investigated in (facultatively) photoorganotrophic halophilic bacteria of the genus Rhodovibrio. In addition to a small amount of a soluble c-type cytochrome, three b-type and three c-type cytochromes were detected in the membranes of Rhodovibrio sodomensis cells grown aerobically in the dark as chemoheterotrophs. Inhibitor studies suggested that this organism has a branched electron transport chain (Bonora et al., 1998). The components of the electron flow chain of Rhodovibrio salinarum have been characterized as well. Two soluble cytochromes c' were found, one of which has a midpoint potential of 265 mV (the midpoint potential of the second could not be determined), in addition to cytochrome c-551 (midpoint potential -143 m V, monomer size 12 kDa). There are also two types of membrane-bound cytochrome c with peaks at 552 and 553 nm, respectively. Two high-redox potential ferredoxins are also present. Cells grown aerobically in the dark have three b-type cytochromes (+180, +72, and –5 mV), two c-type cytochromes (+244 and +27 mV), and two a-type cytochromes (+325 and +175 mV); cells grown anaerobically in the light have two btype cytochromes (+60 and –45 mV) and 5 c-type cytochromes (+290, +250, +135, -20, and –105 mV) (Meyer et al., 1990b; Moschettini et al., 1997, 1999). Rhodothalassium salexigens has membrane-bound cytochrome c-558 and c-551 and soluble cytochrome midpoint potential, 13 kDa), (95 mV, being a dimer of 14 kDa subunits), and c-551 (-170 mV, 94 kDa) (Meyer et al., 1990a).

4.3. PHYSIOLOGY OF HALOPHILIC EUCARYA Most physiological studies performed with the halophilic green alga Dunaliella deal with the metabolism of glycerol (accumulated as osmotic solute) or with the accumulation of These aspects are discussed in Chapter 7 and 5, respectively. When Dunaliella salina is transferred from low salt media to high salt concentrations, two proteins (60 and 150 kDa) appear in the cytoplasmic membrane in large amounts. Neither of these salt-induced proteins is directly related to the ways the

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cells cope with osmotic or bioenergetic problems caused by the salt upshock, but instead they are involved in nutrient acquisition. The 60 kDa protein is a carbonic anhydrase. Increased carbonic anhydrase activity is necessary to supply sufficient amounts of to the interior of the cell for photosynthetic fixation (Fischer et al., 1996). The 150 kDa salt-induced protein has been identified as a transferrin, and mediates the uptake of inorganic iron (Fischer et al., 1997, 1998).

4.4. METABOLIC DIVERSITY AMONG THE HALOPHILES A BIOENERGETIC APPROACH A survey of the known halophilic microorganisms (see also Chapter 2) shows that not all metabolic types that exist in nature also function in the presence of high salt concentrations. Oxygenic and anoxygenic photosynthesis, aerobic respiration and denitrification occur at or close to NaCl saturation, but many other physiological groups have never been shown to thrive at high salinities. For example, methanogens growing autotrophically with hydrogen as electron donor, aceticlastic methanogens, dissimilatory sulfate reducers that perform complete oxidation of their substrates, and autotrophic ammonia and nitrite oxidizers do not appear to occur at salt concentrations above 100A bioenergetic explanation has suggested forward for the apparent lack of certain metabolic types within the halophile world (Oren, 1999, 2001, 2002). This explanation is based on the fact that life at high salt concentrations is energetically expensive as it involves the build-up and maintenance of steep ion concentration gradients across the cell membrane, whether or not accompanied by the biosynthesis or accumulation of organic osmotic compounds (see Chapters 6 and 7). Calculations show that de novo biosynthesis of organic osmotic solutes, the strategy commonly used in the aerobic halophilic Bacteria and in Eucarya, as well as in the halophilic methanogens (see Chapter 7) is energetically more expensive than the accumulation of and the mode of osmoadaptation used by the aerobic Halobacteriaceae (Archaea) and the anaerobic Halanaerobiales (Bacteria) (see Chapter 6). It was therefore hypothesized that the upper salt concentration limit at which a dissimilatory process can occur is determined primarily by bioenergetic constraints, and that therefore the important factors that determine whether a certain type of microorganism can make a living in the presence of high salt concentrations are (1), the amount of energy generated in its dissimilatory metabolism, and (2), the mode of osmotic adaptation used. Following are a few examples. Autotrophic oxidation of to does not appear to occur above salt, and the salt limit for the oxidation of to may even be lower (Rubentschik, 1929). The most halophilic autotrophic nitrifier isolated, described as "Nitrosococcus halophilus", grows optimally at NaCl and only tolerates up to (Koops and Möller, 1992; Koops et al., 1990). No nitrification was observed in a microcosm simulation of the nitrogen cycle in the Great Salt Lake, Utah at a total salt concentration above (Post and Stube, 1988) (see Section 12.4.2). Ammonia is often abundant in hypersaline lakes and saltern ponds (Javor, 1983a, 1983b; Litchfield et al., 1999; Nissenbaum et al., 1990; Post, 1977; Stephens and Gillespie, 1976), so lack of an

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available energy source cannot be the reason for the absence of nitrification activity. Ammonia oxidizing bacteria have been detected in Mongolian soda lakes varying in salinity between 5 and and with pH values between 9.7 and 10.5. Bacteria resembling Nitrosomonas halophila in their 16S rDNA sequence were isolated from these lakes. None of them, however, grew above NaCl (Sorokin et al., 2001). Nitrifying bacteria obtain only little energy during their dissimilatory metabolism. Their electron donors (ammonia, nitrite) are relatively oxidized, so that only little energy can be gained from their oxidation. In addition, most of the energy generated is used to drive uphill electron transport to produce NADPH, the reducing power required for autotrophic fixation. The energetic burden of haloadaptation may therefore be too great for ammonia or nitrite-oxidizing autotrophs. Oxidation of reduced sulfur compounds is energetically more favorable, as substrates such as sulfide and elemental sulfur are more reduced. The process can accordingly occur up to reasonably high salt concentrations. Halothiobacillus halophilus, which oxidizes thiosulfate, tetrathionate and elemental sulfur, grows optimally at NaCl, but growth is possible up to (Wood and Kelly, 1991). Dissimilatory sulfate reduction is possible up to very high salt concentrations, and halophilic or highly halotolerant sulfate reducing bacteria have been isolated. Examples are Desulfovibrio halophilus (optimum NaCl concentration maximum 180 g (Caumette et al., 1990) and Desulfovibrio oxyclinae (optimum maximum (Krekeler et al., 1977). The most halophilic strain described to date is Desulfohalobium retbaense from Lake Retba in Senegal, which grows optimally at NaCl and tolerates up to (Ollivier et al., 1991; see also Ollivier et al., 1994 and Oren, 1988). Little information is available on the nature of the electron donors used by the communities of sulfate reducing bacteria in the sediments of salt lakes. Acetate was found to be the main substrate for sulfate reduction in Lake Eliza, South Australia, at the relatively low salt concentration of (Skyring, 1988). However, all truly halophilic and halotolerant strains of sulfate reducers isolated thus far are incomplete oxidizers that grow on lactate as preferred electron donor. The most halotolerant acetate-oxidizing sulfate reducer known, Desulfobacter halotolerans, was recently isolated from the sediments of the Great Salt Lake, Utah (Brandt and Ingvorsen, 1997). It performs poorly at high salt concentrations: its optimum is at and no growth was obtained above NaCl. The phenomenon may have a bioenergetic basis. Desulfovibrio halophilus, and probably the other sulfate reducers as well, uses organic solutes to provide osmotic balance (Welsh et al., 1996), a strategy that is energetically relatively costly. The oxidation of lactate to acetate and yields much more energy than oxidation of acetate (Table 4.2). The main methanogenic reactions in freshwater environments are the reduction of with hydrogen and the aceticlastic split. Neither of these have yet been shown to occur at high salt concentrations (Oremland and King, 1989; Oren, 1988). No methanogenesis from acetate or from could be demonstrated in sediments of Solar Lake (Sinai) salt) (Giani et al., 1984) (Section 17.2). Mono Lake, CA salt) is the most hypersaline environment in which methanogenesis from has been observed (Oremland and King, 1989) (Section 16.2). The most halotolerant methanogen known that grows on is Methanocalculus

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halotolerans, originally isolated from an oil well. This organism grows up to NaCl with an optimum at (Olivier et al., 1998). There is a report on the isolation

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of a methanogen from the saltern ponds salt) near Alicante, Spain, that grows on hydrogen + with an optimum at salt, and can grow on trimethylamine as well (Pérez-Fillol et al., 1985). Unfortunately this strain has not been characterized further. The upper salinity limit at which acetate can be used as methanogenic substrate is probably even lower, but few data are available. Methanogenesis at the highest salt concentrations is based on conversion of methylated amines, methanol, and dimethylsulfide (Oremland and King, 1989;Zhilina and Zavarzin, 1987, 1990b; see also Ollivier et al., 1984). A number of species with a high salt tolerance are known, such as Methanohalobium evestigatum and the different Methanohalophilus species (Lai and Gunsalus, 1992; Zhilina and Zavarzin, 1987; see also Section 2.2). Halophilic methanogens produce a variety of organic osmotic compounds, including and glycine betaine (Lai and Gunsalus, 1992; Lai et al., 1991; Robertson et al., 1990, 1992; Sowers et al., 1990) (see also Section 8.1). Accordingly they need a relatively large amount of energy for osmotic adaptation, Comparison of the free energy changes associated with different methanogenic reactions (Table 4.2) is not straightforward. Reaction stoichiometries are complex, and a process such as methanogenesis from involving reduction reactions only, is difficult to compare with the disproportionation reactions that lead to methanogenesis from methanol or from methylated amines. However, it is clear that methanogenesis from acetate yields only very little energy (-31.1 kJ per mol acetate). Thus, also in this case the apparent lack of halophilic representatives may be due to energetic constraints. Methanogenesis from acetate has been reported to occur in the monimolimnion of Soap Lake, Washington total dissolved salts, pH 9.7) (Oremland and Miller, 1993). This observation deserves a more in-depth study, including attempts to isolate the organism responsible for the process. The standard free energy change during growth on hydrogen is -34 kJ per mol of hydrogen, not very different from that on acetate. The energy yield on those methanogenic substrates that do enable growth at high salt concentrations is much larger: between -79 and -191 kJ per mol of substrate transformed. While methanogens that grow on appear to be largely absent in hypersaline environments, halophilic homoacetogenic bacteria that use the same substrates for the production of acetate have been isolated (Zavarzin et al., 1994). Indications for the occurrence of homoacetogens in the sediments of the Dead Sea have also been obtained (Oren, 1990). Acetohalobium arabaticum grows between 100-250 g NaCl with an optimum at (Zhilina and Zavarzin, 1990a; Zavarzin et al., 1994). The acetogenic reaction yields even less energy than the methanogenic process (see Table 4.2). However, the halophilic homoacetogens belong to the order Halanaerobiales, and all representatives of this group examined thus far accumulate inorganic ions to establish osmotic balance rather than producing organic compatible solutes (Oren, 1986; Oren et al., 1997; Rengpipat et al., 1988) (see also Section 6.5). Unfortunately, no analyses of the intracellular salt and solute content of the halophilic homoacetogens have yet been reported. The apparent absence of aerobic methane oxidation in many hypersaline environments is less easy to explain. Methane oxidation is a highly exergonic process

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While methane is often produced in hypersaline anaerobic sediments (see above), little or no methane oxidation can be demonstrated in the aerobic overlying waters. Even in environments with relatively low salinities, such as the cyanobacterial mats of Solar Lake, Sinai (about salt) (see Section 17.2) and saltern evaporation ponds in Eilat, Israel salt), no methane oxidation could be measured, in spite of the availability of both methane and oxygen (Conrad et al., 1995). Interestingly, anaerobic methane oxidation, an as yet little understood process that that yields very little energy, and that involves cooperation of an archaeal methane oxidizer and a sulfate-reducing bacterium, appears to be the main sink for methane in the hypersaline Mono Lake, California, and Big Soda Lake, Nevada (see also Sections 16.2.3 and 16.3.3). Recent reports on the occurrence of aerobic methane oxidation in sediments of hypersaline reservoirs in Ukraine and Tuva (up to salts) and the isolation of halophilic methanotrophs from these environments (Doronina and Trotsenko, 1997; Khmelenina et al., 1996, 1997; Sokolov and Trotsenko, 1995) indicate that halophilic or halotolerant methanotrophs do exist. The earlier reported lack of methane oxidation in hypersaline environments (Conrad et al., 1995; Slobodkin and Zavarzin, 1992) may in part be due to the low solubility of gases in hypersaline brines. Genes coding for key enzymes of the methane oxidation process, such as mxaF (methanol dehydrogenase), mmoX (soluble methane monooxygenase), and pmoA (particulate methane monooxygenase) have been detected in DNA isolated from the biomass of several inland basins with salinities up to (Kalyuznaya et al., 1998; Sokolov and Trotsenko, 1995). Few isolates of halophilic or halotolerant aerobic methanotrophs are available as yet. Methylomicrobium modestohalophilum grows optimally at NaCl (Kalyuznaya et al., 1998), and Methylomicrobium sp. N1 has its optimum at Some soda lake isolates tolerate up to NaCl (Kalyuznaya et al., 1999; Khmclenina et al., 1999; Trotsenko and Khmclenina, 2002a, 2002b).

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Zoratti, M., and Lanyi, J.K. 1987. Phosphate transport in Halobacterium halobium depends on cellular ATP levels. J. Bacteriol. 169: 5755-5760. Zvyagintseva, I.S., Belyaev, S.S., Borzenkov, I.A., Kostrikina, N.A., Mileklhina, E.I., and Ivanov, M.V. 1995a. Halophilic archaebacteria from the Kalamkass oil field. Mikrobiologiya 64: 83-87 (Microbiology 64: 67-71). Zvyagintseva, I.S., Gerasimenko, L.M., Kostrikina, N.A., Bulygina, E.S., and Zavarzin, G.A. 1995b. Interaction of halobacteria and cyanobacteria in a halophilic cyanobacterial community. Mikrobiologiya 64: 252-258 (Microbiology 64: 209-214).

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CHAPTER 5 PIGMENTS OF HALOPHILIC MICROORGANISMS

The Microbe is so very small You cannot make him out at all. … His seven tufted tails with lots Of lovely pink and purple spots, On each of which a pattern stands, Composed of forty separate bands; His eyebrows of a tender green; All these have never yet been seen .... (Hilare Belloc, More Beasts for Worse Children, 1897)

Pink is the color of and other carotenoid pigments of the halophilic Archaea, the retinal pigments bacteriorhodopsin and halorhodopsin produced by Halobacterium and relatives are purple, and many Dunaliella strains, halophilic cyanobacteria, and some halophilic photosynthetic purple bacteria such as Halorhodospira halochloris are colored green. The "Microbe" described in Mr. Belloc's poem may thus well have been a halophile! The presence of dense communities of halophilic microorganisms in hypersaline environments can often be observed with the unaided eye thanks to their bright red, orange, purple, and/or green coloration. Red colored brines have been observed in the north arm of the Great Salt Lake (Post, 1977) (see Section 12.2), the Dead Sea (Oren, 1988) (see Section 13.3), and in hypersaline alkaline lakes such as Lake Magadi, Kenya (Grant and Tindall, 1986) (see Section 15.2.3.1). Red colored brines also form during the final stages of the evaporation of seawater in solar saltern crystallizer ponds (Borowitzka, 1981; Javor, 1989; Oren, 1993, 1994) (see Sections 14.3 and 14.6). In all these cases bacterioruberin pigments in the membranes of red halophilic Archaea of the family Halobacteriaceae are responsible for the pigmentation of the brines. Presence of bacteriorhodopsin may contribute a purple color. The red coloration of saltern crystallizer brines may also be due in part to the presence of extremely halophilic Bacteria of the genus Salinibacter (Oren and Rodríguez-Valera, 2001). Eukarotic algae of the genus Dunaliella may contribute the green color of chlorophyll a and b, and certain species (Dunaliella salina, Dunaliella bardawil) may also contain large amounts of which adds a red-orange color to crystallizer ponds and

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other hypersaline lakes. In the case of the alkaline hypersaline lakes of the Wadi Natrun (Egypt), photosynthetic purple bacteria of the genus Halorhodospira may be responsible for at least part of the red coloration of the brines (Jannasch, 1957) (see also Section 15.1). This chapter discusses the properties and function of a variety of pigments detected in halophilic microorganisms. These include both the carotenoids of halophilic Archaea, Bacteria and eukaryotic algae, and the different retinal pigments of the Halobacteriaceae. In addition, the photoreactive yellow protein of Halorhodospira and other anoxygenic photosynthetic Bacteria is discussed here in view of the resemblance of its function to the sensory rhodopsins of the Halobacteriaceae.

5.1. ALGAL CAROTENOIDS Certain strains of the unicellular green alga Dunaliella, notably Dunaliella salina and Dunaliella bardawil, may accumulate large amounts of under suitable conditions. Such cells then obtain a bright orange-red color. Massive production of carotenoid pigments is mainly a reaction to high light intensities, but other factors such as nutrient limitation also play a role (see below). In Dunaliella bardawil up to 8% of the dry weight may be (Ben-Amotz and Avron, 1981), and a Dunaliella strain from Chile was reported to have an even higher carotene content (Gomez et al., 1999). In this strain, was unusually abundant, as it accounted for about 20% of the total carotenoids. In the Dunaliella salina community of Pink Lake, Victoria, Australia, was estimated to constitute as much as 13.8% of the total dry organic matter (Aasen et al., 1969). The is accumulated in globules, located in the interthylacoid space within the cell's single chloroplast (Ben-Amotz, 1999; Ben-Amotz et al., 1982, 1988) (Figure 5.1). A 38 kDa protein was found associated with the globules, and may serve to stabilize the structure of the globules (Katz et al., 1995). The oily globules contain a mixture of the all-trans and the 9-cis isomers of (Figure 5.2), together with minor amounts of other mono-cis and di-cis stereoisomers (BenAmotz, 1999; Ben-Amotz et al., 1982). When grown under high light intensity, Dunaliella bardawil produces about equal amounts of all-trans and 9-cis The ratio between the amount of the 9-cis to the all-trans form increases with the integral light intensity to which the algae had been exposed during a division cycle. When grown in continuous white light of 2,000 quanta this ratio reached 1.5, at quanta the ratio was about 0.2. At quanta phytoene made up 8% of the carotenoids, with a 9-cis to all-trans ratio of 1.0, which decreased to 0.1 in cells grown at quanta This observation suggests that the isomerization leading to the formation of the 9-cis isomer occurs early in the pathway of carotene formation (BenAmotz et al., 1988). The ratio between the 9-cis and the all-trans stereoisomer of the in thylacoids of the chloroplast differs from that in the interthylacoid globules. Incubation of Dunaliella bardawil at high light intensities induced a 20 to

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30-fold increase in the amount of in the globules and a 2.5-fold increase in thylacoid carotene (Jimenez and Pick, 1994). In that study no great effect of the light intensity was found on the stereoisomeric composition of the pools. However, in Dunaliella salina incubation at high light induced a reduction of the 9cis/all-trans ratio. Growth at low temperatures led to an increase in the ratio of the 9cis and the all-trans isomer of the in the globules. However, the composition of the pool in the thylacoids was hardly affected by growth phase, light intensity, or temperature (Jimenez and Pick, 1994).

The biochemical pathway that leads to the formation of and phytoene in Dunaliella bardawil has been largely elucidated (Figure 5.3). The branching point for the formation of the all-trans and the 9-cis isomers of was suggested to occur early in the pathway, probably at the level of phytoene (Ebenezer and Pattenden, 1993). The content of Dunaliella bardawil cells is mainly determined by the total irradiance the alga receives during a cell division cycle. High light intensities and stress conditions such as nutrient limitation or supraoptimal salt concentrations cause a decrease in the amount of chlorophyll per cell and an increase in its content (Ben-Amotz, 1999; Ben-Amotz and Avron, 1983). Nitrogen deficiency and low growth temperatures both induce massive intracellular accumulation of under any light intensity (Ben-Amotz, 1999). Non-dividing sulfate- and phosphatedeficient cells also accumulate as a reaction to increased light intensity

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(Ben-Amotz, 1987). Ultraviolet radiation has a strong inducing effect on biosynthesis in Dunaliella bardawil: when quanta UV-A. light of 320400 nm was given in addition to photosynthetically effective visible wavelengths, the carotenoid/chlorophyll ratio increased by 80-310%. The effect was observed at all salinities and at all levels of photosynthetically available radiation tested (Jahnke, 1999). Photoinduction of is abolished by antibiotics such as actinomycin-D, chloramphenicol or cycloheximide (Lers et al., 1990).

A correlation was found between the level of synthesis and the amounts of triacylglycerol in Dunaliella bardawil. produced during nutrient starvation is accumulated in newly formed triacylglycerol droplets within the chloroplast. Formation of the sequestering triacylglycerol droplets and accumulation are interdependent. When synthesis of triacylglycerol is blocked by the herbicide sethoxydim, no overproduction of occurs (Rabbani et al., 1998). Inorganic pyrophosphatase probably acts as a regulator of synthesis in Dunaliella salina. Its activity increases with increasing medium salinity. The enzyme is responsible for the hydrolysis of the pyrophosphate released when isopentenyl pyrophosphate building blocks are condensed in the biosynthetic pathway that leads to the production of carotenoids (Mortain-Bertrand et al., 1994). Dunaliella cells with a high content tolerate much higher light intensities than green, cells. Thus, in Dunaliella salina, green cells were already inhibited at light intensities above quanta of visible light, while red cells continued to evolve oxygen up to quanta (Gómez-Pinchetti et al., 1992). The action spectrum of the photoprotection parallels the absorption spectrum of the carotenoids: protection is strong with blue light, intermediate with white light, and no protection is provided against high levels of red

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light. When cells are irradiated by supraoptimal intensities of blue light, oxygen evolution is rapidly inhibited. This is followed by the photodestruction first of 9-cis carotene, then of all-trans Only afterwards photodestruction of chlorophyll occurs, whereafter the cells die (Ben-Amotz et al., 1989).

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The protective effect of the accumulated may be based on the destruction of singlet oxygen radicals produced during photosynthesis under high radiation. However, electron micrographs (see Figure 5.1) indicate that the massive accumulation of occurs in globules in the interthylacoid space rather than in the thylacoids themselves. The large distance between the globules and the thylacoid-bound chlorophyll would not allow effective quenching of singlet oxygen or of any other chlorophyll-generated radicals (Ben-Amotz, 1999). A possible function of the globules as carbon storage can be eliminated, as the cells do not degrade the pigment under limitation. The most widely accepted hypothesis suggests that the carotene globules protect the cells against injury by high light intensity under limiting growth conditions by acting as a screen to absorb excess radiation (Ben-Amotz, 1999). Dunaliella salina and Dunaliella bardawil are extensively exploited in biotechnological operations for the commercial production of This aspect is discussed in further detail in Section 11.4.1. Dunaliella salina also accumulates When grown at low temperature (17 °C), the level of increased 7.5-fold as compared to the control culture grown at 34 °C, while the level remained unaltered. The total accumulation was not affected by the level of irradiance, but the proportion of 9-cis increased from 15% to 45% of the total when irradiance was decreased from 260 to quanta (Orset and Young, 1999). Pigments of the xanthophyll cycle may also be involved in stress protection in Dunaliella. High light-treated Dunaliella salina cells accumulated zeaxanthin, whereas salt-stressed cells formed violaxanthin at the expense of zeaxanthin. Highlight-treated cells maintained a high zeaxanthin content in addition to the accumulation of (Cowan et al., 1995).

5.2. PIGMENTS OF OXYGENIC AND ANOXYGENIC PHOTOSYNTHETIC BACTERIA Cyanobacteria often contribute to the color of hypersaline waters and surface sediments. They rarely abound in the plankton of lakes with salt concentrations exceeding However, benthic microbial mats dominated by cyanobacteria cover the shallow sediments of many hypersaline water bodies such as the Great Salt Lake (Section 12.2) and solar saltern evaporation ponds (Section 14.2). Microcoleus mats, rich in chlorophyll and phycocyanin, often impart a blue-green color to the sediment. Unicellular cyanobacteria, designated Aphanothece halophytica. Cyanothece or Halothece (Oren, 2000) are frequently found in the upper layer of the sediment, and they may be colored yellow to brightly orange due to the abundance of different carotenoid pigments, mainly myxoxanthophylls and echinenone. Cells exposed to high light intensities have a low chlorophyll content (Caumette et al.. 1994; Oren, 2000; Oren et al., 1995). Such hypersaline Halothece mats may also be rich in mycosporine-like amino acids that contribute a strong absorbance in the UV-A range (320-380 nm) (Oren, 2000; Oren et al., 1995). A blue-green layer of

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filamentous cyanobacteria, often in combination with unicellular species, is generally present below the orange layer of unicellular cyanobacteria (Caumette, 1993; Caumette et al., 1994; Oren et al., 1995). Anoxygenic phototrophic bacteria often develop below the cyanobacterial layers in these stratified benthic hypersaline communities. Their massive growth can be visible as a distinct red-pink layer. Microscopic examination and culture experiments shows the presence of organisms such as Halochromatium, Thiohalocapsa, and Ectothiorhodospira or Halorhodospira (Caumette et al., 1994; Ollivier et al., 1994). Their coloration is due to bacteriochlorophyll a and to different carotenoids. Anoxygenic phototrophic purple bacteria occur even more prominently in hypersaline soda lakes. The red color of the highly alkaline, salt-saturated brines of the Wadi Natrun was attributed to a large extent to the presence of dense communities of bacteria of the Ectothiorhodospira/Halorhodospira group (Jannasch, 1957) (see Section 15.1.2). Representatives of the genus Halorhodospira such as Halorhodospira halochloris and Halorhodospira halophila can be found up to the highest salt concentrations. Halorhodospira halophila has bacteriochlorophyll a as photosynthetic pigment, while Halorhodospira halochloris possesses bacteriochlorophyll b. Halorhodospira halophila has caroteonoids of the spirilloxanthin series (spirilloxanthin, with minor amounts of hydroxyspirilloxanthin, rhodovibrin, and chloroxanthin) (Schmidt and Trüper, 1971). A novel class of carotenoids, dihydroxylycopene diglucoside diesters, was recently characterized from Halorhodospira abdelmalekii and Halorhodospira halochloris. Methoxyhydroxylycopene glucoside was the major component, with minor amounts of dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester (Takaichi et al., 2001). Halorhodospira halophila and many other halophilic purple bacteria also contain the photoactive yellow protein photosensor. This interesting pigment is discussed in further detail in Section 5.5.

5.3. CAROTENOIDS OF AEROBIC HETEROTROPHIC ARCHAEA AND BACTERIA Most representatives of the Halobacteriaceae are colored brightly red-orange due to a high content of carotenoid pigments in their cell membrane. Only rarely do halophilic archaeal isolates lack this red pigmentation. Natrialba asiatica is a rare exception to the rule (Kamekura and Dyall-Smith, 1995). The reddish-pink color of saltern crystallizer brines is mainly caused by these archaeal carotenoids (Oren and Dubinsky, 1994; Oren et al., 1992). Most carotenoids of the halophilic Archaea are straight-chain derivatives of bacterioruberin (Kelly et al., 1970) (Figure 5.4). Also present are mono-anhydrobacterioruberin and bisanhydrobacterioruberin (Kelly et al., 1970; Kushwaha et al., 1972, 1974, 1975). Several other derivatives have been found in minor amounts, such as the dodecaene carotenoids 3',4'-dihydromonoanhydrobacterioruberin, haloxanthin (a 3',4'-dihydromonoanhydrobacterioruberin derivative with a peroxide

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end group), and 3',4'-epoxymonoanhydrobacterioruberin, identified in Haloferax volcanii (Rønnekleiv et al., 1995) (Figure 5.4). The content of bacterioruberin pigments in the biomass has been used to monitor the density of halophilic archaeal communities in the Dead Sea (Oren and Gurevich, 1995).

The pigment content of the cells of certain members of the Halobacteriaceae depends on their nutritional status (Gochnauer et al., 1972; Kushwaha and Kates, 1979a). The medium salinity may be important as well: certain Haloferax species are brightly pigmented when grown in the lower salinity range enabling growth or below), while at higher salt concentrations they may appear almost

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colorless (Kushwaha et al., 1982; Rodriguez-Valera et al., 1980). Haloferax mediterranei, a species that is only weakly pigmented at high salinity, produces massive amounts of bacterioruberin pigments when incubated at salt, a concentration too low to support growth (D'Souza et al., 1997). Small amounts of other carotenoid pigments and related isoprenoid compounds have been found in Halobacterium, including phytoene, and lycopene. Together these compounds made up less than 5% of the total amount of carotenoid pigments (Kushwaha et al., 1974). It was suggested that bacterioruberin is synthesized by addition of isoprene units to each end of the lycopene chain, followed by introduction of four hydroxyl groups. Evidence for such a pathway was obtained from inhibitor studies in which nicotine was used to inhibit the formation of bacterioruberin (Kushwaha and Kates, 1979b). The presence of multiple genes for several of the steps in Halobacterium NRC-1 carotenoid production suggests that there may be more than one biosynthetic pathway (DasSarma et al., 2001). Lycopene is a precursor of carotene, but the amounts of lycopene and in the cells were not correlated. There was also no consistent correlation between the amounts of and carotenoids, implying that their biochemical pathways may be independent to a large extent (Kushwaha and Kates, 1979c). Retinal, the prosthetic group of bacteriorhodopsin, halorhodopsin, and the sensory rhodopsins, is synthesized via lycopene and (Sumper et al., 1976). Other minor carotenoid compounds identified in halophilic Archaea are lycopersene, cis- and trans-phytoene, cis- and trans-phytofluene, and The low concentrations of these compounds suggest that they may serve as precursors for the synthesis of retinal, lycopene, and the pigments of the bacterioruberin group (Kushwaha et al., 1982; Tindall, 1992). A halophilic archaeon isolated from a seawater evaporation pond near Alexandria, Egypt, produces considerable amounts of the ketocarotenoid canthaxanthin (Asker and Ohta, 1999). As much as 0.34 mg of canthaxanthin was found per mg total carotenoids. In addition, was present (0.03 mg per mg carotenoids) (Asker and Ohta, 1999), as are 3hydroxyechinenone, cis-astaxanthin, lycopene, trisanhydrobacterio-ruberin, monoanhydrobacterioruberin, and bacterioruberin isomers. The proportions in which bacterioruberins, and canthaxanthin are present depends on the salt concentration of the growth medium (Asker et al., 2002). This interesting isolate will soon be described as a new species of Haloferax, Haloferax alexandrinus (Asker and Ohta, in press). Calo et al. (1995) stated that major amounts of the carotenoids 3hydroxyechinenone and trans-astaxanthin occur in Halobacterium salinarum, Haloarcula hispanica, and Haloferax mediterranei; in Halobacterium salinarum 24% of the pigment on a per weight basis was 3-hydroxyechinenone, and 11% was identified as trans-astaxanthin. However, identification was based on HPLC analysis only, and no additional chemical techniques were employed to confirm the structure of these pigments. Two functions have been attributed to the bacterioruberin pigments of Halobacterium and other members of the Halobacteriaceae: protection against

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damage by high intensities of light in the visible and ultraviolet range of the spectrum, and reinforcement of the cell membrane. White mutants lacking bacterioruberins can easily be isolated from species such as Halobacterium salinarum. In the dark such mutants grow as well as the red wild type. However, when incubated at high light intensities approaching those of full sunlight, the white mutants are outcompeted by the pigmented parent strain, demonstrating the role of the carotenoid pigments in protecting the cells against light damage (Dundas and Larsen, 1962; Larsen, 1973). Even more dramatic differences between the survival of the red wild type and the white mutants were obtained when photosensitizers such as toluidine blue or phenosafranine were added: the white variety was rapidly killed, but the carotenoid-rich cells showed excellent survival (Dundas and Larsen, 1963). When Haloferax mediterranei cells grown at (weakly pigmented) and NaCl (strongly pigmented) were exposed to strong illumination, the pigmented cells grew normally, while in the weakly pigmented cells growth ceased and part of the cells lysed. Nutrient-starved cells, however, were only little affected by high light intensities, independent of their bacterioruberin content (Rodriguez-Valera et al., 1982). The carotenoid pigments of Halobacterium salinarum were also claimed to protect the cells against UV radiation and aid in photoreactivation (Wu et al., 1983). However, no dramatic difference in survival between the red wild type strain and the colorless mutant used can be seen in the data presented in that study. A protective role of bacterioruberin by providing resistance to DNA damaging agents (ionizing radiation, exposure to or to mitomycin-C) was also shown in Halobacterium salinarum. A colorless mutant proved more sensitive to DNA damaging agents (except for mitomycin-C). The frequency of DNA strand-breaks induced by ionizing radiation was significantly reduced by the presence of bacterioruberin (Shahmohammadi et al., 1998). A completely different role that the bacterioruberin pigments may play in the physiology of the Halobacteriaceae is a structural function, reinforcing the cytoplasmic membrane. The incorporation of bacterioruberins in reconstituted Halobacterium lipid membranes was found to greatly increase their rigidity and to decrease their water permeability (Lazrak et al., 1988). A novel group of aerobic red extremely halophilic prokaroytes has recently been discovered. Salinibacter, a representative of the domain Bacteria phylogenetically associated with the Cytophaga - Flavohacterium - Bacteroides group, was recognized as an important component of the microbial community in Spanish saltern crystallizer ponds. Its pigment has been identified as (all-E, 2’,S)-2’-hydroxy-1’-[6-O-(13methyltetradecanoyl) caroten-4-one, now named salinixanthin (Lutnæs et al., 2002) (Figure 5.5). The pigment shows an absorbance maximum at 482 nm with a shoulder at 506-510 nm. About 5-7.5% of the total prokaryote-derived pigment extracted from these salterns originated from bacterial, rather than from archaeal extreme halophiles (Oren and Rodriguez-Valera, 2001).

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5.4. THE RETINAL PIGMENTS: BACTERIORHODOPSIN, HALORHODOPSIN, AND SENSORY RHODOPSINS The retinal pigments present in halophilic Archaea of the family Halobacteriaceae have received much interest since the discovery of the structure and function of bacteriorhodopsin in the early 1970s. Four such retinal-containing proteins have been identified: the outward proton pump bacteriorhodopsin (absorption maximum 568 nm), the inward chloride pump halorhodopsin (absorption maximum 578 nm), and two sensory rhodopsins involved in light sensing for phototaxis (Figure 5.6). These proteins all consist of seven transmembrane helixes, the retinal group being covalently attached through the formation of a protonated Schiff base between the aldehyde function of the chromophore and the group of a lysine in the seventh helix. The retinal proteins are unusual among the proteins of the halophilic Archaea as they do not require salt for conformational stability and function. During the three decades that have passed since retinal was identified as the prosthetic group of the pigment in the purple membrane of Halobacterium salinarum, more publications have appeared on the retinal pigments than on all other aspects of the biology of the Halobacteriaceae combined. A database search (ISI Web of for papers dealing with bacteriorhodopsin between the years 1988 and 2001 yielded no less than 3,581 entries. Only a small part of the articles that have been published on the retinal pigments has been reviewed in the following sections. More in-depth information on the biology, biochemistry, and biophysics of the retinal proteins can be found in specialized reviews such as those by Bickel-Sandkötter et al. (1996), Lanyi (1998, 1999a, 1999b, 2000a, 2000b), Lanyi and Luecke (2001), Lanyi and Váró (1995), Mukohata et al. (1999), Oesterhelt (1995, 1998), Schäfer et al., (1999), and Stoeckenius and Bogomolni (1982). Until recently the presence of a light-driven retinal-based proton pump was believed to be a unique feature of the halophilic Archaea. However, a similar pigment has now been discovered in the genome of a (yet-to-be-cultured) member of the Proteobacteria that is abundant in the oceans (Béjà et al., 2000). Moreover, it has been demonstrated that this pigment indeed functions to make light energy available to

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the marine bacterial community (Béjà et al., 2001). Retinal pigments arc thus more widespread in nature. Homologues of the archacal rhodopsins are also found in plants, animals, and fungi. The microbial rhodopsins are distantly related to a family of proteins that includes the human lysosomal cystine transporter and a putative fungal chaperone protein (Zhai et al., 2001).

5.4.1. Bacteriorhodopsin Under the proper conditions Halobacterium salinarum produces large quantities of the purple pigment bacteriorhodopsin. The color of the cultures then changes from redorange (due to the presence of and derivatives, see Section 5.3) to brightly purple. The pigment present in the purple membrane of Halobacterium salinarum was identified as a retinal-containing protein in 1971 (Oesterhelt and Stoeckenius, 1971). The protein serves as an outward proton pump (for reviews see e.g. Lanyi, 1993, 1997; Lanyi and Luecke, 2001; Schäfer et al., 1999). Its function in the energy metabolism of the cell was rapidly recognized: excitation of bacteriorhodopsin drives the formalion of ATP, and the bacteriorhodopsin proton

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pump thus presents the cell with an additional mode of the ATP generation to supplement the respiration-driven ATP generation based on electron transfer through the cytochrome chain (Danon and Stoeckenius, 1974; Hartmann and Oesterhelt, 1977). Historical surveys providing information on the early research that has led to the elucidation of the structure and function of bacteriorhodopsin were given by Stoeckenius (1976, 1994). Most studies on light-driven proton pumps in the Halobacteriaceae have been performed with Halobacterium salinarum (a species that includes strains designated in the past as Halobacterium halobium). This is one of the few representatives of the family that produces massive amounts of purple membrane with bacteriorhodopsin. Another species that may produce large amounts of the pigment, probably also located in differentiated purple membrane, is Halorubrum sodomense (Oren, 1983a). Smaller amounts of retinal protein proton pumps have been identified in other genera within the Halobacteriaceae, but not all members of the family produce bacteriorhodopsin. Bacteriorhodopsin genes have been detected in all Haloarcula isolates tested (Kamekura et al., 1998; Kitajima et al., 1996; Otomo et al., 1992; Sugiyama et al., 1994; Tateno et al., 1994). Haloarcula isolates from the salterns of Baja California, Mexico and southern California ("Haloarcula californiae") showed both bacteriorhodopsin and halorhodopsin activities (Javor et al., 1982). The bacteriorhodopsin gene was further found in Halorubrum sodomense, Halorubrum coriense, and Halorubrum distributum (Kamekura et al., 1998), as well as in a number of as yet unidentified isolates from Australia and China (Li et al., 2000; Mukohata, 1994; Mukohata et al., 1988, 1991). A gene coding for bacteriorhodopsin has even been found in a halophile isolate (strain HT, JCM 9743) that never appears to express it. When this gene was placed under the promoter region of the bop gene in a bacteriorhodopsin-deficient host strain, a functional bacteriorhodopsin proton pump was produced (Kamekura et al., 1998). All these retinal proteins differ in their amino acid sequence from the Halobacterium bacteriorhodopsin, and they have therefore sometimes been designated by other names (archaerhodopsin from the Australian strains, cruxrhodopsin-1 of Haloarcula vallismortis and Haloarcula argentinensis). Comparative sequence studies of all these genes have been made to elucidate the taxonomic, the evolutionary, and the functional aspects of the rhodopsin family of pigments (Ihara et al., 1999; Mukohata, 1994, 1999). It has been speculated that first halophilic archaeal retinal pigment had the closest resemblance to Halobacterium bacteriorhodopsin (Mukohata, 1999). Halorubrum lacusprofundi and Halorubrum vacuolatum do not possess bacteriorhodopsin genes. Such genes are also absent in the genera Haloferax, Halococcus, Natronococcus, Natronobacterium, Natronomonas, and Natrialba (Kamekura et al., 1998). When bacteriorhodopsin is excited with light of a suitable wavelength, a complex photocycle results (Figures 5.7, 5.8). In the course of this photocycle the retinal group undergoes isomerization from the all-trans to the 13-cis isomer (Oesterhelt et al., 1973). Another important feature of the photocycle is the alternating deprotonation and protonation of the Schiff base between the retinal and lysine-216. A number of intermediates have been identified, designated K, L, N, and O, each with its

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characteristic absorption spectrum. Some of the conversions are reversible, others are irreversible reactions. The photocycle can be schematically written as:

(Lanyi, 1992; see also Figure 5.7). The cycle is complete after approximately 10 milliseconds with the re-establishment of the parent state of the molecule. During each turnover, one proton is transported across the cell membrane. The transition was in the past thought irreversible, but is nowadays considered to be in a "flickering" equilibrium between extracellular and cytoplasmatic accessibility of the Schiff base (see the in-depth discussion by Haupts et al., 1999).

The three-dimensional structure of bacteriorhodopsin in the purple membrane of

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Halobacterium salinarum was elucidated to 7 Å resolution already in 1975, based on electron microscopical data (Fourier analysis of defocused bright field micrographs and electron diffraction patterns) (Unwin and Henderson, 1975). The electron crystallographic model was later refined to 3.5 Å resolution (Grigorieff et al., 1996). The pathway of the proton transfer through the molecule in the course of the photocycle has now been elucidated at high resolution, based on crystallographic studies (electron cryomicroscopy, X-ray diffraction) and the examination of the properties of variants of the protein obtained by site-directed mutagenesis (Bickel-Sandkötter et al., 1996; Haupts et al., 1999; Krebs and Khorana, 1993; Lanyi, 1998, 1999a, 1999b, 1999c, 2000a, 2000b, 2000c; Luecke et al., 1998, 1999a, 1999b) (Figure 5.9). Efficient protocols for the production of recombinant bacteriorhodopsins have been developed (Ferrando et al., 1993; Ni et al., 1990), enabling the elucidation of the role of specific amino acids in the photocycle.

Site-directed mutagenesis studies have been performed to classify the amino acids of the bacteriorhodopsin protein into groups according to how their substitution affects proton pumping. Asp-85 serves as the acceptor for the proton from the isoinerized retinylidene Schiff base, and Asp-96 participates in the reprotonation of this group (Krebs and Khorana, 1993).

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Using X-ray diffraction of bacteriorhodopsin crystals, the three-dimensional structure of the molecule has now been resolved to a resolution of 1.55 Å, sufficient to show the location of water molecules bound within the protein. The structural changes during ion transport could be identified at 2 Å resolution (Luecke et al., 1999a). The

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three-dimensional structure shows a pore across the membrane in the center of the helix bundle of bacteriorhodopsin. The retinal chromophore protrudes into this watercontaining channel and controls the transport of protons through its conformational changes. Asp-85 (in dissociated form in the resting state of the molecule) is located on the extracellular side in close proximity to the chromophore. This aspartate acts as proton acceptor when the retinal molecule adopts the M-intermediate conformation. Asp-96 (in undissociated form) is located relatively far from the chromophore at the cytoplasmic side of the proton channel, and has been identified as the proton donor during reprotonation of the Schiff base. A network of interacting residues and bound water molecules at the inner wall of the channel bridges the 12 Å distance between the two Asp residues. The proton transport cycle can be described in terms of specific and wellunderstood displacements of hydrogen-bonded water and main-chain and side-chain atoms of the amino acid chain that lower the of the proton release group in the extracellular region and Asp-96 in the cytoplasmic region. Thus, local electrostatic conflict of the photoisomerized retinal with Asp-85 and Asp-212 causes deprotonation of the Schiff base, resulting in a cascade of events culminating in the release of a proton to the extracellular surface. Local steric conflict of the 13-methyl group with Trp-182 causes, in turn, a cascade of movements in the cytoplasmic region, which leads to reprotonation of the Schiff base. The directionality of the proton translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96. Bound water plays a crucial role in proton conduction in both the extracellular and the cytoplasmic regions. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. Elucidation of the crystal structure of bacteriorhodopsin in which Asp-96 has been mutated to Asn and the M intermediate was produced by illumination showed that displacements of the side chains near the retinal induced by the photoisomerization to 13-cis, 14-anti and the extensive rearrangement of the threedimensional network of hydrogen-bonded residues and bound water account for the changed values of the Schiff base and of Asp-96 (Luecke et al, 1999a). The crucial intermediate in the photocycle is the M state, the state in which the Schiff base is deprotonated, Asp-85 to the extracellular side is protonated, and a proton has been released to the surface, but Asp-96 to the cytoplasmic side has not yet become deprotonated. The sequential order of events during the photocycle can be described as: (1). deprotonation of the Schiff base, protonation of Asp-85; (2), proton release to the extracellular surface; (3), reprotonation of the Schiff base, deprotonation of Asp-96; (4), reprotonation of Asp-96 from the cytoplasmic surface, and (5), deprotonation of Asp-85, together with reprotonation of the proton release site. Comparison of the crystallographic structures of the M states in the D96N and E204Q mutants (the latter to 1.8 Å resolution) suggested that the retinal progressively relaxes after deprotonation

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of the Schiff base, and that its motions are coupled to displacements of protein groups and bound water (Luecke et al., 2000). A general model proposed by Haupts et al. (1997), named the Isomerization/Switch/Transfer model, describes the course of events, the switch (i.e. the change in accessibility of the relevant residues) being a time-dependent process in the millisecond time range. Electron crystallographic analyses of light-driven structural changes in wild-type bacteriorhodopsin and a number of mutants has shown that a significant conformational change occurs within 1 ms after illumination, coincident with the formation of the intermediate in the photocycle (Subramaniam and Henderson. 1999, 2000a, 2000b; Subramaniam et al., 1999, 2002). The synthesis of bacteriorhodopsin in Halohacterium salinarum is directed by the bop gene cluster. This cluster contains at least three genes: bop – the gene encoding the bacterio-opsin (the protein backbone of bacteriorhodopsin), brp – a bacterio-opsinrelated protein, and bat – the bacterio-opsin activator. Expression of the bop gene cluster is induced by low oxygen tension and by light. When grown under high oxygen tensions in the dark, the transcript levels of bop and bat were low during the exponential growth phase, and they increased about 29 and 45-fold, respectively, upon entering the stationary phase. The brp gene transcription level remained low during all stages of growth. Exposure of to high light intensities stimulated expression of all three genes, even in the presence of high oxygen levels (Betlach et al., 1984, 1989; Shand and Betlach, 1991). A regulatory model was proposed involving two different mechanisms: (1), expression of the bat gene is induced under conditions of low oxygen tension, whereafter the bat gene product activates expression of the bop gene, and (2). light induces transcription of brp, which stimulates or modulates bat transcription. Study of deletion mutants have shown that when the bop gene alone was present, purple membrane was synthesized constitutively. Induction by low oxygen requires the presence of the bop gene cluster. It was postulated that the bat gene encodes a transacting factor that is necessary and sufficient to confer inducibility of purple membrane synthesis by low oxygen tension (Betlach et al., 1989; Gropp and Betlach, 1994; Shand and Betlach, 1991). Mutants that express bacteriorhodopsin and produce purple membrane constitutively have been isolated (Juez and Rodriguez-Valera, 1984). The genes brp and blh are required for the synthesis of the retinal cofactor, Mutant strains affected in brp produce normal bacterio-opsin, and they contain levels about 3.8-fold of those found in the wild type. These observations suggest that the brp and the blh genes encode similar proteins that catalyze or regulate the conversion of carotene to retinal (Peck et al., 2001). The availability of the complete genomic sequence of Halobacterium salinarum has now enabled a detailed analysis of the genes involved in bacteriorhodopsin synthesis and its regulation. The production of the protein and the retinal moiety are coordinated in a regulon, dependent on light intensity and oxygen concentration (Baliga et al., 2001; DasSarma et al., 2001). Presence of bacteriorhodopsin in halophilic Archaea is of obvious ecological advantage, as it enables the cells to directly use light energy when organic energy sources are in short supply. Experiments in nutrient-limited chemostat cultures have shown that light can relieve energy starvation at low nutrient and low oxygen

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concentrations in Halobacterium cells that contain the pigment (Brock and Petersen, 1976). Under low oxygen, light enhanced growth rate and growth yields. It was thus suggested that the bacteriorhodopsin may be important in low-oxygen habitats (Rodriguez-Valera et al., 1983; Rogers and Morris, 1978). Anaerobic growth driven by light absorbed by bacteriorhodopsin is possible (Hartmann et al., 1980; Oesterhelt, 1982; Oesterhelt and Krippahl, 1983). Only very minute amounts of oxygen are required for sustained anaerobic growth with bacteriorhodopsin synthesis. In view of the obvious ecological advantage of bacteriorhodopsin as an alternative mode of energy generation in members of the Halobacteriaceae it is surprising that there are only few indications that natural communities of halophilic Archaea indeed may contain high concentrations of the pigment and that they use light to obtain energy in situ. Blooms of halophilic Archaea are generally colored pink-red due to their high content of bacterioruberin derivatives, rather than purple due to retinal pigments. Massive occurrence of bacteriorhodopsin was first reported in a natural community of halophilic Archaea in the Dead Sea in 1980-1982. The biomass collected from the lake had the typical purple color of bacteriorhodopsin, and concentrations of the pigment were estimated to be in the range of 0.6-0.7 nmol bacteriorhodopsin per liter in a prokaryote community of cells (Oren and Shilo, 1981). Cells resembling Halorubrum sodomense, a species known to produce purple membrane, were abundant in the community. It was also suggested that most of the photoassimilation that occurred in the lake at the time was driven by light absorbed by bacteriorhodopsin rather than by chlorophyll (Oren, 1983b) (see Sections 4.1.4 and 13.3). Javor (1983) found bacteriorhodopsin in high concentrations (2.2 in the archaeal community in the crystallizer ponds of an oligotrophic saltern of Exportadora de Sal, Guerrero Negro, Baja California, Mexico. Presence of bacteriorhodopsin in a natural community of halophilic Archaea was also documented in the Gavish sabkha, a shallow coastal hypersaline salt flat on the Sinai peninsula, Egypt. Flash spectroscopy of cells collected from the biomass collected from the site provided evidence for the presence of both bacteriorhodopsin and halorhodopsin activity (Stoeckenius et al., 1985). The above-cited reports are probably the only cases in which the presence of bacteriorhodopsin in the natural environment has been assessed qualitatively and quantitatively.

5.4.2. Halorhodopsin The second retinal protein, also first discovered in Halobacterium salinarum, is halorhodopsin. Its structure is quite similar to that of bacteriorhodopsin. However, this pigment acts as a chloride pump: excitation by light (absorption maximum 578 nm) drives transport of chloride ions from the medium into the cells (Schobert and Lanyi, 1982). Inward chloride transport is important for maintaining the proper ionic balance, and is essential for cell growth. The following short discussion of the properties of halorhodopsin should be supplemented by the far more in-depth treatises in specialized reviews (Lanyi, 1986, 1990; Oesterhelt, 1995, and others).

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Although the function of halorhodopsin as a chloride pump was only recognized in 1982 (Schobert and Lanyi, 1982), the presence of a second retinal protein in Halobacterium salinarum had been known for a number of years already (see Lanyi, 1990, and Oesterhelt, 1995 for historical information). The first indications for the existence of a second retinal pigment (although not recognized as such by the authors) can be found in a report by Kanner and Racker (1975), who worked with Halobacterium salinarum envelope vesicles for transport experiments. They observed light-induced extrusion of protons from the vesicles due to action of bacteriorhodopsin. However, upon addition of low concentrations of uncouplers the direction of proton movement was reversed. Matsuno-Yagi and Mukohata (1977) found that Halobacterium salinarum strain a red strain deficient in bacteriorhodopsin biosynthesis, was observed to pump protons inwards, leading to an alkalinization of the medium and an increase in cellular ATP levels. Light-dependent proton uptake and ATP formation in cells occurred even after the majority of the bacteriorhodopsin present had been bleached by treatment with hydroxylamine in the light. However, a short heat treatment abolished these processes (halorhodopsin is sensitive to heating for 5 min at 75 °C, a treatment that does not affect bacteriorhodopsin). The action spectrum of the proton pumping activity in strain in hydroxylamine-treated cells of strain or in cells grown in the presence of nicotine to inhibit carotenoid synthesis followed by reconstitution of the pigment with externally supplied retinal, showed maximum activity between 580-600 nm (Matsuno-Yagi and Mukohata, 1980). Such membranes show an absorption band at 588 nm (Lanyi and Weber, 1980). The presence of a second retinal pigment was also demonstrated when membrane fragments were separated in the ultracentrifuge. While the faster sedimenting bactcriorhodopsin-containing membrane vesicles excrete protons when excited by light, a more slowly sedimenting minor fraction of membrane vesicles of Halobacterium strain took up protons upon illumination, as do vesicles of a purplemembrane-depleted strain (Greene and Lanyi, 1979; MacDonald et al., 1979). At the time the effect was postulated to be due to the presence of a light-dependent electrogenic primary sodium pump rather than an inward-directed chloride pump (Lindlay and MacDonald, 1979). The final proof for the mode of action of halorhodopsin as a chloride pump was obtained when purified halorhodopsin was reconstituted in an artificial membrane system (Bamberg et al., 1984). The halorhodopsin content of Halobacterium salinarum strain is about 5 percent of the amount of bacteriorhodopsin in these cells. Halorhodopsin is probably more widely distributed than bacteriorhodopsin, although no systematic survey has yet been performed of the presence of halorhodopsin among the different genera and species within the Halobacteriaceae. Halorhodopsin has been detected in several haloalkaliphilic Archaea (Bivin and Stoeckenius, 1986), and the halorhodopsin of Natronomonas pharaonis has been studied in detail (Lanyi et al., 1990). Spectroscopic measurements provided evidence for the presence of halorhodopsin (and of bacteriorhodopsin as well) in a Haloarcula isolate (tentatively named "Haloarcula californiae" but never formally described) obtained from saltern crystallizer ponds in southern California and in Baja California, Mexico (Javor et al., 1982). Flash

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spectroscopic measurements with concentrates of cells collected from the Gavish sabkha on the shore of the Sinai peninsula also suggested presence of both retinal ion pumps (Stoeckenius et al., 1985). When excited, halorhodopsin undergoes a complex photocycle that is in all properties comparable to that of bacteriorhodopsin (Schobert et al., 1983). Lightdependent isomerization of the all-trans form of the bound retinal to the 13-cis form also occurs in halorhodopsin (Lanyi, 1984). Detailed flash photolysis and fast difference spectra measurements enabled the elucidation of the absolute spectra of five intermediates and and the first correct description of the photocycle (Tittor et al., 1987). The photocycle of the Natronomonas pharaonis halorhodopsin has been characterized in detail. Treatment with sodium azide transforms the Natronomonas halorhodopsin photocycle from a chloride-transporting one to a proton-transporting one, which is very similar to that of bacteriorhodopsin: after going through the K and L intermediates, the M intermediate is formed (an intermediate missing in the halorhodopsin photocycle) in which the retinal Schiff base deprotonates (Kulcsar et al., 2000). The structure of halorhodopsin is now known at high-resolution, thanks to studies involving electron cryo-microscopy of two-dimensional crystals followed by electron diffraction at the temperature of liquid nitrogen (resolution 6 Å) (Havelka et al., 1993), electron crystallography of two-dimensional crystals (resolution 5 Å) (Kunji et al., 2000), atomic force microscopy of two-dimensional crystals (14 Å resolution) (Persike et al., 2001), and X-ray diffraction (resolution 1.8 Å) (Kolbe et al., 2000). The molecule assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys-242 and the retinal chromophore, a single chloride ion occupies the transport site. The transport function of the two aspartates D-85 and D-96 that donate and accept protons in bacteriorhodopsin is replaced by two arginines (R-108 and R-200) which form the anion-binding sites. Halorhodopsin can act either as light-driven chloride pump or as a proton pump, depending on light conditions. Green light drives chloride transport, while addition of blue light induces proton pumping. In the living cell, both these vectorial processes would be directed toward the cytoplasm; compared to ion transport by bacteriorhodopsin, this represents an inversed proton flow. Azide, which catalyzes a reversible deprotonation of halorhodopsin, enhances proton transport (Bamberg et al., 1993). On the basis of a comparative study of bacteriorhodopsin and halorhodopsin, Oesterhelt et al. (1992) have formulated a unifying concept for the mechanism of ion translocations by retinal proteins. Structural changes in the protein molecule lead to a change in ion affinity and accessibility, and this change determines the vectoriality of the ion transport. The light-triggered switch between the cis and the trans form or between the trans and the cis form is the key element for vectorial transport, while ion binding sites (at least in the case of bacteriorhodopsin) appear to serve the main purpose of making the proton pump optimally efficient and pH-independent. Halorhodopsin is not the only inward chloride pump present in halophilic Archaea. Halobacterium salinarum also possesses a light-independent chloride transporter,

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which probably acts by coupling the inward transport of chloride with the influx of sodium ions (Duschl and Wagner, 1986). No information is available on the relative importance of the two chloride pump in the ion metabolism of Halobacterium cells and on the factors that regulate the synthesis of the halorhodopsin chloride pump.

5.4.3. Sensory rhodopsins and signal transduction to the flagellar motor Halobacterium salinarum contains in addition to bacteriorhodopsin and halorhodopsin two more retinal-containing membrane proteins, and these are involved in light sensing for phototaxis. Sensory rhodopsin I is a green light receptor that responds to a color to which the cells are attracted. Sensory rhodopsin II, also termed phoborhodopsin, is sensitive to blue light that acts as a repellent. The amount of sensory rhodopsins in the cell membrane is small, and the presence of these pigments does not contribute significantly to the pigmentation of the cells. However, they are discussed here because of their structural resemblance to bacteriorhodopsin and halorhodopsin. The identification of a third rhodopsin-like protein in Halobacterium salinarum in addition to bacteriorhodopsin and halorhodopsin was first reported in 1982 (Bogomolni and Spudich, 1982), although the involvement of a retinal protein in phototaxis had already been suggested a few years earlier, when it was shown that presence of nicotine in the growth medium abolished phototactic behavior, and that phototaxis could be restored by addition of retinal to the medium (Dencher and Hildebrand, 1979; Hildebrand and Dencher, 1975; Sperling and Schimz, 1980; Spudich and Stoeckenius, 1979). Bogomolni and Spudich (1982) described a sensory pigment in which two photoactive states with absorption maxima at 580-590 nm and at 373 nm alternate. Illumination of the long wavelength species generated the 373-nm intermediate, which was found to convert back to the ground state with a half-life time of about one second. Reviews on the properties of the sensory rhodopsins were given by Bickel-Sandkötter et al. (1996), Spudich (1993), Spudich and Bogomolni (1984, 1988), and Spudich et al. (1995). A systematic survey of the presence of sensory rhodopsins in the different genera and species of the Halobacteriaceae has never been made. It is clear that synthesis of such photosensors would be useful only for those members of the family that are motile. Sensory rhodopsin II is present in of Natronomonas pharaonis (Hirayama et al., 1992; Kunji et al., 2001; Tomioka and Sasabe, 1995). In-depth characterizations of the sensory rhodopsin II of Halobacterium salinarum have been published (Seidel et al., 1995; Zhang et al., 1996), and the structure of Halobacterium salinarum and Natronomonas pharaonis sensory rhodopsin II has been resolved by X-ray diffraction to 2.1-2.4 Å (Luecke et al., 2001; Royant et al., 2001). The action spectrum of the phototactic behavior of Halobacterium salinarum (repulsion by blue and long-wavelength ultraviolet radiation, attraction by green-red light) corresponds with the absorption spectrum of the sensory rhodopsins, the photoreceptor for negative phototaxis being the long-lifetime intermediate in the photocycle of sensory rhodopsin II (Takahashi et al., 1985; see also Bogomolni and

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Spudich, 1982). The primary reactions of the sensory rhodopsins follow the reaction schemes of bacteriorhodopsin and halorhodopsin (Lutz et al., 2001), and they undergo similar photocycles. However, these photocycles are much slower than those of the specialized light-driven proton and chloride pumps: the photocycles of the latter are completed within tens of milliseconds, while the sensory rhodopsin photocycle takes seconds to complete. Sensory rhodopsin II takes up a proton in M-to-O conversion and releases it during O decay. The uptake and release are from and to the extracellular side, and thus no protons are transported during this photocycle (Sasaki and Spudich, 1999). The photocycle of phoborhodopsin of Halobacterium salinarum (absorption maximum of the ground state 490 nm) has been characterized by low temperature spectrophotometry (Imamoto et al., 1991). The intermediates and probably correspond to the K, M, and O intermediates of bacteriorhodopsin on the basis of their absorption spectra; no intermediates corresponding to the L and N intermediates of the bacteriorhodopsin photocycle have been identified. The photocycle of the Natronomonas pharaonis phoborhodopsin (absorption maximum of the ground state 498 nm) has been characterized by flash photolysis (Miyazaki et al., 1992). Intermediates with absorption maxima at 390 and 550 nm and correspond to the M and O intermediates of bacteriorhodopsin. The structure of Natronomonas pharaonis phoborhodopsin has been resolved to 6.9 Å resolution by electron microscopical analysis of two-dimensional crystals (Kunji et al., 2001). The positions of the transmembrane helices match the seven-helix arrangement of bacteriorhodopsin and halorhodopsin. Distinctive properties of sensory rhodopsin II of Natronomonas pharaonis, as compared with bacteriorhodopsin and halorhodopsin, are the largely unbent conformation of the retinal, the outward orientation of the guanidinium group of Arg-72, and the presence of a positively charged surface path for a probable interaction with its transducer protein HtrII (sec below) (Royant et al., 2001; Luecke et al., 2001). The changes in conformation that occur in the transducer protein upon illumination have been determined by probing disulfide formation from introduced cysteines in adjacent helices. (Yang and Spudich, 2001). The light sensing functions of the sensory rhodopsins enable the cells to actively position themselves in an optimal light environment. Halobacterium salinarum cells accumulate in a spot of 565 nm light on a dark background, as the light spot acts as a trap for cells entering randomly. When a background light field of 392 nm is supplied, photoaccumulation is abolished (Dencher, 1978). A decrease in the light intensity of the spot leads to increased photoaccumulation, an increase in photodispersal (Nultsch and Häder, 1978). The action spectra of these phenomena correspond with the absorption spectra of the two sensory rhodopsins (Hildebrand and Dencher, 1975). Carotenoids have been suggested to participate as accessory pigments in the sensory rhodopsin II reaction (Dencher, 1978; Dencher and Hildebrand, 1979). The sensory rhodopsins are the first link in a chain of signal transduction, from the light sensor to the flagellar motor. The second component in this chain is formed by the methyl-accepting transducers (HtrI and HtrII), which are located in the cell membrane and form stable complexes with sensory rhodopsin I and II, respectively

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(Krah et al., 1994; Spudich et al., 1995). The methylation sites in these proteins have been identified by site-directed mutagenesis (Perazzona and Spudich, 1999). A balance in the SRI/HtrI and SRII/HtrII signaling system maintains the cells in a spectrum of light optimal for light-mediated energy production. In both systems the receptor (SRI and SRII) senses the appropriate stimulus and propagates the signal to two distinct sites in the transducer (HtrI and HtrII). The first site to receive the signal is the signaling domain, which causes an alteration of autophosphorylation of a bound histidine kinase (CheA). The CheA histidine kinase of Halobacterium salinarum was the first such transmitter recognized in the Archaea. Deletion of part of of its gene leads to loss of both phototactic and chemotactic responses (Rudolph and Oesterhelt, 1995). The second site is the methylation site which undergoes a conformational change altering its susceptibility to methylation. A balance in methylation and demethylation of this site resets the activity of CheA, which modulates the activity of the cytoplasmic motility regulator protein CheY by controlling its phosphorylation state. The phototactic system mediated by sensory rhodopsin I and II and their respective membrane-bound transducer proteins makes part of a more extensive systems of sensors and signal transduction systems that integrate the signals received from the environment and govern the direction of movement of the cells by modulating the flagellar motor. The membrane also contains chemosensors that monitor the presence of nutrients (see also Section 4.1.3). The information transferred to the cytoplasmic components of the signal transduction machinery by these chemosensors also influences the behavior of the cell (Kokoeva and Oesterhelt, 2000). The frequency of spontaneous reversal of the flagellar rotation direction is modulated both by changes in attractant and repellent light intensity and by concentrations of chemicals. The integrated signal from all photosensory and chemosensory systems modulates reversal of swimming direction (Spudich and Stoeckenius, 1979). The methylation and demethylation reactions connected with the processing of phototactic and chemotactic signals in Halobacterium salinarum have been studied indepth. When the cells are stimulated by an attractant, the level of methylation of the proteins of the sensory transduction system rises, the methyl groups added being derived from methionine; repellants cause demethylation (Sehimz, 1981; Spudich et al., 1989). Calcium ions regulate the methyltransferase and methylesterase activities: methylation in vitro is inhibited by calcium, demethylation is activated (Schimz, 1982). Following demethylation the methyl groups are released in the form of methanol. Release of methanethiol (methylsulfide) observed in Halobacterium salinarum is not connected with phototaxis or chemotaxis phenomena (Nordmann et al., 1994).

5.5. THE PHOTOACTIVE YELLOW PROTEIN OF HALORHODOSPIRA AND OTHER HALOPHILIC PURPLE BACTERIA Another interesting photopigment used for light sensing is present in many halophilic anoxygenic phototrophic Bacteria. This pigment, known as the photoactive yellow protein, was first observed in Halorhodospira halophila in 1985 (Meyer, 1985). The

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pigment is a water soluble, very stable protein of 14 kDa molecular mass, and it shows a broad absorption maximum around 446 nm. The protein is a photoreceptor that mediates negative phototaxis to blue light (Hendriks et al., 1999). The photoactive yellow protein of Halorhodospira halophila is located in the cytoplasm. Similar proteins have been detected in other halophilic photosynthetic bacteria such as Rhodothalassium salexigens and Halochromatium salexigens (Thiemann and Imhoff, 1995). The protein from Halorhodospira halophila shows immunological crossreactions with the protein from other photosynthetic bacteria (Hoff et al., 1995).

The photochemistry of the Halorhodospira photoactive yellow protein has certain points of resemblance with that of the sensory rhodopsins of the halophilic Archaea. However, it was rapidly recognized that the protein's chromophore is not identical with retinal (McRee et al., 1989; Meyer et al., 1987). It was later identified as a 4hydroxycinnamyl (p-coumaric acid) chromophore (Figure 5.10) (Hoff et al., 1994). It is covalently bound to Cys-69 of the protein via a thiol ester bond. Upon excitation the photoactive yellow protein undergoes a photocycle that involves transient proton

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uptake and release. When excited with a laser flash at 445 nm, the pigment is bleached with a red shift in its absorption spectrum in less than 10 nanoseconds. Further bleaching occurs in about 200 microseconds, whereafter the original color reappears in the next 200 milliseconds. One proton is taken up during the formation of the fully bleached second intermediate, and an equivalent proton release takes place upon return to the ground state (Meyer et al., 1987, 1993). During this photocycle also a isomerization takes place of the 4-hydroxycinnamyl chromophore of the photoactive yellow protein in its pB state (Hendriks et al., 1999; Hoff et al., 1996). Mutant proteins have been used to probe which amino acids interact with the chromophore (Imamoto et al., 2001). The step critical to entry in the photocycle was identified as the flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail photocycle, as shown by monitoring of the changes that occur in crystals of the protein following a 7 ns laser pulse (Ren et al., 2001). The three-dimensional structure of the photoactive yellow protein has been resolved by X-ray diffraction to 2.4 Å resolution (McRec et al., 1989). The molecule is composed of two perpendicular that form a so-called structure (Hoff et al., 1995; McRee et al., 1989). The structure of the photoactive yellow pigment exhibits all the major structural and functional features characteristic of the PAS domain. PAS domain sequences are found in many multidomain protein sensors and transcription factors involved in signal transduction. They contain a shared modular domain fold of ~ 125-150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly. The photoactive yellow protein has now become a paradigm for PAS domains (Pellequer et al., 1998).

5.6. REFERENCES Aasen, A.J., Eimhjellen, K.E., and Liaaen-Jensen, S. 1969. An extreme source of Acta Chem. Scand. 23: 2544-2545. Asker, D., and Ohta, Y. 1999. Production of canthaxanthin by extremely halophilic bacteria. J. Biosci. Bioengin. 88: 617-621. Asker, D., and Ohta, Y. 2002. Haloferax alexandrinus sp. nov., an extremely halophilic canthaxanthinproducing archaeon from a solar saltern in Alexandria (Egypt). Int. J. Syst. Evol. Microbiol., in press. Asker, D., Awad, T., and Ohta, Y. 2002. Lipids of Haloferax alexandrinus strain TMT: an extremely halophilic canthaxanthin-producing archaeon. J. Biosci. Bioengin. 93: 37-43. Baliga, N.S., Kennedy, S.P., Ng, W.V., Hood, L., and DasSarma, S. 2001. Uenomic and genetic dissection of an archaeal regulon. Proc. Natl. Acad. Sci. USA 98: 2521-2525. Bamberg, E., Tittor, J., and Oesterhelt, D. 1993. Light-driven proton or chloride pumping by halorhodopsin. Proc. Natl. Acad. Sci. USA 90: 639-643. Bamberg, E., Hegemann, P., and Oesterhelt, D. 1984. The chromoprotein of halorhodopsin is the light-driven electrogenic chloride pump in Halobacterium halobium. Biochemistry 23: 6216-6221. Béjà, O., Spudich, E.N., Spudich, J.L., Leclere, M., and DeLong. E.F. 2000. Proteorhodopsin in the ocean. Nature 411: 786-789. Béjà, O., Arvind, L., Koonin, R.V., Suzuki, M.T., Hadd, A.. Nguyen, L.P., Jovanovich, S., Gates, C.M., Feldman, R.A., Spudich, J.L., Spudich, E.N., and DeLong, E.F. 2001. Bacterial rhodopsin: evidence for a new type of photosynthesis in the ocean. Science 289: 1902-1906.

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Ben-Amotz, A. 1987. Effect of irradiance and nutrient deficiency on the chemical composition of Dunaliella bardawil Ben-Amotz and Avron (Volvocales, Chlorophyta). J. Plant Physiol. 131: 479-487. Ben-Amotz, A. 1999. Dunaliella From science to commerce, pp. 401-410 In: Seckbach, J. (Ed.), Enigmatic microorganisms and life in extreme environments. Kluwer Academic Publishers, Dordrecht. Ben-Amotz, A., and Avron, M. 1981. Glycerol and metabolism in the halotolerant alga Dunaliella: a model system for biosolar energy conversion. Trends Biochein. Sci. 6: 297-299. Ben-Amotz, A., and Avron, M. 1983. On the factors which determine massive accumulation in the halotolerant alga Dunaliella bardawil. Plant Plysiol. 72: 593-597. Ben-Amotz, A., Katz, A., and Avron, M. 1982. Accumulation of in halotolerant algae: purification and characterization of globules from Dunaliella bardawil (Chlorophyceae). J. Phycol. 18: 520537. Ben-Amotz, A., Lers, A., and Avron, M. 1988. Stereoisomers of and phytoene in the alga Dunaliella bardawil. Plant Physiol. 86: 1286-1291. Ben-Amotz, A., Shaish, A., and Avron, M. 1989. Mode of action of the massively accumulated of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol. 91: 10401043. Betlach, M., Friedman, J., Boyer, H.W., and Pfeifer, F. 1984. Characterization of a halobacterial gene affecting bacterio-opsin gene expression. Nucleic Acids Res. 12: 7949-7959. Betlach, M.C., Shand, R.F., and Leong, D.M. 1989. Regulation of the bacterio-opsin gene of a halophilic archaebacterium. Can. J. Microbiol. 35: 134-140. Bickel-Sandkötter, S., Gärtner, W., and Dane, M. 1996. Conversion of energy in halobacteria: ATP synthesis and phototaxis. Arch. Microbiol. 166: 1-11. Bivin, D.B., and Stoeckenius, W. 1986. Photoactive retinal pigments in haloalkaliphilic archaebacteria. J. Gen. Microbiol. 132: 2167-2177. Bogomolni, R.A., and Spudich, J.L. 1982. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proc. Natl. Acad. Sci. USA 79: 6250-6254. Borowitzka, L.J. 1981. The microflora. Adaptations to life in extremely saline lakes. Hydrobiologia 81: 33-46. Brock, T.D., and Petersen, S. 1976. Some effects of light on the viability of rhodopsin-containing halobacteria. Arch. Microbiol. 109: 199-200. Calo, P., de Miguel, T., Sieiro, C., Velazquez, J.B., and Villa, T.G. 1995. Ketocarotenoids in halobacteria: 3hydroxy-echinenone and trans-astaxanthin. J. Appl. Bacteriol. 79: 282-285. Caumette, P. 1993. Ecology and physiology of phototrophic bacteria and sulfate-reducing bacteria in marine salterns. Experientia 49: 473-481. Caumette, P., Matheron, R., Raymond, N., and Relexans, J.-C. 1994. Microbial mats in the hypersaline ponds of Mediterranean salterns (Salins-de-Giraud, France). FEMS Microbiol. Ecol. 13: 273-286. Cowan, A.K., Logie, M.R.R., Rose, P.D., and Phillips, L.G. 1995. Stress induction of zeaxanthin formation in the alga Dunaliella salina Teod. J. Plant Physiol. 146: 554-562. Danon, A,, and Stoeckenius, W. 1974. Photophosphorylation in Halobacterium halobium. Proc. Natl. Acad. Sci. USA71: 1234-1238. DasSarma, S., Kennedy, S.P., Berquist, B., Ng, W.V., Baliga, N.S., Spudich, J.L., Krebs, M.P., Eisen, J.A., Johnson, C.H., and Hood, L. 2001. Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon. Photosynth. Res. 70: 3-17. Dencher, N.A. 1978. Liglit-induced behavioural reactions of Halobacterium halobium: evidence for two rhodopsins acting as photopigments, pp. 67-85 In: Caplan, R.S., and Ginzburg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam. Dencher, N.A., and Hildebrand, E. 1979. Sensory transduction in Halobacterium halobium: retinal protein pigment controls UV-induced behavioral response. Z. Naturforsch. 34c: 841-847. D'Souza, S.E., Altekar, W., and D'Souza, S.F. 1997. Adaptive response of Haloferax mediterranei to low concentrations of NaCl (<20%) in the growth medium. Arch. Microbiol. 168: 68-71. Dundas, I.D., and Larsen, H. 1962. The physiological role of the carotenoid pigments of Halobacterium salinarium. Arch. f. Mikrobiol, 44: 233-239. Dundas, I.D., and Larsen, H. 1963. A study on the killing by light of photosensitized cells of Halobacterium salinarium. Arch. f. Mikrobiol. 46: 19-28. Duschl, A., and Wagner, G. 1986. Primary and secondary chloride transport in Halobacterium halobium. J. Bacteriol. 168: 548-552. Ebenezer, W.J.. and Pattenden, G. 1993. cis-Stereoisomers of and its congeners in the alga Dunaliella

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Schmidt, K., and Trüper, H.G. 1971. Carotenoid composition in the genus Ectothiorhodospira Pelsh. Arch. f. Mikrobiol. 80: 38-42. Schobert, B., and Lanyi, J.K. 1982. Halorhodopsin is a light-driven chloride pump. J. Biol. Chem. 257: 1030610313. Schobert, B., Lanyi, J.K., and Cragoe, E.J., Jr. 1983. Evidence for a halide-binding site in halorhodopsin. J. Biol. Chem. 258: 15158-15164. Seidel, R., Scharf, B., Gautel, M., Kleine, K., Oesterhelt, D., and Engelhard, M. 1995. The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II. Proc. Natl. Acad. Sci. USA 92: 3036-3040. Shahmohammadi, H.R., Asgarani, E., Terato, H., Saito, T., Ohyama, Y., Gekko. K., Yamamoto, O., and Ide, H. 1998. Protective roles of bacterioruberin and intracellular KC1 in the resistance of Halobacterium salinarium against DNA-damaging agents. J. Radiat. Res. 39: 251-262. Shand, R.F., and Betlach, M.C. 1991. Expression of the bop gene cluster of Halobacterium halobium is induced by low oxygen tension and by light. J. Bacteriol. 173: 4692-4699. Sperling, W., and Schimz, A. 1980. Photosensory retinal pigments in Halobacterium halobium. Biophys. Struct. Mech. 6: 165-169. Spudich, J.L. 1993. Color sensing in the Archaea: a eukaryotic-like receptor coupled to a prokaryotic transducer. J. Bacteriol. 175: 7755-7761. Spudich, J.L., and Bogomolni, R.A. 1984. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature 312: 509-513. Spudich, J.L., and Bogomolni, R.A. 1988. Sensory rhodopsins of halobacteria. Ann. Rev. Biophys. Biophys. Chem. 17: 193-215. Spudich, J.L., and Stoeckenius, W. 1979. Photosensory and chemosensory behavior of Halobacterium halobium. Photobiochem. Photobiophys. 1: 43-53. Spudich, E.N., Takahashi, T., and Spudich, J.L. 1989. Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis. Proc. Natl. Acad. Sci. USA 86: 7746-7750. Spudich, J.L., Zacks, D.N., and Bogomolni, R.A. 1995. Microbial sensory rhodopsins: photochemistry and function. Israel J. Chem. 35: 495-513. Stoeckenius, W. 1976. The purple membrane of salt-loving bacteria. Sci. Am. June 1966: 38-46. Stoeckenius, W. 1994. From membrane structure to bacteriorliodopsin. J. Membr. Biol. 139: 139-148. Stoeckenius, W., and Bogomolni, R.A. 1982. Bacteriorhodopsin and related pigments of halobacteria. Ann. Rev. Biochem. 51: 587-616. Stoeckenius, W., Bivin, D., and McGinnis, K. 1985. Photoactive pigments in halobacteria from the Gavish sabkha, pp. 288-295 In: Friedman, G.M., and Krumbein, W.E. (Eds.), Hypersaline ecosystems. The Gavish sabkha. Springer-Verlag, Berlin. Subramaniam, S., and Henderson, R. 1999. Electron crystallography of bacteriorhodopsin with millisecond time resolution. J. Struct. Biol. 128: 19-25. Subramaniam, S., and Henderson, R. 2000a. Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle. Biochim. Biophys. Acta 1460: 157-165. Subramaniam, S., and Henderson, R. 2000b. Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406: 653-657. Subramaniam, S., Lindahl, M., Bullough, P., Faraqui, A.R., Tittor, J., Oesterhelt, D., Brown, L., Lanyi, J.K., and Henderson, R. 1999. Protein conformational changes in the bacteriorhodopsin photocycle. J. Mol. Biol. 287: 145-161. Subramaniam, S., Hirai, T., and Henderson, R. 2002. From structure to mechanism: electron Crystallographic studies of bacteriorhodopsin. Phil. Trans. R. Soc. London A 360: 859-874. Sugiyama, Y., Yamada, N., and Mukohata, Y. 1994. The light-driven pump, cruxrhodopsin-2 in Haloarcula sp. arg-2 (bR+, hR-), and its coupled ATP formation. Biochim. Biophys. Acta 1188: 287-292. Sumper, M., Reitmeier, H., and Oesterhelt, D. 1976. Zur Biosynthese der Pupermembran von Halobakterien. Angew. Chem. 88: 203-210. Takahashi, T., Watanabe, M., Kamo, N., and Kobatake. Y. 1985. Negative phototaxis from blue light and the role of third rhodopsinlike pigment in Halobacterium cutirubrum. Biophys. J. 48: 235-240. Takaichi, S., Maoka, T., Hanada, S., and Imhoff, J.F. 2001. Dihydroxylycopene diglucoside diesters: a novel class of carotenoids from the phototrophic purple sulfur bacteria Halorhodospira abdelmalekii and Halorhodospira halochloris. Arch. Microbiol. 175: 161-167. Tateno, M.. Ihara. K., and Mukohata, Y. 1994. The novel ion pump rhodopsins from Haloarcula form a family

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CHAPTER 6 INTRACELLULAR SALT CONCENTRATIONS AND ION METABOLISM IN HALOPHILIC MICROORGANISMS

In all species the concentration was much higher than in the medium, although it was lower than the cell concentration in two instances. The value found in H. salinarium was close to the limit of solubility of KCl. (Christian and Waltho, 1962, on

Halobacterium salinarum) .... adaptation at the cellular level, in a mechanism that is probably energy-dependent that maintains the intracellular salt concentration at a level considerably below that of the environment. (Baxter and Gibbons, 1954, on Halomonas halodemtrificans)

6.1. INTRODUCTION Biological membranes are permeable to water. No microorganism living at high salt concentrations can therefore maintain a hypoosmotic intracellular environment relative to the osmotic pressure of the medium in which it lives. This is true also for halophilic microorganisms that thrive in environments characterized by high osmotic pressures. When in addition a turgor pressure is to be maintained, the cytoplasm should even be kept hyperosmotic with respect to the outer medium. No significant turgor pressure is present in halophilic Archaea of the family Halobacteriaceae (see Section 3.1.1), but other halophilic microorganisms may be expected to have a turgor pressure similar to that of their non-halophilic counterparts. Osmotic adjustment or osmoadaptation (terms to be preferred over the much used "osmoregulation") (Reed, 1984) can be achieved in several ways. Different strategies have evolved that enable halophiles to cope with the osmotic stress exerted by their hypersaline environment. One option is to accumulate inorganic ions intracellularly to provide the osmotic balance with the high salt concentration of the medium. This chapter will explore the intracellular ionic concentrations and ionic composition of a variety of halophilic microorganisms, archaeal, bacterial, as well as eukaryotic. Only a few groups of halophiles use inorganic ions as sole or principal osmotic stabilizers. This strategy is used by the aerobic Archaea of the family Halobacteriaceae and by the anaerobic Bacteria of the order Halanaerobiales. It was recently discovered that also the aerobic red extremely halophilic bacterial genus Salinibacter uses inorganic salts to provide osmotic balance. Potassium rather than sodium is the main intracellular cation

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in all cases, and chloride is used as the dominant intracellular anion. Presence of molar concentrations of inorganic ions requires special adaptations of the entire intracellular enzymatic machinery, as all proteins should remain soluble and retain their activity in a high salt environment. The special properties of such salt-adapted proteins are discussed in Chapter 7. The "salt-in" strategy permits little flexibility and adaptability to changing conditions, as most salt-adapted enzymes and structural proteins require the continuous presence of high salt for activity and stability. Another option, much more widespread in nature, is the use of organic "compatible" solutes to increase the osmotic pressure of the cells' cytoplasm to provide osmotic balance and to enable the establishment of a proper turgor pressure. The variety of organic solutes used for this purpose in the different groups of halophiles will be explored in Chapter 8. General overviews of the issues concerned with osmotic adaptation and use of different ions and/or organic solutes to achieve optimal osmotic conditions enabling growth up to the highest salt concentrations can be found in a number of review papers and books (e.g. Bayley and Morton, 1978; Brown, 1976, 1990; Csonka, 1989; Kushner, 1978; Lanyi, 1974; Oren, 1999; Ventosa et al., 1998; Vreeland, 1987).

6.2. METHODS USED TO ESTIMATE INTRACELLULAR IONIC CONCENTRATIONS IN HALOPHILIC MICROORGANISMS The assessment of the nature of the true intracellular environment of halophilic microorganisms depends on our ability to obtain reliable measurements of the concentrations of inorganic ions and other compounds within the cells' cytoplasm. Such measurements are generally far from easy, and many of the controversies that exist in the literature on this issue can be tracked down to possible methodological problems. Kushner (1988) has devoted an excellent discussion to this issue. One of the recurring problems when attempting to calculate intracellular ionic concentrations is the reliable estimation of the cell volume. Intracellular solute concentrations are generally determined by analyses of ions in extracts of cell pellets. The fraction of the pellet volume that is occupied by the intracellular space has therefore to be accurately assessed. To discriminate between intracellular and extracellular volumes, radioactive marker molecules are often used. or labeled ethylene glycol can be employed to label the total water space. When in addition membrane-impermeable molecules labeled e.g. with are added, information can be obtained on the proportion of the water space contained inside and outside the cells (Imhoff, 1993). Cell-impermeable markers that have become popular in this type of studies include inulin (Hamaide et al., 1983; Matheson et al., 1976; Shindler et al., 1977; Vreeland et al., 1983), dextran (De Médicis et al., 1986; Miguelez and Gilmour, 1994; Sadler et al., 1980), and blue dextran (Kamekura and Onishi, 1982). Accurate concentration estimations using this approach are often difficult, especially in the case of and which are present at very high concentrations in the media used to grow halophiles: small errors in the determination of the size of the intracellular and extracellular water spaces result in large errors in the apparent concentrations of these ions. The presence of a periplasmic space in Gram-negative type Bacteria provides an additional complication. Reliable methods to differentiate between the periplasmic

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space and the osmotically active cytoplasmic space are still lacking (Imhoff, 1993). Use of small molecules such as raffinose (Hamaide et al., 1983) or sorbitol or sucrose (Shindler et al., 1977) has been attempted for this purpose, compounds expected to penetrate the outer membrane and to become distributed in the periplasmic space as well. The volume of the periplasmic space may be far from being negligible: in Salinivibrio costicola up to 38% of the total cellular volume was estimated to be occupied by the periplasmic space (Shindler et al., 1977). It should also be taken into account that the validity of the calculations depends on the (not necessarily true) assumption that the molecules used as markers are neither taken up and metabolized by the cells, nor become bound to the envelopes and to other cellular structures. Dextran and inulin may indeed bind to cell envelopes. Smaller polysaccharide fragments that can be present in dextran preparations may be taken up by the cells, and this may also lead to large errors in the calculation of the apparent cytoplasmic solute concentrations. A simple comparative approach, based on the expression of concentrations of intracellular ions or organic solutes relative to cellular protein is sometimes convenient (Antón et al., 2002; Pérez-Fillol and Rodríguez-Valera, 1986). However, it should be kept in mind that the ratio between cellular volume and cell protein may not be constant. This ratio may even depend on the salinity of the medium. Thus, in Halomonas elongata the cellular volume per unit of protein was found to be inversely related to salinity, decreasing from 2.62 to per mg of protein in cells grown from 10 to NaCl (Miguelez and Gilmour, 1994). A similar phenomenon was observed in Chromohalobacter canadensis: cells grown in NaCl had a volume of permgprotein,decreasing to 2.69 µl per mg protein in NaClgrown cells (Matheson et al., 1976). Another potential problem when estimating intracellular salt and solute concentrations in cell pellets is the stress exerted on the cells during the centrifugation step that is generally included in the procedure. Anaerobic conditions rapidly develop in the densely packed cells during handling and washing, and this may lead to a loss of substantial amounts of potassium and a gain in the sodium in those halophiles that depend on oxygen for their energy metabolism (Shindler et al., 1977). Variations in harvesting and handling procedures may therefore explain the differences in the estimates of the intracellular ion concentrations reported by different authors (see e.g. the data for Halomonas halodenitrificans presented by different authors (Christian and Waltho, 1962; Sadler et al., 1980), as given in Table 6.2. Centrifugation of cells through a layer of silicone oil to separate intracellular and extracellular water may bypass some of the problems involved in conventional centrifugation techniques. Oils of different densities must then be used to adjust for the density of the cells, which in its turn depends on the salt concentration of the medium. This approach has not been widely applied in the study of the intracellular salt and solute concentrations in halophilic microorganisms. The phase of growth may also have a major influence on the results of the internal ion concentration measurements: stationary phase cells often show much higher intracellular sodium concentrations than do exponentially growing cells. This may also explain some of the high apparent intracellular sodium concentrations reported in the literature.

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A different approach used to estimate intracellular ion concentrations is X-ray microanalysis in the electron microscope. In this method, the ion content and cell volume are estimated in single cells collected on an electron microscope grid (Oren et al., 1997). Stressful handling of the cells is minimal, and the method has the additional advantage that the extent of variation of the parameters measured can be assessed in a large number of single cells, this in contrast to the analysis of cell pellets, in which only mean concentrations are obtained. A possible artifact of the electron microscopic procedure may be caused by the flattening of the cells during sample preparation, which may distort cell shape and cause erroneous calculations of the true cell volume. NMR-based techniques have been used to probe the concentrations of intracellular sodium ions in halophilic microorganisms (Gilboa et al., 1991; Goldberg and Gilboa, 1978; Melamud et al., 1981; Nagata et al., 1995; Sakhnini and Gilboa, 1993). The approach also provides information on the state of binding of the intracellular sodium ions.

6.3. ION METABOLISM IN THE HALOBACTERIACEAE 6.3.1. Measurements of intracellular ion concentrations Analyses of intracellular ion concentrations in different aerobic halophilic Archaea of the family Halobacteriaceae show that these microorganisms maintain extremely high salt concentrations inside their cells. Potassium and chloride are the main intracellular cations, and the intracellular sodium concentrations are maintained at a level much below that of the outside medium (Table 6.1). A survey including Haloferax volcanii. Haloferax mediterranei and Haloferax gibbonsii in addition to Halobacterium salinarum yielded estimates of intracellular concentrations between 1.9 and 5.5 M, depending on the species and the growth stage of the cultures (Pérez-Fillol and Rodríguez-Valera, 1986). Potassium was found to make up at least 30% of the dry weight of Halobacterium salinarum; cultures that contained less were not viable (Goclmauer and Kushner, 1971). To examine to what extent potassium and other intracellular ions are present in a freely dissolved state in the cytoplasm or whether the potassium may be bound, calorimetry studies have been performed with Halobacterium salinarum, measuring the heat of dilution of the cytoplasm (Brown and Duong, 1982) and examining the freezing transitions by differential scanning calorimetry (Brown and Sturtevant, 1980). No evidence was obtained for the presence of an unusual state of the water inside the cells. It was therefore assumed that at least most of the intracellular ions are in a freely dissolved state. NMR measurements, however, suggested that the intracellular sodium ions may be bound to a significant extent. studies in Halobacterium salinarum provided evidence for the existence of three types of extracellular present in free solution, present between the cell wall and the cytoplasmic membrane, and intracellular bound which is undetectable by NMR. The intracellular concentration in resting cells was estimated at 1.9 M (Melamud et al., 1981). Conflicting reports exist on the state of binding of intracellular potassium. It

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has been claimed that in Haloarcula marismortui most of the intracellular was tightly and specifically bound to the cell matrix (M. Ginzburg, 1978, Ginzburg et al., 1971). In Haloferax volcanii only about half of the intracellular could be exchanged (Meury and Kohiyama, 1989). However, no bound was detected in lysates of Halobacterium salinarum cells, which suggests that in this organism most of the internal is osmotically active and is held inside only due to the properties of the cell membrane (Lanyi and Silverman, 1972).

It is obvious that the huge potassium concentration gradient over the cytoplasmic membrane (often up to three orders of magnitude) and the generally large sodium gradient present as well can only be created and maintained at the expense of energy. The following sections describe the different mechanisms used by the Halobacteriaceae to regulate their intracellular ionic concentrations and discuss the energy sources used to generate and to maintain the concentration gradients.

6.3.2. Sodium metabolism in the Halobacteriaceae The primary energy source for the extrusion of and accumulation of Halobacterium and related organisms is the proton electrochemical gradient

in

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across the cytoplasmic membrane. This proton electrochemical gradient can be derived from respiratory electron transport during aerobic growth or from anaerobic respiration. It may also be generated at the expense of ATP through the activity of membrane-bound ATPases during fermentation (see Section 4.1.4), or by the use of light energy absorbed by the bacteriorhodopsin proton pump (see Section 5.4.1). Chapter 4 provides more information on the energy metabolism of the Halobacteriaceae; an excellent overview of the subject has been given by BickelSandkötter et al. (1996). The gradient is established at the expense of the proton gradient via antiporters. Active efflux is mediated by the NhaC proteins. Interestingly, one of the three copies of nhaC in the Halobacterium NRC-1 genome is located on megaplasmid pNRC200 (Ng et al., 2000). The membranes of all halophiles investigated possess high antiporter activity, which uses the proton electrochemical gradient as the driving force for the extrusion of from the cell (Lanyi and MacDonald, 1976; Luisi et al., 1980; Speelmans et al., 1995). In Halobacterium salinarum the antiporter was shown to be electrogenic, and probably has a stoichiometry of (Lanyi and Silverman, 1979). In addition to its function of keeping intracellular concentrations at the desired low levels, the antiporter activity plays an important role in the homeostasis of the intracellular pH, which is kept approximately neutral (7.2) in Halobacterium salinarum (Tsujimoto et al., 1988). The activity of the antiporter is therefore regulated both by the pH gradient and by the membrane potential (Murakami and Konishi, 1990). The sodium gradient built up is also used as an energy reservoir to drive certain energy-requiring transport processes. Many of the membrane transport systems for amino acids and other compounds in the aerobic halophilic Archaea are energized by co-transport with ions (see also Section 4.1.2). The passive permeability of halophilic archaeal membranes for is very low (10 to 100-fold lower than the proton permeability), as was shown in liposomes prepared from Halobacterium salinarum and Halorubrum vacuolatum (van de Vosseberg et al., 1995, 1999). This low permeability explains why the sodium gradient is so well suited to serve as energy source to drive active transport processes in the halophiles. The Halobacterium salinarum antiporter activity is the target of the killing action of halocin H6, an archaeocin excreted by Haloferax gibbonsii (Meseguer et al., 1995) (see Section 9.2).

6.3.3. Potassium metabolism in the Halobacteriaceae Accumulation of potassium ions in halophilic Archaea was in the past considered as a simple passive process, driven by the membrane potential, so that extrusion of is compensated by uptake. The membrane of Halobacterium salinarum was found relatively permeable to potassium ions (Lanyi and Hilliker, 1976). can thus enter the cells via a uniport system in response to the membrane potential (Garty and Caplan, 1977; Wagner et al., 1978). The nature of the influx as a simple diffusion process was demonstrated in bacteriorhodopsin-containing membrane vesicles of

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Halobacterium salinarum, which upon illumination accumulated proportionally to the external concentration (Lanyi et al., 1979, see also Lanyi, 1979). entersthe cell as is ejected by the electrogenic antiporter, thus maintaining electroneutrality. Evidence for such a mechanism was found in experiments that showed accumulation of radioactively labeled rubidium (a potassium analog) ions in right-side-out vesicles of Halobacterium salinarum as a reaction to sodium extrusion, following excitation of the light-driven primary proton pump bacteriorhodopsin (Kanner and Racker, 1975). This simple uniport-mediated diffusion model may require some modification. Studies with Haloferax volcanii showed that the gradient over the membrane is not in equilibrium with the membrane potential and transport can thus not be accounted for uniquely by a passive process. When the of the cation is more negative than the of the membrane, either of two processes should be active: transport has to be energized by symport or antiport with other ions, or primary transport should occur driven by ATP hydrolysis. ATP is required to enable Haloferax volcanii to accumulate up to 3.6 M KC1, which represents a 500-1,000-fold gradient of (Meury and Kohiyama, 1989). Cells can be partially depleted of their intracellular down to about 1.5 M. The accumulation of the remainder is energy-dependent. The presence of an energy-dependent active transport system with a of about 1-2 mM has been documented. The system showed a requirement for ATP, and is comparable with the low-affinity transporter (the Trk system) of Escherichia coli, which transports in symport with mediated by ATP. ATP is not used here as the energy source for the transporter but rather as a regulator of its activity. In Haloferax volcanii part of the intracellular content (up to about 1.5 M) could be in an osmotically non-active bound form, and remainder is probably accumulated using a transport system, whose activity is regulated by ATP (Meury and Kohiyama, 1989). Analysis of the Halobacterium strain NRC-1 genome (Ng et al., 2000) has now shown the presence of multiple transport systems. These include KdpABC, an ATP-driven transport system, and five copies of TrkAH, a low-affinity transporter driven by the membrane potential. Both kdpABC and three out of the five copies of trkA are located on megaplasmid pNRC200.

6.3.4. Chloride metabolism in the Halobacteriaceae The finding that the intracellular chloride concentrations in Halobacterium cells are similar to those in the outside medium signifies that the distribution of chloride is far from equilibrium in the presence of the inside-negative membrane potential. Thus, electrical potential-driven passive chloride movement can result only in a loss of chloride from the cells, rather than in the required uptake. An increase in the amount of intracellular at the expense of energy is essential if the cells should increase their volume during growth and cell division. During growth the net flux of ions should result in uptake in excess of loss, and uptake should be equal to the difference, so as to provide net gain of intracellular KCl commensurate with the gain in intracellular volume (Lanyi, 1986; Schobert and Lanyi, 1982).

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Two energy-dependent inward chloride pumps have been identified in Halobacterium cells. The first is a light-independent transport system, which is probably driven by symport with (Duschl and Wagner, 1986). The second is the light-driven chloride pump halorhodopsin, discussed in detail in Section 5.4.2.

6.3.5. Divalent ion metabolism in the Halobacteriaceae Little is known about the intracellular concentrations and the mechanisms of uptake and/or extrusion of magnesium ions by halophilic Archaea (Oren, 1986a). Much of the intracellular magnesium is probably bound to macromolecular structures such as DNA and ribosomes. An outward calcium transport system that probably acts as a antiporter has been identified in Halobacterium salinarum (Belliveau and Lanyi, 1978). There is little information on the functions and concentrations of intracellular calcium in Halobacterium, except for the fact that most of it is probably present in a bound state.

6.3.6. Porins and mechanosensitive channels in the cell envelope of halophilic Archaea Haloferax volcanii was shown to possess voltage-dependent porin-like ion channels in its cell envelope. There may be at least two types of porin-like channels, one type being preferentially used for the translocation of the other for the translocation of ions (Besnard et al., 1997). These channels were postulated to be located in the cell envelope outside the cytoplasmic membrane. If true, this implies that the outer layers of the cell may have a more complex organization than was previously believed. Two mechanosensitive ion channels were identified in the membrane of Haloferax volcanii. Both these channels are blocked by low concentrations of gadolinium ions. One of these channels is formed by a 37 kDa protein which has been purified (Kloda and Martinac, 2001; Le Dain et al., 1998). These mechanosensitive channels show a great resemblance to bacterial ones, which are used e.g. for the rapid release of osmotic solutes upon salt downshock.

6.4. ION METABOLISM IN AEROBIC HALOPHILIC BACTERIA 6.4.1. Intracellular ion concentrations in halophilic Bacteria Estimates of intracellular and concentrations of halophilic Bacteria are summarized in Table 6.2. Intracellular concentrations have only rarely been determined, and data on intracellular divalent cation concentrations are scarce. Among the halophilic Bacteria there are great variations in the apparent intracellular ion concentrations, both among different species and within the same species, depending on the growth conditions and on the analytical method used. A few

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general trends are clear, however (Kushner and Kamekura, 1988; Ventosa et al., 1998): The intracellular concentration is generally considerably higher than that in the medium. In most cases is accumulated to a few tenths of a molar. Halomonas elongata appears to be an exception, with as low as 20 mM reported (Vreeland et al., 1983). In contrast to the situation in the archaeal halophiles (see section 6.3), cytoplasmic potassium contributes relatively little to the achievement of osmotic balance. The concentration inside the cells is generally lower than that outside. The apparent intracellular concentrations increase with increasing external NaCl concentration in a nonlinear fashion. Generally the sum of the intracellular concentrations measured is insufficient to balance the osmotic pressure of the medium. As discussed in Chapter 8, the remainder of the osmotic balance is provided by the accumulation of organic compatible solutes. In the Halomonadaceae (genera Halomonas and Chromohalobacter), the sum of the apparent intracellular and concentrations is much lower than their concentration in the medium. In Halomonas halodenitrificans, the sum of intracellular and remained low and constant (about and in exponentially growing cells) over a wide range of medium NaCl concentrations. Stationary phase cells showed a drastic increase in the intracellular concentration (up to 0.5 and in cells grown in 1 and 3 M NaCl, respectively), while the intracellular concentration decreased to about 0.1 M (Sadler et al., 1980). In Chromohalobacter canadensis, the apparent intracellular concentration increased when the cells reached the stationary phase (Matheson et al., 1976). In "Pseudomonas halosaccharolytica" the intracellular cation concentrations measured was quite high (1.4 to and did not change greatly when the NaCl concentration at

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which the cells were grown was varied in the range from 1 to 3 M NaCl. Much of the cell-associated was suggested to be bound to the cells' surface layers rather than to occur freely dissolved in the cytoplasm (Masui and Wada, 1973; see also Imhoff, 1993). In Salinivibrio costicola the intracellular cation concentrations were significantly lower than those in the outside medium only when grown at the highest medium salinities. In this organism the ions may in part occur in a bound state (Shindler et al., 1977). The subject of cation binding to the negative charges present on proteins, cell envelopes, and other macromolecules in halophilic Bacteria has been discussed in-depth by Kushner (1988, 1989a, 1989b). Bulk analysis of ions in cell pellets does not provide information on what part of the sodium and other cations may be tightly bound to different cellular structures. studies have indicated that a significant fraction of the intracellular sodium does not occur in a freely dissolved state in the cytoplasm but is rather strongly bound. Measurements of sodium NMR relaxation times can be used to assess the intracellular environment in which the ions are embedded. A drawback of the methods is the need for thick cell suspensions, which easily leads to the development of anoxic conditions and artifacts resulting from the lack of oxygen (Gilboa et al., 1991), By using to probe the ionic environment inside Chromohalobacter israelensis cells, three types of cell-associated sodium were detected. One fraction (about 40% of the total) is dissolved in the cytosol, and can exchange with the extracellular sodium. The other 60% is bound. Only part of this bound sodium could exchange with the "free" intracellular sodium, while the remainder could not exchange, and was "invisible" to the NMR. These measurements could be performed up to about 2 M salt only, as the shift reagent [dysprosium bis(tripolyphosphate)] used to discriminate between extracellular and cell-associated sodium was not effective at higher NaCl concentrations. In the concentration range tested the "NMR-visible" intracellular sodium concentration never exceeded 15-20% of the medium concentration (Goldberg and Gilboa, 1978; Sakhnini and Gilboa, 1993). The state of intracellular lithium was also investigated by NMR. Lithium can replace part of the intracellular sodium. In cells grown in low salt (0.5 M), almost all (92%) of the was free and "visible" by NMR, while in 3 M salt grown cells only 55% was detected. Three types of lithium ions were observed: free, weakly bound and strongly bound (Goldberg et al., 1983). NMR methods have also been applied to study the intracellular in Salinivibrio costicola grown at NaCl concentrations between 0.6 and 2 M. Apparent intracellular concentrations (excluding strongly bound which is not detected by NMR) were always below 25% of the concentration in the medium (Gilboa et al., 1991). The fraction of the free intracellular was about 40% of the total. The total intracellular concentration in Salinivibrio costicola was thus estimated between 0.14 and 0.44 M (Gilboa et al., 1991), values much lower than those obtained from bulk analyses of cell pellets (Shindler et al., 1977). Nagata et al. (1995) also reported low intracellular concentrations in Salinivibrio costicola (around 0.1 M internal in cells grown in 0.8 - 2 M NaCl) on the basis of NMR experiments. A third halophilic bacterium in which has been used to probe the intracellular environment is an organism designated as Brevibacterium sp. strain JCM 6894, which is a halotolerant isolate obtained from seawater that can grow from freshwater up to 3 M NaCl or KCl. The

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taxonomic status of this strain remains to be ascertained. Intracellular concentrations as low as 170 mM were found in cells grown in 2.5 M NaCl, and cells grown in 0.1-1 M NaCl contained less than Bulk methods to estimate intracellular ion concentrations yielded much higher values: about 1.3 M apparent intracellular in addition to about were measured in cells grown in 2 M NaCl. In cells grown in 0.5 M NaCl the concentrations of free and bound in the cytosol were 0.14 and per mg protein, respectively. When suitable organic osmoprotectants (1 mM of proline, glycine betaine, or were added to the medium, the intracellular free concentration dropped from 118 mM to 50 mM, 87 mM, and 74 mM, respectively (Nagata et al., 1991, 1995). Additional evidence for the presence of substantial amounts of sodium ions bound to cellular structures came from a study in which cell envelopes of "Pseudomonas halosaccharolytica" were incubated in 2 M NaCl and 5.5 mM KCl. Per gram of envelope protein 149 mg and 14 mg were bound. This value for is about twice as high and the value for is about eight times as low as found per gram of total cellular protein (Masui and Wada, 1973). Intracellular magnesium concentrations have only seldom been determined in halophilic Bacteria. In Halomonas elongata grown in medium containing 24 mM the intracellular magnesium concentrations varied from 9 to 23 mM depending on the growth conditions (Vreeland et al., 1983). These values are low when compared to values of 30 mM and 102 mM measured in Escherichia coli and in Halobacterium salinarum, respectively (De Médicis, 1986; De Médicis et al., 1986). Halomonas elongata, when grown in medium with 0.7 mM in the presence of 0.05-3.4 M NaCl, was found to contain between 3.1 and 12 mM intracellularly, most of it being probably bound (Vreeland, 1993). Manganese was present in Salinivibrio costicola cells at a concentration of 0.6 mmol per kg of cell water (De Médicis et al., 1986). Estimates of intracellular chloride concentrations within cells of halophilic Gramnegative Bacteria are greatly variable, from relatively low values (55 and 139 mM in Halomonas halodenitrificans and Salinivibrio costicola grown in 1 M NaCl) (Christian and Waltho, 1962) to values as high as 0.7-1 M in "Pseudomonas halosaccharolytica" grown at NaCl concentrations between 1-3 M (Masui and Wada, concentrations are in most cases much lower 1973). The measured intracellular than the combined and concentrations (Imhoff, 1993). Thus, Salinivibrio costicola grown in 1 M NaCl and 6 mM KCl had a combined intracellular and concentration of about 0.6 M, but contained only about 0.1-0.2 M (Kushner, 1989a). When grown at higher salt concentrations, the apparent intracellular chloride concentration increased (1.5 M in cells grown in 3 M salt) (Kamekura and Kushner, 1984). Older studies have suggested that the intracellular ionic concentrations in halophilic cyanobacteria may be quite high. Miller et al. (1976) reported intracellular concentrations as high as 1 M in Aphanothece halophytica. The intracellular concentration measured increased with the salt concentration of the growth medium. Potassium was therefore claimed to be one of the main osmotic compounds in this organism (Miller et al., 1976; Yopp et al., 1978b). However, the intracellular

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enzymatic systems of halophilic cyanobacteria are salt sensitive. Thus, the ribulose bisphosphate carboxylase of Aphanothece halophytica does not function at chloride concentrations above 150 mM (Incharoensakdi and Takabe, 1988). Later studies have shown that the ionic concentrations inside halophilic cyanobacteria are indeed relatively low. Intracellular chloride concentrations in Aphanothece halophytica were found to increase from 35 mM to 150 mM when the medium NaCl concentration increased from 0.5 to 2 M (Incharoensakdi and Takabe, 1988). Reed et al. (1984) detected only low internal potassium concentrations (170-310 mM) in Aphanothece halophytica and in a number of other halophilic cyanobacteria growing in salt concentrations up to four times that of seawater. Cells of Synechocystis PCC 6803 grown at 0.65 M and 1.03 M NaCl had apparent intracellular concentrations of 0.22 and 0.51 M, and concentrations of 0.15 and 0.30 M, respectively (Hagemann et al., 1994). Upon a sudden increase in salt concentration in the outside medium, salts may be transiently accumulated in halophilic cyanobacteria to achieve a rapid osmotic balance before sufficient amounts of organic osmotic solutes are produced (see Section 8.3.1). Synthesis of organic solutes is a relatively slow process that can take many hours. Thus, when the euryhaline Synechocystis PCC 6714 was transferred from freshwater to 0.5 M NaCl, a rapid entry of NaCl was observed within 2 minutes, followed by an exchange of by in 20 minutes. Accumulation of the organic osmotic solutes glucosylglycerol and sucrose over a period of 24 hours then allowed the extrusion of excess potassium (Reed et al., 1985). Transient accumulation of salt was also documented to occur in Spirulina subsalsa (probably a strain of the newly described genus Halospirulina tapeticola, an organism that grows between 0.25 and 2.5 M NaCl. Following an increase in medium salinity, intracellular and were temporarily greatly increased. was subsequently extruded by the antiporter in a process driven by the respiration-generated proton gradient (Gabbay-Azaria and Tel-Or, 1991, 1993; Gabbay-Azaria et al., 1992). During this adaptation phase, the intracellular concentration of glycine betaine, the osmotic solute produced by this organism, increased to achieve a new osmotic equilibrium. Influx of ions following salt upshock may have a temporary inhibitory effect on the photosynthetic system. When Agmenellum quadruplicatum, a marine coccoid cyanobacterium that grows from 0 to over NaCl, was transferred from 18.5 to 70 NaCl, photosynthesis was severely depressed. Activity recovered only after several hours, during which excess was pumped out of the cells (Batterton and van Baalen, 1971). Little effort has been devoted to the study of intracellular ion concentrations in halophilic Gram-positive Bacteria. Apparent intracellular cation concentrations as high as those present in the growth medium were reported in the haloalkaliphile Bacillus haloalkaliphilus (Weisser and Trüper, 1985). However, this species is now known to produce ectoine as main osmotic solute, with minor amounts of glycine betaine and (see Chapter 8). A renewed examination of its intracellular ionic concentrations may therefore be warranted. Halobacillus halophilus accumulates chloride to concentrations almost as high as the those in the medium (Roeßler and Müller, 1998). In "Micrococcus varians subsp. halophilus", the apparent

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intracellular concentration was approximately equal to that in the medium over the range from 1 to 2 M, while in cells grown at 4 M NaCl a concentration of 2.1 was measured intracellularly (Chan et al., 1979). The presence of high intracellular concentrations in this organism was confirmed by Kamekura and coworkers, who also showed that other monovalent cations added to the growth medium were not excluded from the cytoplasm (Kamekura and Onsihi, 1982; Kushner and Kamekura, 1988).

6.4.2. Sodium metabolism in aerobic halophilic Gram-negative Bacteria As shown above, the aerobic halophilic Bacteria keep their intracellular concentrations at levels considerably below those present in their medium. Sodium extrusion mechanisms are present in the cell membrane to achieve such relatively low intracellular concentrations against a constant influx of ions leaking inwards through a not completely impermeable membrane, this in addition to the entering of ions during cotransport with amino acids and other substrates (see Section 4.1.2). extrusion is based either on activity of antiporters and/or on respirationdriven primary sodium pumps (see also Ventosa et al., 1998). The antiporter activity of Salinivibrio costicola has been well characterized (Hamaide et al., 1983, 1985; Udagawa et al., 1986). Cells maintain a proton motive force (the combined action of the pH gradient over the membrane and the membrane potential) varying from 170 to 100 mV (inside negative) in cells grown at pH values varying from 5.7 to 9.0. The intracellular pH is maintained close to 7.5. The reversed pH gradient present in high pH-grown cells is compensated at least in part by an elevated membrane potential. The primary respiration-driven outward proton pump supplies the driving force for efflux through the antiporter. Protonophores such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 3,3',4',4tetrachlorosalicylanilide (TCS), respiration inhibitors, and monensin, which normally serves to facilitate exchange, dissipate the proton motive force and abolish antiport activity (Hamaide et al., 1983). When Salinivibrio costicola cells that had been kept anaerobically at pH 6.5 were given a pulse of oxygen, protons were extruded, both in the presence and in the absence of ions. At pH 8.5 an acidic response was observed in absence of but an alkalinization in its presence, suggesting that the antiport mechanism is functional at pH 8.5, and that the establishment of a gradient participates in pH homeostasis. Protonophores abolish all these effects. CCCP (at the rather high concentration of see below) and TCS abolish growth at pH 8.5 (Hamaide et al., 1985). When Salinivibrio costicola was grown on lactate as energy source, translocation was inhibited by CCCP, but valinomycin had little effect. Amiloride, an inhibitor of the eukaryotic antiporter system, inhibited lactate-dependent translocation. It was concluded that lactate-dependent extrusion enables the generation of a gradient mediated by the antiporter (Udagawa et al., 1986). These data support the idea that extrusion is the primary process that enables the secondary conversion of the proton motive force into a motive force via the antiporter.

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However, gradients may also be established by primary pumps without the use of a proton electrochemical gradient as an intermediary. Primary respirationdriven pumps occur in certain marine Bacteria such as the slightly halophilic Vibrio alginolyticus (Tokuda and Unemoto, 1983). Such primary pumps may be present in Salinivibrio costicola and in other moderately halophilic Bacteria as well (Tokuda and Unemoto, 1983; Udagawa et al., 1986). The bioenergetics of the alkaliphilic moderately halophilic sulfate reducer Desulfonatronovibrio hydrogenovorans is also based on primary electron-transport driven translocation (Sydow et al., 2002). Evidence for the activity of primary pumps in halophilic Bacteria is in part based on the fact that in alkaline media growth is not inhibited by the protonophore CCCP. It was postulated that in this case a primary pump which functions optimally in the alkaline pH range maintains the intracellular cation environment, this in addition to the antiporter which is mainly operative at low pH (Tokuda and Unemoto, 1983). The site of the primary outward sodium pump in Salinivibrio costicola was identified to be the NADH oxidase (NADH:quinone reductase) segment of the respiratory chain (Udagawa et al., 1986). At pH 8.5 the formation of a membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline (HQNO), which inhibits the NADH oxidase. When NADH served as electron donor, translocation was resistant to CCCP and stimulated by valinomycin; when lactate was used as electron donor, translocation was inhibited by CCCP but affected only little by valinomycin. Amiloride, which inhibited the lactate-dependent translocation, had little effect on NADH-dependent translocation (Udagawa et al., 1986). In a comparative study of eight moderate halophilic Bacteria, all six Gram-negative species tested (Salinivibrio costicola, Halomonas variabilis, Halomonas halophila, Chromohalobacter canadensis, Pseudomonas beijerinckii and "Pseudomonas halosaccharolytica") showed NADH:quinone reductase activity. CCCP alone did not completely inhibit the build-up of a membrane potential linked to NADH oxidation in inverted membrane vesicles, but a combination of CCCP and the conducting ionophore monensin completely abolished the formation of a membrane potential. Succinate oxidation and the terminal oxidase step of the respiratory chain did not show any specific requirement for ions (Unemoto et al., 1992). Extensive studies of the ion metabolism in Chromohalobacter israelensis also suggested the importance of a primary pump in the regulation of its intracellular concentrations (Ken-Dror and Avi-Dor, 1985; Ken-Dror et al., 1984, 1986a, 1986b). The existence of an uncoupler-stimulated pump may explain the drop in respiration rate of up to 80% observed when the pH was increased from 6.5 to 8.5 in the absence of but not in the presence of catalytic amounts of The ratelimiting step in the electron transport at high pH was located in the NADH-quinone oxidoreductase section of the respiratory chain, and was suggested to release this kinetic control (Ken-Dror and Avi-Dor, 1985). In the presence of a pH gradient of reversed polarity was formed driven by the antiporter activity, both in the absence and the presence of the uncoupler carbonyl cyanyl methyoxyphenylhydrazone (FCCP). Inverted membrane vesicles responded as expected from the behavior of intact cells. Extrusion of ions may thus be the

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primary event, and proton counterflow and symport phenomena are secondary events that minimize the build-up of the membrane potential generated by the primary pump (Ken-Dror et al., 1984). Respiration initiates efflux of from preloaded cells, driven by a pump that is insensitive to uncouplers such as FCCP and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD) (Ken-Dror et al., 1986a, 1986b). Movement of is thus directly coupled to electron transport. The uncoupler-insensitive primary pump may play an important role in the regulation of intracellular salt concentrations. When ions were omitted from the medium, a shift in pH from 6.5 to 8.5 caused an increase in respiration rate in Chromohalobacter israelensis, as compared to the above-described drop in respiration in the absence of The respiration rate was not affected by pH when both and were present (Ken-Dror and Avi-Dor, 1985). Stimulation of respiration by was most pronounced at acidic pH values, and was accompanied by an alkalinization of the cytoplasm. More protons are extruded by the primary proton pump, possibly to compensate for the electrogenic entry of which leads to internal alkalinization. The effect of was only observed in intact cells. Which particular step in the electron flux is affected by has not been elucidated (Ken-Dror and Avi-Dor, 1985). The exclusion of salt and the maintenance of steep ionic gradients in the halophilic Bacteria can only be achieved by the constant pumping of ions by energy-dependent mechanisms, as ions will constantly enter the cell, e.g. as a result of transport processes. Little is known about the passive ion permeability of the cell membrane. However, continuous culture experiments with Halomonas elongata showed that the maintenance energy of this bacterium is relatively independent of the salinity of the medium, thus providing evidence in favor of an ion-tight membrane (Galinski, 1995; see also Ventosa et al., 1998).

6.4.3. Metabolism of other ions in aerobic halophilic Gram-negative Bacteria A potassium transport system has been characterized in a isolate designated Halomonas strain SPC1, which was isolated as a contaminant from a Dunaliella culture. This transporter enables cells to rapidly accumulate the necessary following hyperosmotic shock. The of this transporter for was which signifies an affinity much lower than that of the well-characterized Kdp system of Escherichia coli. High-affinity transport systems are probably not essential for Halomonas and other halophilic Bacteria which generally live in seawater-derived brines, rich in potassium (Cummings et al., 1993). A extrusion system was described in Chromohalobacter israelensis, probably based on antiporter activity. and ions were found to rapidly enter the cell membrane. Under energized conditions a powerful pump extrudes the penetrated and At low pH values the presence of is not required for extrusion. However, millimolar concentrations of are needed for effective and extrusion at high pH values, again pointing to the possible involvement of primary pumps (see above) (Shnaiderman and Avi-Dor, 1982). Little is known about the mechanisms that extrude anions from the cells of halophilic Bacteria. It has been suggested that the membrane potential generated by

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efflux may be the driving force for the efflux of permeant anions (Ken-Dror et al., 1986a, 1986b).

6.4.4. Ion metabolism in aerobic halophilic Gram-positive Bacteria The presence of a primary pump in "Micrococcus varians subsp. halophilus" has been postulated on the basis on the low level of inhibition of alanine transport by CCCP at alkaline pH (Nikolaev and Mateeva, 1990). No NADH oxidase activity could be demonstrated in Kocuria halophila and in "Micrococcus varians subsp. halophilus", as in these organisms the membrane potential generated during NADH oxidation was completely dissipated by CCCP (Unemoto et al., 1992). Halobacillus halophilus has a specific demand for chloride. Flagellar motility and the synthesis of flagella in this organism are strictly chloride-dependent (Roeßler et al., 2000). Its endospores do not germinate unless chloride is present; germination is optimal at Chloride can be replaced by bromide, but not by sulfate or nitrate (Dohrmann and Müller, 1999). Five more chloride-induced proteins were identified in Halobacillus halophilus. Chloride may be an important environmental signal in this organism (Roeßler and Müller, 2002). Energy-dependent uptake of decreasing the gradient from >10 at to a nearly constant value of 2 at high external concentrations, allowed optimal grown (Roeßler and Müller, 1998).

6.5. ION METABOLISM IN THE HALANAEROBIALES The Halanaerobiales (low G+C Gram-positive branch of the Bacteria) display many physiological and biochemical properties that are characteristic of the halophilic aerobic Archaea. No organic osmotic solutes have yet been found in these anaerobic fermentative Bacteria (Mermelstein and Zeikus, 1998; Oren, 1986b; Oren et al., 1997; Rengpipat et al., 1988). High concentrations of and have been measured inside the cells of Halanaerobium praevalens, Halanaerobium acetethylicum, and Halobacteroides halobius, high enough to be approximately isotonic with the medium (Table 6.3). In exponentially growing cells was the major cation. The intracellular ionic concentrations of individual cells of Halanaerobium praevalens were assayed by means of X-ray microanalysis with the transmission electron microscope. Apparent intracellular cation concentrations between 1.22 and 1.91 M and chloride concentrations of 0.93-1.57 M were measured in cells growing exponentially in 2.6 M total salts. was the major cation (70% of the cation sum). Stationary phase cells showed a high variability among individual cells, some of the cells containing high concentrations of NaCl rather than of KCl (Oren et al., 1997).

6.6. ION METABOLISM IN DUNALIELLA Reliable estimations of the true intracellular cation concentrations in the halophilic algal genus Dunaliella have been achieved only relatively recently, with the development of suitable techniques to determine the true intracellular volume and/or to

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separate the cells from the extracellular salt solution. Older studies have quoted quite high apparent ion concentrations. Thus, Ehrenfeld and Cousin (1982) stated that Dunaliella cells grown in medium containing 11 contained intracellular concentrations 6 to 13 times that of the medium. Intracellular and were lower than outside. The existence of at least two compartments within the cell was postulated. The larger of these compartments (possibly to be identified as the chloroplast) was suggested to regulate its ion concentrations while maintaining low and and high values, whereas the second compartment (the cytoplasm) was thought to equilibrate with the external medium. B.Z. Ginzburg (1978) and M. Ginzburg (1981) also presented such two-compartment models. The sodium ionaccessible space in Dunaliella parva was estimated at 51% of total cell volume (Zmiri and Ginzburg, 1983). Rapid influx was reported when cells were subjected to a sudden increase in salinity (Ginzburg, 1981).

Three novel approaches have enabled more realistic estimates of the true ionic concentrations in Dunaliella. Lithium was found to be a reliable marker for extracellular water for the estimation of the intracellular volume. Intracellular concentrations, both in cells grown in 0.5 M and in 4 M NaCl, were now found to be below 100 mM. This value was not exceeded when the extracellular salt concentration was increased (Katz and Avron, 1985). It proved also possible to physically separate the cells from the extracellular medium by passing the algae through cation-exchange minicolumns (Karni and Avron, 1988; Pick et al., 1986a). Analyses of intracellular ion concentrations of cells grown in NaCl concentrations between 1 and 4 M in the presence of 5 5 mM and 0.3 mM yielded the following values: 20100 mM 150-300 200-350 mM 1-3 mM 30-50 mM and

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20 mM Part of the intracellular is bound to storage polyphosphate (Pick et al., 1987). With these intracellular concentrations, inorganic ions contribute no more than 5 to 20% to the intracellular osmotic pressure (Pick et al., 1986a), the remainder being provided by glycerol (see Section 8.4). The third approach was based on NMR, using dysprosium tripolyphosphate complex as a sodium shift reagent to discriminate between intracellular and extracellular Concentrations of 88 ± 28 mM were thus measured in Dunaliella salina. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the medium, in parallel to the increase in intracellular glycerol, indicating that ions and glycerol are codistributed within the cell volume (Bental et al., 1988). The low intracellular concentrations measured agree with the observation that interferes strongly with the activity of many Dunaliella enzymes that use anionic substrates such as glycerol-3phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphoenolpyruvate carboxylase, acid phosphatase, and others (Gimmler et al., 1984).

The

concentration within Dunaliella cells is kept low by the activity of a antiporter in the cytoplasmic membrane. This antiporter is sensitive to inhibition by amiloride, as are other eukaryotic antiporters. Its activity is increased when the cells are growing at high NaCl concentrations (Katz et al., 1989, 1992). This antiporter also has a key function in the regulation of the intracellular pH (Katz et al., 1991, 1992). It is energized by the proton gradient generated by a

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vanadate-sensitivc in the plasma membrane (Pick et al., 1987). Gimmler (2000) provided a thermodynamic model explaining the possible mechanisms of homeostasis in Dunaliella (Figure 6.1). This model included the possibility of the existence of a respiration-driven primary pump in Dunaliella or the presence of a ATPase in charge of transport. In such a model the antiporter may be used primarily for pH homeostasis. Recent evidence has shown that electron transport in the Dunaliella salina plasma membrane is coupled to extrusion (Katz and Pick, 2001). Elevated levels of intracellular enhanced the extracellular reduction of ferricyanide. Quinone analogs, NQNO, and dicumarol inhibited ferricyanide reduction and led to the accumulation of Anaerobic conditions elevated, ferricyanide partially decreased the intracellular level. All these observations are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to extrusion in the plasma membrane. Treatment with the ionophores monensin and ETH-157 elevated the content of the cell and stimulated ferricyanide reduction (Figure 6.2).

The uptake systems for potassium and calcium ions in Dunaliella salina have been partially characterized (Pick et al., 1986b, 1987). Two distinct carriers were identified, driven by the transmembrane electrical potential generated by activity of the ATPase within the plasma membrane (Pick et al., 1987). Phosphate and sulfate are co-

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transported with ions in Dunaliella salina, and their transport is energized by the gradient (Weiss et al, 2001).

6.7. REFERENCES Antón, J., Oren, A., Benlloch, S., Rodríguez-Valera, F., Amann, R., and Rosselló-Mora, R. 2002. Salinibacter ruber gen. nov., sp. nov., a novel extreme halophilic Bacterium from saltern crystallizer ponds. Int. J. Syst. Evol. Microbiol. 52: 485-491. Batterton, J.C., and van Baalen, C. 1971. Growth responses of blue-green algae to sodium chloride concentration. Arch. Mikrobiol. 76: 151-165. Baxter, R.M., and Gibbons, N.E. 1956. Effects of sodium and potassium chloride on certain enzymes of Micrococcus halodenitrificans and Pseudomonas salinaria. Can. J. Microbiol, 2: 599-606. Bayley, S.T., and Morton, R.A. 1978. Recent developments in the molecular biology of extremely halophilic bacteria. CRC Crit. Rev. Microbiol. 6: 151-205. Belliveau, J.W., and Lanyi, J.K. 1978. Calcium transport in Halobacterium halobium envelope vesicles. Arch. Biochem. Biophys. 186: 98-105. Bental, M., Degani, H., and Avron, M. 1988. studies of the intracellular sodium ion concentration in the halotolerant alga Dunaliella salina. Plant Physiol. 87: 813-817. Besnard, M., Martinac, B., and Ghazi, A. 1997. Voltage-dependent porin-like ion channels in the archaeon Haloferax volcanii. J. Biol. Chem. 272: 992-995. Bickel-Sandkötter, S., Gärtner, W., and Dane, M. 1996. Conversion of energy in halobacteria: ATP synthesis and phototaxis. Arch. Microbiol. 166: 1-11. Brown, A.D. 1976. Microbial water stress. Bacteriol. Rev. 40: 803-846. Brown, A.D. 1990. Microbial water stress physiology. Principles and perspectives. Jolm Wiley & Sons, Chichester. Brown, A.D., and Duong, A. 1982. State of water in extremely halophilic bacteria: heat of dilution of Halobacterium halobium. J. Membr. Biol. 64: 187-193. Brown, A.D., and Sturtevant, J.M. 1980. State of water in extremely halophilic bacteria: freezing transitions of Halobacterium halobium observed by differential scanning calorimetry. J. Membr. Biol. 54: 21-30. Chan, K., Leung, O.C., and Lee, L.H. 1979. Influence of temperature on ionic sparing effect and cell-associated cations in the moderate halophile, Micrococcus varians var. halophilus. Microbios 24: 81 -91. Christian, J.H.B., and Waltho, J.A. 1962. Solute concentrations within cells of halophilic and non-halophilic bacteria. Biochim. Biophys. Acta 65: 506-508. Csonka, L.N. 1989. Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53: 121147. Cummings, S.P., Williamson, M.P., and Gilmour, D.J. 1993. Turgor regulation in a novel Halomonas species. Arch. Microbiol. 160: 319-323. De Médicis, E. 1986. Magnesium, manganese and mutual depletion systems in halophilic bacteria. FEMS Microbiol. Rev. 37: 137-143. De Médicis, E., Paquette, J., Gauthier, J.-J., and Shapcott, D. 1986. Magnesium and manganese content of halophilic bacteria. Appl. Environ. Microbiol. 52: 567-573. Dohrmann, A.-B., and Müller, V. 1999. Chloride dependence of endospore germination in Halobacillus halophilus. Arch. Microbiol. 172: 264-267. Duschl, A., and Wagner, G. 1986. Primary and secondary chloride transport in Halobacterium halobium. J. Bacteriol. 168: 548-552. Ehrenfeld, J., and Cousin, J.-L. 1982. Ionic regulation of the unicellular alga Dunaliella. J. Membr. Biol. 70: 4757. Gabbay-Azaria, R., and Tel-Or, E. 1991. Regulation of intracellular content during NaCl upshock in the marine cyanobacterium Spirulina subsalsa cells. Biores. Technol. 38: 215-220. Gabbay-Azaria, R., and Tel-Or, E. 1993. Mechanisms of salt tolerance in eyanobacteria, pp. 123-132 In: Gresshoff, P.M. (Ed.), Plant responses to the environment. CRC Press, Boca Raton. Gabbay-Azaria, R., Schonfeld, M., Tel-Or, S., Messinger, R., and Tel-Or, E. 1992. Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance. Arch. Microbiol. 157: 183-190. Galinski, E.A. 1995. Osmoadaptation in bacteria. Adv. Microb. Physiol. 37: 273-328. Garty, H., and Caplan, S.R. 1977. Light-dependent rubidium transport to intact Halobacterium halobium cells. Biochim. Biophys. Acta 459: 532-545.

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Gilboa, H., Kogut, M, Chalamish, S., Regev, R., Avi-Dor, Y., and Russell, N.J. 1991. Use of nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium. J. Bacteriol. 173: 7021-7023. Gimmler, H. 2000. Primary sodium plasma membrane ATPases in salt-tolerant algae: facts and fictions. J. Exp. Bot. 51: 1171-1178. Gimmler, H., Kaaden, R., Kirchner, U., and Weyand, A. 1984. The chloride sensitivity of Dunaliella parva enzymes. Zeitschr. Pflanzenphysiol. 114: 131-150. Ginzburg, B.Z. 1978. Regulation of cell volume and osmotic pressure in Dunaliella, pp. 543-560 In: Caplan, S.R., and Ginzburg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier, Amsterdam. Ginzburg, M. 1978. Ion metabolism in whole cells of Halobacterium halobium and H. marismortui, pp. 561577 In: Caplan, S.R, and Ginzbu rg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam. Ginzburg, M. 1981. Measurements of ion concentrations in Dunaliella parva subjected to hypertonic shocks. J. Exp. Bot. 32: 333-340. Ginzburg, M., Sachs, I., and Ginzburg, B.Z. 1970. Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations. J. Gen. Physiol. 55: 187-207. Ginzburg, M., Sachs, L., and Ginzburg, B.Z. 1971. Ion metabolism in a Halobacterium. II. Ion concentrations in cells at different levels of metabolism. J. Membr. Biol. 5: 78-101. Gochnauer, MB., and Kushner, D.J. 1971. Potassium binding, growth, and survival of an extremely halophilic bacterium. Can. J. Microbiol. 17: 17-23. Goldberg, M., and Gilboa, H. 1978. Sodium exchange between two sites. The binding of sodium to halotolerant bacteria. Biochim. Biophys. Acta 538: 268-283. Goldberg, M., Risk, M., and Gilboa, H. 1983. Lithium nuclear magnetic resonance measurements in halotolerant bacterium Biochim. Biophys. Acta 763: 35-40. Hagemann, M., Fulda, S., and Schubert, H. 1994. DNA, RNA, and protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803 adapted to different salt concentrations. Curr. Microbiol. 28: 201-207. Hamaide, F., Kushner, D.J., and Sprott, G.D. 1983. Proton motive force and antiport in a moderate halophile. J. Bacteriol. 156: 537-544. Hamaide, F., Kushner, D.J., and Sprott, G.D. 1985. Proton circulation in Vibrio costicola. J. Bacteriol. 161: 681686. Imhoff, J.F. 1993. Osmotic adaptation in halophilic and halotolerant microorganisms, pp. 211-253 In: Vreeland, R.H., and Hochstein, L.I. (Eds.), The biology of halophilic bacteria. CRC Press, Boca Raton. Incharoensakdi, A., and Takabe, T. 1988. Determination of intracellular chloride ion concentration in a halotolerant cyanobacterium Aphanothece halophytica. Plant Cell Physol. 29: 1073-1075. Kamekura, M., and Kushner, D.J. 1984. Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola. J. Bacteriol. 160: 385-390. Kamekura, M., and Onishi, H. 1982. Cell-associated cations of the moderate halophilic Micrococcus varians ssp. halophilus grown in media of high concentrations of LiCl, NaCl, KCl, RbCl, or CsCl. Can. J. Microbiol. 28: 155-161. Kanner, B.I., and Racker, E. 1975. Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium. Biochem. Biophys. Res. Commun. 64: 1054-1061. Karni, L., and Avron, M. 1988. Ion content of the halotolerant alga Dunaliella salina. Plant Cell Physiol. 29: 1311-1314. Katz, A., and Avron, M. 1985. Determination of intracellular osmotic volume and sodium concentration in Dunaliella. Plant Physiol. 78: 817-820. Katz, A., and Pick, U. 2001. Plasma membrane electron transport coupled to extrusion in the halotolerant alga Dunaliella. Biochim. Biophys. Acta 1504: 423-431. Katz, A., Pick, U., and Avron, M. 1989. Characterization and reconstitution of the antiporter from the plasma membrane of the halophilic alga Dunaliella. Biochim. Biophys. Acta 983: 1-14. Katz, A., Bental, M., Degani, H., and Avron, M. 1991. In vivo pH regulation by antiporter in the halotolerant alga Dunaliella salina. Plant Physiol. 96: 110-115. Katz, A., Pick, U., and Avron, M. 1992. Modulation of antiporter activity by extreme pH and salt in the halotolerant alga Dunaliella salina. Plant Physiol. 100: 1224-1229. Ken-Dror, S., and Avi-Dor, Y. 1985. Regulation of respiration by and in the halotolerant bacterium Arch. Biochem. Biophys. 243: 238-245. Ken-Dror, S., Shnaiderman, R., and Avi-Dor, Y. 1984. Uncoupler-stimulated pump and its possible role in the halotolerant bacterium, Arch. Biochem. Biophys. 229: 640-649.

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praevalens and Halobacteroides halobius. Can. J. Microbiol. 32: 4-9. Oren, A. 1999. Life at high salt concentrations, In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H. and Stackebrandt, E. (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. 3rd. Ed. Springer-Verlag, New York (electronic publication). Oren, A., Heldal, M., and Norland, S. 1997. X-ray microanalysis of intracellular ions in the anaerobic halophilic eubacterium Haloanaerobium praevalens. Can. J. Microbiol. 43: 588-592. Pérez-Fillol, M., and Rodríguez-Valera, F. 1986. Potassium ion accumulation in cells of different halobacteria. Microbiología SEM 2: 73-80. Pick, U., Karni, L., and Avron, M. 1986a. Determination of ion content and ion fluxes in the halotolerant alga Dunaliella salina. Plant Physiol. 81: 92-96. Pick, U, Ben-Amotz, A., Karni, L., Seebregts, C.J., and Avron, M. 1986b. Partial characterization of and uptake systems in the halotolerant alga Dunaliella salina. Plant Physiol. 81: 875-881. Pick, U., Katz, A., Weiss, M., and Avron, M. 1987. Dunaliella – a model system for cellular ion regulation in plants and algae, pp. 241-255 In: Leaver, C.J., and Sze, H. (Eds.), Plant membranes: structure, function, biogenesis. Alan R. Liss, New York. Reed, R.H. 1984. Use and abuse of osmo-terminology. Plant Cell Environ. 7: 165-170. Reed, R.H., Chudek, J.A., Foster, R., and Stewart, W.D.P. 1984. Osmotic adjustment in cyanobacteria from hypersaline environments. Arch. Microbiol. 138: 333-337. Reed, R.H., Warr, S.R.B., Richardson, D.L., Moore, D.J., and Stewart, W.D.P. 1985. Multiphasic osmotic adjustment in a euryhaline cyanobacterium. FEMS Microbiol. Lett. 28: 225-229. Rengpipat, S., Lowe, S.E., and Zeikus, J.G. 1988. Effect of extreme salt concentrations on the physiology and biochemistry of Halobacteroides acetoethylicus. J. Bacteriol. 170: 3065-3071. Roeßler, M., and Müller, V. 1998. Quantitative and physiological analysis of chloride depenence of growth in Halobacillus halophilus. Appl. Environ. Microbiol. 64: 3813-3817. Roeßler, M., and Müller, V. 2002. Chloride, a new environmental signal molecule involved in gene regulation in a moderately halophilic bacterium, Halobacillus halophilus. J. Bacteriol., submitted for publication. Roeßler, M., Wanner, G., and Müller, V. 2000. Motility and flagellum synthesis in Halobacillus halophilus are chloride dependent. J. Bacteriol. 182: 532-535. Sadler, M., McAninch, M., Alico, R., and Hochstein, L.I. 1980. The intracellular and composition of the moderately halophilic bacterium, Paracoccus halodenitrificans. Can. J. Microbiol. 26: 496-502. Sakhnini, A., and Gilboa, H. 1993. Double quantum sodium NMR studies of the halotolerant bacterium. Biophys. Chem. 46: 21-25. Schobert, B., and Lanyi, J.K. 1982. Halorhodopsin is a light-driven chloride pump. J. Biol. Chem. 257: 1030610313. Shindler, D.B., Wydro, R.M., and Kushner, D.J. 1977. Cell-bound cations of the moderately halophilic bacterium Vibrio costicola. J. Bacteriol. 130: 698-703. Shnaiderman, R., and Avi-Dor, Y. 1982. The uptake and extrusion of salts by the halotolerant bacterium, Arch. Biochem. Biophys. 213: 177-185. Speelmans, G., Poolman, B., and Konings, W.N. 1995. as coupling ion in energy transduction in extremophilic Bacteria and Archaea. World J. Microbiol. Biotechnol. 11: 58-70.

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CHAPTER 7 PROPERTIES OF HALOPHILIC PROTEINS

A comparison between similar enzymes from normal and halophilic organisms would be especially valuable, since in halophiles the enzymes resist salts even in vivo Whatever the cause of this difference, it is clearly of fundamental importance to the understanding of the halophilic mode of life. (Ingram, 1947) The second mechanism occurs in P. salinaria [= Halobacterium salinarum] and probably in the red halophiles in general. These organisms grow over a more restricted, and very high, range of salt concentrations. Here the adaptation appears to be at the molecular level and to involve some change in the enzyme molecule which not only makes it most active in the presence of high concentrations of salt but also renders it inactive in the absence of salts. (Baxter and Gibbons, 1954) It was found that the bulk protein of the extreme halophiles contains and excess acidic over basic amino acids of about 10 mole-%, whereas the bulk protein of the corresponding non-halophilic bacteria is chemically about neutral The present findings rather support the idea that an acidic nature is a general characteristic of the proteins of the extremely halophilic cell. (Reistad, 1970)

7.1. INTRODUCTION In a now classic paper, Baxter and Gibbons (1956) compared the effect of salt on different enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, 2-oxoglutarate dehydrogenase, cytochrome oxidase, and others) from an extreme halophilic archaeon (Halobacterium salinarum, at the time named Pseudomonas salinaria) with their counterparts from a moderately halophilic (Halomonas halodenitrificans, then designated Micrococcus bacterium halodenitrificans). The enzymes from Halobacterium salinarum were most active at high concentrations of KCl. The succinate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase required 3-4 M and more for optimal activity, and 2oxoglutarate dehydrogenase performed best at 1 M KCl (Figure 7.1). In many cases KCl supported higher activities than NaCl. However, the enzymes from Halomonas reached their optimal activity at salt concentrations ranging from less than 10 mM (malate dehydrogenase) to about 0.6 M (2-oxoglutarate dehydrogenase). An earlier paper on the salt relationships of glycerol dehydrogenase from different halophilic and

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non-halophilic prokaryotes (Baxter et al., 1954) documented a similar phenomenon. These early reports nicely demonstrate the unusual behavior of the proteins of the extremely halophilic Archaea. A few years earlier, Ingram (1947) had already suggested that the proteins of halophiles may be less readily salted out than those of other bacteria.

After it had become known that certain proteins of halophilic Archaea show a markedly acidic behavior, Reistad (1970) determined the frequency of occurrence of the different amino acids in the bulk protein of Halobacterium salinarum as compared

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with non-halophilic microorganisms. He reported an unusually high excess of the acidic amino acids glutamate and aspartate in the Halobacterium proteins. The above cited papers set the stage for all subsequent work on the adaptation of the enzymes of halophilic microorganisms to the presence of salt. Salt-adapted and salt-dependent enzymes occur especially in those halophiles that use inorganic ions to provide osmotic balance, i.e. the halophilic Archaea of the order Halobacteriales, the anaerobic Bacteria of the order Halanaerobiales, and the recently discovered aerobic red halophilic Bacterium Salinibacter (see Chapter 6). Most halophilic Bacteria use organic compatible solutes to maintain the proper osmolarity of their cytoplasm as dictated by the salt concentration of their medium (see Chapter 8). Their intracellular salt concentrations are accordingly much lower than those of their medium. The same is true for halophilic Eucarya such as the green unicellular alga Dunaliella. However, even in such organisms salt-adapted proteins should be expected to occur in the cytoplasmic membrane exposed to the outer medium. Also any extracellular enzymes excreted by such "low-salt-in" organisms have to be fully functional at the high salinities that prevail in the environment in which such halophiles live. The enzymatic activities and metabolic pathways present in the different types of halophilic microorganisms have been discussed in Chapter 4. This chapter provides information on the special properties of the enzymes as dictated by the need to function at the salt concentrations to which these enzymes are exposed at their site of action inside or outside the cell.

7.2. HALOPHILIC BEHAVIOR OF ENZYMES FROM HALOPHILIC ARCHAEA To be active, stable, and soluble in high salt are major challenges facing proteins in halophilic microorganisms. The presence of molar concentrations of salts is generally devastating to proteins and other macromolecules. It causes aggregation or collapse of the tertiary structure because of enhancement of hydrophobic interactions, it interferes with essential electrostatic interactions within or between macromolecules due to charge shielding, and because of salt ion hydration it reduces the availability of free water below the level required to sustain essential biological processes (Madern et al., 2000a; Zaccai and Eisenberg, 1991). The presence of high intracellular salt concentrations thus requires special adaptations of the whole enzymatic machinery of the cell. Cells thus adapted are able to function in the presence of high salt. However, these adaptations often make the cells strictly dependent on the continuous presence of high salt concentrations for the maintenance of structural integrity and viability (Ebel et al., 1999a; Eisenberg, 1995; Eisenberg and Wachtel, 1987; Eisenberg et al., 1992; Lanyi, 1974). As a result, the aerobic halophilic Archaea display little flexibility and adaptability to changes in the external salt concentration. Most enzymes and other proteins of the Halobacteriales (including the cell envelope glycoprotein – see Section 3.1.1) denature when suspended in solutions that contain less than 1-2 M salt. Some of these enzymes are much more active in the presence of KCl than of NaCl (see also Figure 7.1), a phenomenon that agrees well

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with the finding that is intracellularly the dominating cation. The Halobacterium salinarum cysteine desulfurylase and the ribosomal proteins are good examples. In other enzymes (e.g. glycerol dehydrogenase, citrate synthetase) is the preference for over less pronounced, and still others function as well in NaCl as in KC1 (e.g. malate dehydrogenase, lactate dehydrogenase, cytochrome oxidase, and fumarate dehydrogenase) (Lanyi, 1974). "Salting-out" salts stabilize, while "salting-in" salts inactivate halophilic enzymes. The behavior of different salts coincides with the lyotropic Hofmeister series (Lanyi, 1974). Most proteins of the Halobacteriales contain a large excess of the acidic amino acids glutamate and aspartate and a low content of the basic amino acids lysine and arginine (Dennis and Shimmin, 1997, Lanyi, 1974, and many other references cited in this chapter). The high content of acidic side groups was first fully recognized during analyses of the bulk protein of Halobacterium and Halococcus (Reistad, 1970). The malate dehydrogenase of Haloarcula marismortui has a 10.4 mol percent excess of acidic residues, and the cell envelope glycoprotein of Halobacterium salinarum even 19-20 mol percent. Analysis of protein sequences deducted from the Halobacterium strain NRC-1 genome showed that among the acidic amino acids aspartate is much more abundant than glutamate, and lysine is much more underrepresented than arginine in the cytoplasm-exposed surface of the proteome of Halobacterium (Ng et al., 2000). Another prominent feature of the proteins of the Halobacteriales is their low content of hydrophobic amino acid residues, and this is generally offset by an increased content of the borderline hydrophobic amino acids serine and threonine (Lanyi, 1974). There are certain exceptions to the rule that Halobacterium proteins are highly acidic and require high salt concentrations for activity and structural stability. Some well-known exceptions are the gas vesicle proteins (see Section 3.1.7) and the retinal proteins bacteriorhodopsin and halorhodopsin (see Section 5.4.1). The ribulose bisphosphate carboxylase of Haloferax mediterranei, Haloferax volcanii, and Haloarcula marismortui is also not a halophilic protein (Altekar and Rajagopalan, 1990). It has been argued that the excess of acidic residues may be a major factor determining the halophilic character of the proteins. Excess of negative charges on the protein surface makes the structure unstable because of the mutual repulsion of the side groups. Only when high concentrations of cations are added to shield the negative charges can the protein maintain its proper conformation required for structural stability and enzymatic activity. Shielding of negative charges by cations may indeed play some part in the effects of salt on the enzymes and other proteins of the halophiles. A theoretical analysis has been made of the contribution of electrostatic interactions in Haloarcula marismortui ferredoxin and malate dehydrogenase, based on the use of the equations of classical electrostatics applied at an atomic level of detail to crystal structures of the proteins. The repulsive interactions between the acidic residues at the protein surface were shown to be a major factor in the destabilization of halophilic proteins in low-salt conditions. These electrostatic interactions remain destabilizing even at high salt concentrations. As a consequence the role of acidic residues in halophilic proteins may be more to prevent aggregation than to make a

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positive contribution to intrinsic protein stability (Elcock and McCammon, 1998). It is now becoming clear that ions may not be the primary factor responsible for the shielding of the excess negative charges. High resolution structure elucidation of the Haloarcula marismortui ferredoxin, a highly acidic protein, using X-ray diffraction analysis of crystals, showed only six bound ions; two of these are coordinated between side chains from symmetry-related protein molecules across the crystal interface and these might therefore not occupy the same sites in solution. The other four ions mainly interact with oxygen in the protein and not with acidic side chains. Close examination at 1.9 Å resolution revealed the presence of a layer of tightly bound water in the first hydration shell that interacts through hydrogen bonds and dipole moments with the side chain carboxylic acid groups. Presence of up to five organized water layers could be shown that extend into the bulk solution (Mevarech et al., 2000). Lanyi (1974) and Lanyi and Stevenson (1970) stated that all the effects of salts cannot be due to charge-shielding action alone, as the concentrations required are too high. Maximal electrostatic charge shielding would be achieved already in about 0.1 M salt or 0.5 M at most, and even much lower concentrations of divalent cations should provide sufficient protection. They argued that especially a high content of glutamate may be favorable as glutamate has the greatest water binding ability of any amino acid residue. This may have important implications when considering the need of any protein to maintain a proper hydration shell to remain functional. It has also been shown that certain acidic residues can be used to form salt bridges with strategically positioned basic (lysine, arginine) residues, thus providing further stabilization of the protein structure. Another prominent feature of the proteins of the Halobacteriaceae is their low content of hydrophobic amino acid residues, and this is generally offset by an increased content of the borderline hydrophobic amino acids serine and threonine (Lanyi, 1974). The requirement for extremely high salt concentrations for structural stability of the proteins can probably to a large extent be attributed to the low content of hydrophobic residues and the accordingly weak hydrophobic interactions within the protein molecules. High salt is then needed to maintain the weak hydrophobic interactions. Entropy increases when non-polar groups turn away from the water phase and interact with each other to form hydrophobic interactions. These interactions seem to be driven more by an avoidance of water than by an active attraction between the non-polar molecules (Lanyi, 1974). At higher salt concentrations new hydrophobic interactions are formed which have insufficient stability in water, and the molecule assumes a more tightly folded conformation. The possible involvement of the weak hydrophobic interactions in the salt requirement of the halophilic proteins is supported by the finding that certain enzymes from halophilic Archaea (including threonine deaminase, aspartate carbamoyltransferase, and alanine dehydrogenase) show cold lability: their maximal stability is reached at temperatures greater than 0 °C, and decreases at lower temperatures. The effect may be considered in terms of water structure: at lower temperature the size of the cluster of water molecules is increased, and hydrophobic

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groups can interact more easily, breaking the hydrophobic interactions (Brown. 1990; Lanyi, 1974). The malate dehydrogenase of Haloarcula marismortui has become a wellinvestigated model for the study of the mechanisms involved in the halophilic behavior of proteins. Techniques such as velocity sedimentation, light scattering, neutron scattering, and circular dichroism measurements have been used to obtain information on the structural changes that occur as a function of changing salt concentrations and the hydration properties of the protein (Eisenberg, 1995; Eisenberg and Wachtel, 1987; Mevarech and Neumann, 1977; Pundak and Eisenberg, 1981; Pundak et al., 1981). These studies have shown that the halophilic properties of the enzyme are related to its capacity of associating with unusually high amounts of salts. This observation has led to a thermodynamic "solvation-stabilization model", in which the halophilic protein has adapted to bind hydrated ions cooperatively via a network of acidic groups on its surface (Ebel et al., 1999a). This model for malate dehydrogenase is based on three key observations (Madern et al., 2000a): 1. Enthalpic mechanisms dominate the kinetic deactivation in molar KCl, NaCl, and on the low concentration side of the stability curve in 2. The folded protein binds relatively large amounts of salt and water in KCl, NaCl, or solvents. 3. The excess of acidic amino acids in the protein composition could provide favored sites for specific water and ion binding to the tertiary or quaternary structure. X-ray diffraction studies on crystals of the halophilic malate dehydrogenase and the ferredoxin of Haloarcula marismortui and the dihydrofolate reductase of Haloferax volcanii have added much important information (Dym et al., 1995; Frolow et al., 1996; Mevarech et al., 2000; Pieper et al., 1998). These studies showed how the carboxylic groups on the acidic residues are used to sequester, organize, and arrange a tight network of water and hydrated ions at the surface of the protein, and to form an unusually large number of internal salt bridges with strategically located basic amino acid residues to provide internal structural rigidity of the protein. These salt bridges appear to be important determinants in the stabilization of the threedimensional structure of halophilic proteins. Intervening solvent molecules shield the negative charges of the carboxylic acid groups on the protein surface from each other. Comparison of the Haloarcula marismortui ferredoxin with the plant-type 2Fe-2S ferredoxin shows that the surface of the halophilic protein is coated with acidic residues except for the vicinity of the iron-sulfur cluster, and that it contains two additional helices near the N-terminus which form a separate hyperacidic domain, postulated to provide extra surface carboxylates for solvation. Bound water molecules on the protein surface have on the average 40% more hydrogen bonds than in a typical non-halophilic protein crystal structure. These water molecules are thus tightly bound within the hydration shell by protein-water and water-water hydrogen bonds and by hydration of interspersed ions (Frolow et al., 1996). A recent study of the glutamate dehydrogenase of Halobacterium salinarum showed the surface of the molecule being decorated with acidic residues. The exposed hydrophobic character is thus significantly reduced as compared to non-halophilic proteins. The difference arises not from a loss of surface-exposed hydrophobic

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residues, as has previously been proposed, but rather from a reduction in surfaceexposed lysine residues. The low lysine content helps to increase the overall negative charge on the protein surface, but also serves to decrease the hydrophobic fraction of the solvent-accessible surface (Britton et al., 1998). Mevarech et al. (2000) have summarized the available data on halophilic adaptation of proteins in a comprehensive model. According to this model, the negative charges on the protein's surface have a dual role. First of all, they provide hydrated carboxylate groups that maintain the solubility of the protein in the presence of high salt concentrations. Secondly, their destabilizing electrostatic repulsion offsets the stabilization gained by the enhancement of the hydrophobic effect by salt. The presence of molar quantities of salt would rigidify the enzyme's hydrophobic core, thereby abolishing the dynamic fluctuations and flexibility of the protein that are necessary for catalytic activity. The electrostatic repulsion allows the enzyme to maintain the marginal stability that is the hallmark of protein structure. The saltinduced stabilization of the protein can arise, in the case of stabilizing ions such as phosphate that are excluded from the protein surface, by ordering of the bulk solvent, but also through a direct interaction of ions with the folded protein. The requirement for high NaCl or KCl concentrations for the stabilization of halophilic malate dehydrogenase can then be explained by specific, but low affinity binding of only a few ions to the folded protein. Because of the low affinity of the binding sites, molar concentrations of salt are necessary to saturate these sites. The different efficacy of stabilization of different salts might reflect a combination of their position in the Hofmeister series in enhancing the hydrophobic effect and the affinity of the specific ion for these sites. It was recently shown that under hyposaline conditions, halophilic Archaea may produce a chaperone-like enzyme that serves to repair proteins damaged as a result of lack of salt. A 45 kDa protein was identified in Haloarcula marismortui that forms complexes displaying chaperone-like activities in vitro. The protein, which does not show significant homology to any known protein, has ATPase activity. It assembles into a large ring-shaped oligomeric complex of about 10 subunits. The oligomer was shown to form complexes with halophilic malate dehydrogenase during salt-dependent denaturation-renaturation, and to decrease the rate of deactivation of the enzyme at low salt, dependent on presence of ATP. The protein does not require high salt for activity and stability, and accumulates in cells exposed to low salt (Franzetti et al., 2001). The examples given above show that considerable progress has been made toward the understanding of the behavior of halophilic proteins in high-salt environments. At least in a limited number of model proteins has a reasonable understanding been achieved of those forces that play a role in the stabilization of halophilic proteins. However, we hardly know anything about the influence of high salt concentrations on the interactions between different proteins and between proteins and other macromolecules in the cell. In a review on the properties of halophilic proteins, written in 1995, Eisenberg wrote: "Whereas the stability and activity of enzymes and other proteins can be modified to perform at high salt concentrations by use of currently known structural concepts, the existence of meaningful protein nucleic acid

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interactions in physiological concentrations of 4 to 5 M KC1 constitutes an unsolved enigma worth intensive investigation". This statement is as true today as it was at the time those words were written. Certain enzymes from halophilic Archaea show markedly thermophilic properties. The alanine dehydrogenase of Halobacterium salinarum is a well-known example (Keradjopoulos and Holldorf, 1977; Keradjopoulos and Wolff, 1974). The assimilatory nitrate reductase of Haloferax mediterranei is optimally active at 80 °C at 3.1 M NaCl (Martinez-Espinosa et al., 2001b). A nitrate reductase of Halobacterium sp. (?) isolated from the Great Salt Lake, Utah, is optimally active at 85 °C when suspended in 4.27 M NaCl, at 73 °C in 2 M KCl, and at 56 °C in 0.5 (Marquez and Brodie, 1973). The amyloglucosidase of Halorubrum sodomense functions optimally at 75 °C (Oren, 1983). Table 7.1 presents a few additional examples of such high temperature optima. However, such a thermophilic behavior is not a general property of the enzymes of the halophilic Archaea. 7.3. PURIFICATION OF HALOPHILIC PROTEINS The requirement for salt for structural stabilization and activity poses severe limitations to the techniques that can be used to purify halophilic enzymes (Eisenberg, 1987; Eisenberg et al., 1992). High salt concentrations should be continuously present to maintain the proteins in their native, active conformation. Thus, ion exchange chromatography with an increasing salt gradient to elute proteins is generally unsuitable for salt-requiring proteins. Techniques that have proven valuable include gel filtration and hydroxylapatite chromatography using high salt buffers, hydrophobic chromatography with a decreasing gradient of ammonium sulfate on Sepharose 4B, carboxymethylcellulose and other column materials, and affinity chromatography (Mevarech et al., 1976, 2000). Electrophoresis of native proteins is generally not feasible either because of the high electrical conductivity of the suspending solution. It also should be noted that the use of denaturing electrophoresis on SDS-polyacrylamide gels to obtain information on the molecular mass of halophilic proteins often overestimates the true molecular mass as the excess of acidic charges causes a retardation of the migration velocity of the proteins in the electric field (Vyazmensky et al., 2000). Table 7.1 provides a number of examples of this phenomenon. Due to the high acidity of the proteins of the halophilic Archaea, isoelectric focusing is generally of little use for protein characterization and isolation. In Halococcus salifodinae all proteins were found to have isoelectric points between 3.8 and 4.5 (Denner et al., 1994). Analysis of the genome of Halobacterium strain NRC-1 confirmed the highly acidic nature of its proteome: an average pI of 5.1 was predicted for the proteins and putative proteins identified within the genome (Ng et al., 2000). It is sometimes possible to purify halophilic proteins in the absence of salt in a denatured state, provided they can refold to the active form under controlled conditions. The first purification of a halophilic protein - the malate dehydrogenase

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of Halobacterium salinarum - was performed at low salt concentration by methods involving ion exchange chromatography and electrophoresis. The pure enzyme was then reactivated by dialysis against 4.3 M NaCl. The yield obtained, however, was poor (about 0.5%) (Holmes and Halvorson, 1965a). More success is now achieved with the reactivation of the malate dehydrogenase of Haloarcula marismortui. The enzyme can be cloned and expressed in Escherichia coli. The protein is then recovered in an inactive form, but it can be activated with the formation of a functional enzyme by increasing the salt concentration to 3 M (Cendrin et al., 1993). Dialysis against 4 M NaCl refolded Halobacterium salinarum isocitrate dehydrogenase inactivated during purification at low salt (Hubbard and Miller, 1969). Similarly, the genes for dihydrolipoamide dehydrogenase and citrate synthase from Haloferax volcanii could be cloned and expressed in Escherichia coli, the citrate synthase in soluble and inactive form, the dihydrolipoamide dehydrogenase as inclusion bodies. Both enzymes could then be reactivated at high salt (Connaris et al., 1999). The glucose dehydrogenase of Haloferax mediterranei, overexpressed in inactive form in Escherichia coli, could also be refolded and reactivated at increased salt concentrations (Pire et al., 2001). Another example in which reactivation has been successfully applied is the nucleotide diphosphate kinase of Halobacterium salinarum. Although its amino acid composition is typical of that of halophilic enzymes, and the net negative charge of the enzyme (23 mol% excess acidic over basic amino acids) is in the range of halophilic proteins, the active enzyme maintains its conformation also in the absence of salt. The recombinant enzyme (a 24 kDa protein) expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding it no longer requires the presence of salts for stability or activity (Ishibashi et al., 2001). The catalase-peroxidase gene from Halobacterium salinarum was expressed in Escherichia coli as a functional enzyme, displaying both catalase and peroxidase activity (Long and Salin, 2001). There are also cases in which organic solutes can replace the role of salt to some extent in the stabilization of halophilic archaeal proteins. The NADP- dependent glutamate dehydrogenase of Haloferax mediterranei is stabilized by glycerol in the absence of salt (Ferrer et al., 1996). Polyols stabilize the Haloferax mediterranei glucose dehydrogenase as well as salts do. Sorbitol provides the best stabilization, followed by xylitol and erythritol, but glycerol is a relatively poor stabilizer (Obón et al., 1996). A 66 kDa extracellular protease from Halobacterium salinarum could also be protected to some extent by organic solvents against inactivation at low salt. Enzyme stability in aqueous/organic solvent mixtures correlated with the salting-out capacity of the solvent. Solvents that act to increase the apparent hydrophobicity of the enzyme's core stabilize in much the same way as salting-out salts. Dimethylsulfoxide and ethanol acted as stabilizers, while tetrahydrofuran, acetone, and dioxane destabilized (Kim and Dordick, 1997). The zwitterionic glycine betaine at a concentration of 4 to 6 M could be used to stabilize certain Halobacterium salinarum enzymes during non-denaturing electropho-

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resis in which the use of salt at high concentrations is prohibitive (Cadenas and Engel, 1994). The Haloarcula marismortui malate dehydrogenase can be stabilized by sub-millimolar concentrations of NADH; molar concentrations of salt are then no longer required (Mevarech et al., 2000). Use of nondetergent sulfobetaines as mild solubilizing agents has been suggested as a convenient procedure for the purification of active halophilic proteins at low salt. The procedure has been successfully applied to the purification of malate dehydrogenase and elongation factor Tu of Haloarcula marismortui by ion-exchange chromatography (Vuillard et al., 1995).

7.4. SALT RELATIONSHIPS OF SELECTED PROTEINS FROM HALOPHILIC ARCHAEA Many enzymes from halophilic Archaea of the family Halobacteriaceae have been purified and characterized, some of them in great depth. Table 7.1 presents examples of cytoplasmic enzymes that have been studied, and Table 7.2 gives examples of the properties of extracellular enzymes. Details on a few enzymes that have been investigated in great detail or that show properties of special interest are given below. A number of recent developments in this field have been reviewed by Madigan and Oren(1999). The analysis of complete genomes and proteomes, such as already available for Halobacterium salinarum, and such as may become available for other representatives of the family in the near future, will undoubtedly add a wealth of information on the ways halophilic enzymes have adapted to function in the presence of molar concentrations of salt.

7.4.1. Malate dehydrogenase of Haloarcula marismortui The malate dehydrogenase of Haloarcula marismortui has become the best investigated model system used to obtain an understanding of the halophilic nature of proteins (Bonneté et al., 1994; Dym et al., 1995; Eisenberg, 1995; Eisenberg et al., 1992; Madern et al., 2000a, 2000b; Zaccai and Eisenberg, 1991; Zaccai et al., 1986, 1989). On the basis of its sedimentation rate in the ultracentrifuge and its electrophoretic behavior in denaturing gels the protein was originally described as a dimer (84 kDa) of 39 kDa subunits (Mevarech et al., 1977). We now know that the protein is a homotetramer. First evidence for this was obtained after crosslinking of the subunits with glutaraldehyde, followed by SDS gel electrophoresis (Daniel et al., 1993). The subunit polypeptide consists of 303 amino acids, and has a molecular mass of 32,638 Da (Bonneté et al., 1993; Cendrin et al., 1993). Aspartate and glutamate constitute 20.5% of all residues, while the content of lysine and arginine combined is only 7.6%. Small-angle X-ray scattering suggested that the protein has a spheroidal shape with a radius of gyration of 31.8 ± 0.6 Å. No evidence was obtained for the existence of an extensive outer hydration shell (Reich et al., 1982).

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The enzyme requires high salt concentrations for activity and stability. Optimum activity is achieved at about 1 M NaCl (Hecht et al., 1989; Mevarech et al., 2000). The earlier reported apparent activity optimum at 1-1.2 M with decreasing rates at higher concentrations (Mevarech and Neumann, 1977) is an artifact caused by the increase of the enzyme's affinity for oxaloacetate with increasing salt concentration (Hecht et al., 1989). The stabilization by different ions follows the Hofmeister lyotropic series. Anions of high charge density are the most efficient to stabilize the folded form, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond (Ebel et al., 1999b; Pundak et al., 1981). In the absence of salt the enzyme dissociates into monomers, and the content of is reduced to zero (Pundak et al., 1981). When suspended in solutions containing less that 2 M NaCl, the enzyme is inactivated in a first-order reaction. When salt is subsequently increased, reactivation occurs, which follows second-order kinetics (one of the arguments used at the time to postulate a dimeric structure for the active enzyme) (Mevarech and Neumann, 1977). The changes occurring in the protein during low-salt inactivation were investigated indepth using physical parameters such as sedimentation properties, light scattering measurements, neutron scattering, and circular dichroism (Bonneté et al., 1993; Madern et al., 2000b; Pundak and Eisenberg, 1981). It was concluded that upon decreasing the salt concentration below 2 M, the tetrameric enzyme dissociates directly into monomers. A recent neutron scattering study combined with circular dichroism measurements, comparing the halophilic malate dehydrogenase with bovine serum albumin, has revealed information on the fast dynamics of malate dehydrogenase. Complex correlations were found between dynamics and stability, correlations that are different for halophilic and non-halophilic proteins (Tehei et al., 2001). The crystallization of the protein and its structure elucidation at 3.2 Å resolution has provided a detailed picture of the three-dimensional structure of the enzyme (Dym et al., 1995). Comparison with other malate dehydrogenases and the highly homologous dogfish lactate dehydrogenase (also a tetrameric enzyme) shows that the surface of the halophilic tetrameric enzyme is coated with acidic residues. The net charge of the H. marismortui malate dehydrogenase is -156, while the dogfish lactate dehydrogenase has a net charge of +16. The halophilic tetrameric enzyme also has an unusually large number of salt bridges between glutamate or aspartate and arginine residues. The number of arginines per monomer is not excessively high, but compared with other similar enzymes many more are involved in formation of salt bridges, both intramolecular and intermolecular, thus stabilizing the tetrameric structure. All arginine residues that are not involved in the formation of the active site and in catalysis are believed to be involved in ionic interaction with acidic residues. Neutron scattering studies have shown that the hydration of the mesophilic and halophilic protein is similar, but that salt binding is approximately an order of magnitude larger for the halophile enzyme (Dym et al., 1995; Ebel et al., 1999).

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A combination of site-directed mutagenesis and X-ray crystallography has been used to provide information on the importance of selected amino acid residues within the protein (Madern et al., 2000a, 2000b; Richard et al., 2000). The E267R mutant was found to be more halophilic than the wild type protein; it is more sensitive to temperature and requires significantly higher concentrations of NaCl or KC1 for equivalent stability (Madern et al., 1995). The structure of the mutant enzyme E267R (at 2.6 Å resolution) was compared with that of the wild type protein (at 2.9 Å resolution). The tetrameric enzyme appears to be stabilized by an ordered water molecule network and by intersubunit complex salt bridges "locked in" by bound solvent chloride and sodium ions. The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions. It was concluded that halophilic adaptation is not aimed uniquely at "protecting" the enzyme from the extreme salt conditions, as may have been expected, but, on the contrary, consists of mechanisms that harness the high ionic concentrations in the environment (Richard et al., 2000).

7.4.2. Other halophilic dehydrogenases The properties of the glyceraldehyde dehydrogenase of Haloarcula vallismortis (molecular mass 36.0 kDa, as determined by mass spectrometry) have been investigated using physical techniques such as density sedimentation, light scattering, and small-angle neutron scattering measurements. The protein is a tetramer with a radius of gyration of 32 ± 0.5 Å at high salt, and a shape similar to an oblate ellipsoid of axial ratio 0.5, that binds 0.18 ± 0.10 g of water and 0.07 ± 0.02 g of KC1 per g of protein in the native state. Below 1 M salt the tetramer dissociates into unfolded monomers (Ebel et al., 1991, 1995). The cloning and expression of the Haloferax volcanii dihydrofolate reductase in Escherichia coli, followed by reconstitution of the active enzyme by dissolving the protein in 6 M guanidine hydrochloride and dilution in salt solutions, opened the way to the study of this protein and site-specific mutants generated from it (Blecher et al., 1993). The enzyme does not require high salt concentrations: it retains its activity and secondary structure at salt concentrations as low as 0.5 M. By comparative modeling of the protein structure to the known dihydrofolate reductase of Escherichia coli, a model was made explaining the halophilic adaptation of the enzyme. The molecule shows a unique asymmetrical charge distribution with positive charged amino acids centered around the active site, with negative charges at the opposite side of the enzyme. The negative charges on the surface form clusters which are shielded at high salt concentrations, while at low salt they repel each other, thereby destabilizing the protein (Böhm and Jaenicke, 1994). When the Haloferax volcanii dihydrofolate reductase was crystallized and its structure determined at 2.6 Å resolution, it was confirmed that the protein shows the same overall folding structure as other known dihydrofolate reductases. However, the protein shows clusters of non-interacting negatively charged residues, which may account for its instability at salt concentrations lower than 0.5 M because of electrostatic repulsion (Pieper et al., 1997).

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Information has also been obtained on the 2-oxoacid:ferredoxin oxidoreductases of Halobacterium salinarum and the 2-oxoacid dehydrogenase multienzyme complex of Haloferax volcanii. The function of the latter enzyme in halophilic Archaea is not known, as oxidation of pyruvate and 2-oxoglutarate is mediated by the ferredoxinlinked enzyme systems in these organisms. The 2-oxoacid:ferredoxin oxidoreductases of Halobacterium salinarum have been purified and their catalytic mechanism elucidated. The enzyme contains two [4Fe-4S] clusters, and 1.3-1.6 thiamine diphosphate (in the case of pyruvate:ferredoxin oxidoreductase and 2oxoglutarate:ferredoxin oxidoreductase, respectively) per enzyme complex of 250 kDa molecular mass (Kerscher and Oesterhelt, 1981a, 1981b). Haloferax volcanii also has an operon that contains the dihydrolipoamide dehydrogenase gene and three open reading frames that show the highest sequence identities with the (2-oxoacid decarboxylase), and E2 (dihydrolipoyl acyltransferase) genes of the pyruvate dehydrogenase multienzyme complex (Jolley et al., 2000). The physiological function of this enzyme is still unclear. Specific mutations have been introduced in the dihydrolipoamide dehydrogenase of Haloferax volcanii in which a specific glutamate residue that supposedly is involved in the binding of was mutated, and this led to significant changes in salt dependence, kinetic properties and thermal stability (Jolley et al., 1997). The glucose dehydrogenase of Haloferax mediterranei has recently been crystallized (Ferrer et al., 2001a), opening the way toward a detailed structural and functional analysis of the protein.

7.4.3. Haloarchaeal ferredoxins Together with the Haloarcula marismortui malate dehydrogenase, the ferredoxins of Halobacterium salinarum, Haloarcula marismortui, and other members of the Halobacteriaceae have become convenient models to study the effect of salts on halophilic proteins. One of the supposed functions of this ferredoxin is as electron donor during the dissimilatory reduction of nitrite, at least in Haloarcula marismortui (Werber and Mevarech, 1978). The haloarchaeal ferredoxins show a high similarity with plant-type [2Fe-2S] ferredoxins (Kerscher and Oesterhelt, 1981a, 1981b, 1982). The Haloarcula marismortui protein has 128 amino acids (Hase et al., 1980) with a large excess of acidic amino acids. It contains a total of 44 glutamate and aspartate of which only four in the amide form, but only six lysine and arginine residues combined. The ferredoxin gene from Haloarcula japonica has been cloned. The open reading frame codes for a 129 amino acid protein, showing between 84 and 98% identity with sequences of other halophilic Archaea (Matsuo et al., 2001). Halophilic ferredoxins have been studied by methods including Mössbauer and EPR/electron spin resonance spectra (Sugimori et al., 2000; Werber et al., 1978), fluorescence (Bandyopadhyay and Sonawat, 2000; Gafni and Werber, 1979), 1H-NMR (Gochin and Degani, 1985), and circular dichroism measurements (Bandyopadhyay and Sonawat, 2000). The Halobacterium salinarum ferredoxin occurs in beta-sheet

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conformation, and requires at least 1.5-2 M salt for stabilization. At lower salt concentrations the protein unfolds with loss of tertiary structure, leading to the disruption of the [2Fe-2S] center, and eventually resulting in the loss of secondary structural elements (Bandyopadhyay and Sonawat, 2000). The protein is stabilized both by neutralization of electrostatic repulsion among carboxyl groups of the acidic residues and by salting out of hydrophobic residues, leading to their burial and stronger interaction (Bandyopadhyay et al., 2001). The solution structure of the Halobacterium salinarum [2Fe-2S] ferredoxin has been solved by nuclear magnetic resonance methods (Schweimer et al., 2000). The Haloarcula marismortui ferredoxin has been crystallized in crystals diffracting to 1.9 Å and even less (Sussman et al., 1979). A detailed analysis at 1.9 Å resolution showed the surface of this protein to be covered with acidic residues, except for the vicinity of the 2Fe-2S cluster. Two helices near the N-terminus form a hyperacidic domain whose postulated function is to provide extra surface carboxylates for solvation. Bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non-halophilic protein, showing that haloadaptation involves better water binding capacity. A total of 237 molecules of water were found associated with the protein, i.e. an average of 1.9 per amino acid residue or 3.6 molecules per 100 Å2 of accessible surface area (Frolow et al., 1996).

7.4.4. The ribosomal proteins of halophilic Archaea Haloarcula marismortui has become a model organism for the study of the structure and function of prokaryote ribosomes (Ban et al., 2000; Francheschi et al., 1994; Shevack et al., 1985; Yonath, 2002) (see also Section 3.1.5). The ribosomal proteins are highly acidic. Characterization of these proteins has started in the early 1970s (Strøm and Visentin, 1973), and many studies have been devoted to the in-depth analysis of the structure of individual ribosomal proteins (e.g. Arndt et al., 1986). Also other components of the protein synthesis machinery have been subject to intensive studies, such as the elongation factor Tu, which has been used in physical characterizations (Ebel et al., 1992), Protein synthesis by ribosomes of halophilic Archaea is strictly dependent on high salt concentrations. A cell-free amino acid incorporating system for Halobacterium salinarum ribosomes functions optimally in a mixture of 3.8 M KC1, 1 M NaCl, and 0.4 M (Bailey and Griffith, 1968).

7.5. HALOPHILIC BEHAVIOR OF ENZYMES FROM THE AEROBIC HALOPHILIC BACTERIA The aerobic halophilic Bacteria occupy an intermediate position between the nonhalophiles and the extremely halophilic Archaea. They tolerate and sometimes require high salt concentrations for growth, but salt is as much as possible excluded from the cells in favor of the use of organic osmotic solutes to provide osmotic balance. The

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special case of Salinibacter ruber (Oren and Mana, 2002) is probably a rare exception to the rule (see also Table 7.7). However, apparent intracellular salt concentrations can be fairly high in many halophilic Bacteria (see Section 6.4 and Table 6.1), so that a certain extent of adaptation to the presence of elevated salt concentrations may be required at least in some cases. Determinations of the abundance of different amino acids in the bulk protein of Halomonas elongata showed a certain excess of acidic amino acids and a relatively low frequency of basic amino acids, values being intermediate between the non-halophile Escherichia coli and the halophilic archaeon Haloferax mediterranei. Convergent evolution of amino acid usage was suggested to have led to this moderately "halophilic" character of the Halomonas elongata proteins (Gandbhir et al., 1995). However, this convergent evolution did not lead to changes in the frequencies of the hydrophobic amino acids. The ratio of the occurrence of hydrophobic amino acids (alanine, valine, leucine, isoleucine, phenylalanine, methionine) as compared to the borderline hydrophobic amino acids (serine, threonine) was 3.56 in Halomonas elongata, to be compared to 3.58 in Escherichia coli, 2.73 in Haloferax mediterranei, and 2.46 in membranes of Halobacterium salinarum (Oren, 1995). Comparative studies of ribosomal proteins from moderately halophilic Bacteria showed in many cases a slightly higher content of acidic amino acids than the corresponding ribosomal proteins from Escherichia coli and other non-halophilic organisms. The ratio of basic/acidic amino acids for whole ribosomes of Chromohalobacter canadensis was 1.23 or 1.16 for cells grown in 0.5 and 4.25 M NaCl, respectively, as compared to 1.58 in Escherichia coli and 0.69 in Halobacterium salinarum (Falkenberg et al., 1976). The average hydrophobicity of the ribosomal proteins of Salinivibrio costicola and Chromohalobacter canadensis did not greatly differ from the value of Escherichia coli, and was much higher than that of Halobacterium salinarum (Matheson et al., 1978). Intracellular protein turnover has been measured in Salinivibrio costicola by following the breakdown of pulse-labeled proteins. In cells growing exponentially in 1 or 1.5 M NaCl the turnover was about 5% per hour, but in cells growing in 0.5 M NaCl turnover rates as high as 9% per hour were recorded. These values should be compared with 1-2% per hour in Escherichia coli. When Salinivibrio cells grown at high salt were transferred to a low salinity, the turnover rate of pulse-labeled proteins increased greatly. It was suggested that the low salt concentration alters the "native" protein conformation of the slightly halophilic proteins, thereby increasing the susceptibility of these proteins to proteolysis. The high protein turnover at low salinity may possibly set the lower limit to the salt concentrations enabling growth of moderately halophilic Bacteria (Hipkiss et al., 1980). Three distinct categories of enzymatic activities should be discriminated when discussing the salt relationships of enzymes from halophilic Bacteria: (1), true intracellular enzymes, which are not exposed to the salt concentration of the medium, but instead sense the intracellular environment which in most cases is characterized by low ion concentrations and the presence of organic osmotic solutes (see Chapter 8); (2), membrane-bound activities, including transport proteins, which may sense both

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the intracellular environment and the outer medium, and (3), extracellular enzymes, fully exposed to the external hypersaline conditions. A study of the amino acid composition of different protein fractions in photosynthetic Bacteria of the genera Ectothiorhodospira and Halorhodaspira with different salt requirements nicely illustrated this point. These organisms use organic solutes (glycine betaine, ectoine, trehalose) to provide osmotic balance, while maintaining low intracellular ionic concentrations (see Chapter 8). Little difference was found in the amino acid composition of the bulk protein in the different species examined. However, membrane fractions obtained from those species requiring higher salt concentrations are more enriched in polar amino acids and had a lower content of nonpolar amino acids than the membrane proteins from their less salt-requiring relatives (Imhoff et al., 1983). The earliest studies already showed that the intracellular enzymes generally function optimally at low salt concentrations, such as shown for isocitrate dehydrogenase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of Halomonas halodenitrificans (see also Figure 7.1), while membranal enzymes such as lactate dehydrogenase and cytochrome oxidase of the same organism are more active in the presence of salt (Baxter and Gibbons, 1956). Studies of intracellular enzymes of moderate halophiles are of special interest, as the properties of the enzymes may shed light on the nature of their true intracellular environment, which to some extent is still subject to considerable controversy, as discussed in Chapters 6 and 8. The example of Bacillus haloalkaliphilus illustrates this point. This organism was earlier suggested to contain very high intracellular salt concentrations (Weisser and Trüper, 1985; see also Section 6.4 and Table 6.1), concentrations that are incompatible with the existence of salt-sensitive enzymes. However, the intracellular malate dehydrogenase and isocitrate dehydrogenases of Bacillus haloalkaliphilus are inhibited by salt, with only about 20% of the optimal activity remaining above 1-2 M NaCl (Weisser and Trüper, 1985). This interesting organism is now known to accumulate ectoine as osmotic solute (see Table 8.1), and the apparently high intracellular ion concentrations reported in the past may have been the result of experimental artifacts. No consistent picture on the possible properties of the intracellular environments emerges from the data available on the properties of individual enzymes, as shown in the examples given in the following paragraphs and in Table 7.3. Additional information can be found in reviews by Cazzulo (1979), Kushner and Kamekura (1988), and Ventosa et al. (1998). The pyruvate kinase of Salinivibrio costicola has a high content of aspartate and glutamate, a low content of hydrophobic amino acids, and is enriched in glycine and serine, all typical characteristics of halophilic proteins (De Médicis, 1986; De Médicis and Rossignol, 1979). The enzyme is most stable at high salinities. However, while a low concentration (0.25 M) of NaCl or KC1 was somewhat stimulatory, virtually no activity was detected above 1 M salt. If indeed the intracellular and concentrations are as high as 0.5-1.8 M and 0.2-0.8 M, as claimed for this organism (see Table 6.1), the enzyme would be strongly inhibited at the physiological conditions (De Médicis and Rossignol, 1979).

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The phosphoenolpyruvate carboxykinase of Salinivibrio costicola docs not show a great excess of acidic over basic amino acid residues. However, an unusually high content of glycine and serine was found (16.7 and 10.2 mol%, respectively); these amino acids supposedly maintain the balance between hydrophilic and hydrophobic forces. Decarboxylation of oxaloacetate and exchange were activated by NaCl and KCl at concentrations up to 1 M, activities being about 2.5 times as high as in the absence of salt, while the optimum for carboxylation of phosphoenolpyruvate was found at 0.025-0.05 M salt (2 to 2.5 times as high as in the absence of salt). Under physiological conditions the enzyme probably acts in the direction of phosphoenolpyruvate synthesis (Salvarrey et al., 1989, 1995). The complex enzymatic machinery required for protein synthesis by the ribosomes presents an excellent object for the study of the influence of salt on intracellular activities. Extensive efforts have been dedicated to the study of in vitro protein synthesis by Salinivibrio costicola ribosomes (Choquet et al., 1989; Kamekura and Kushner, 1984; Kushner, 1989, 1991; Wydro et al., 1977). These studies were based on the measurement of poly-U-directed phenylalanine incorporation or the incorporation of 14C-valine, using Escherichia coli phage RNA as template. Optimum activity was obtained at low salt concentrations, 0.1-0.3 M ammonium glutamate being most stimulatory. Chloride salts proved especially inhibitory. KCl and NaCl were found increasingly toxic, and hardly any activity was observed in the presence of 0.6 M Chloride prevents the attachment of the 50S ribosomal subunit to the 30S subunit - messenger RNA complex, and also displaces ribosomes already bound. The inhibitory effect of chloride could in part be reversed by addition of glycine betaine or glutamate. When chloride was replaced by other anions such as glutamate, sulfate, or acetate, high in vitro protein synthesis rates were found at salt concentrations as high as 0.6 M, both in Salinivibrio costicola and in Chromohalobacter canadensis (Choquet et al., 1989; Kamekura and Kushner, 1984; Kushner, 1989, 1991; Wydro et al., 1977). The ribosomes of Salinivibrio costicola showed an unusual sedimentation behavior in sucrose gradients: association of subunits to complete ribosomes took only place in high salt (Wydro et al., 1975). A study of the binding of dihydrostreptomycin to the 30S ribosomal subunits of Salinivibrio costicola showed more binding in cells grown at high salt concentrations, suggesting that some ribosomal properties may vary with the salt concentration in which the cells were grown (Kogut and Madira, 1978). Other intriguing cases have been reported in which the properties of certain enzymes depended on the salinity of the growth medium. Thus, the alanine dehydrogenase of Halomonas elongata cells grown in 50 mM salt was optimally active at salt concentrations between 0-20 mM, while the enzyme isolated from 1.37 M and 3.4 M salt-grown cells had its optimum at 340 mM and 500-600 mM, respectively (Bylund et al., 1991). The estimated intracellular salt concentrations of the cells grown at the respective salinities were 42, 312, and 630 mM (Vreeland et al., 1983). The enzyme thus appears to be adapted well to the actual conditions inside the cells. No simple explanation has been suggested to explain the phenomenon, but it was noted that in cells grown at 0.05 and 1.37 M

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salt two alanine dehydrogenase bands were detected on native gels, while in cells grown in 3.4 M only a single band was seen (Bylund et al., 1991). Table 7.4 lists a number of membrane-bound activities studied in the moderate halophiles, including information on their optimum salt concentration. Membranebound enzymes are in contact with the high salt concentrations in the medium, and therefore they should be expected to be active at high salt, or even to require high salt concentrations for full activity. The behavior the transporter of (AIB), a non-metabolizable amino acid analogue, provides a good example. In Salinivibrio costicola, AIB transport is competitively inhibited by glycine, alanine, and to a lesser extent by methionine (Hamaide et al., 1984a). In Halomonas elongata competition was observed by glycine, alanine, L-serine, D-serine, D-alanine, and Lhomoserine, and to a lesser extent by methionine, leucine, phenylalanine, and histidine (Martin et al., 1983). The transport activity is at concentrations below 0.2 M increases the apparent affinity of the Salinivibrio costicola transport system for AIB. Concentrations between 0.2 and 1 M increase the of the transport, while above 1 M NaCl the decreases without affecting the (Hamaide et al., 1984a, 1984b). In cells grown in the presence of 1 or 2 M NaCl, AIB transport was active at higher salinities than in cells grown in 0.5 M NaCl. Low salt-grown cells adapted to AIB transport at high salinities after 6 h incubation at high salt in a process independent of protein synthesis (Kushner et al., 1983). A similar phenomenon was reported for Halomonas elongata, which showed an optimum NaCl for AIB uptake of 0.38 M in low-salt grown cells and 1.37 M in high-salt grown cells (Martin et al., 1983). Adaptation of AIB transport in Salinivibrio costicola to the highest salinities (4 M NaCl) required presence of nutrients, probably due to the need to accumulate compatible solutes; glycine betaine was especially stimulatory (Kushner et al., 1983). The transport shows an optimum at pH 8.5-9, and requires the presence of sodium and a membrane potential (Hamaide et al., 1984a, 1984b; MacLeod, 1986). is required for AIB transport in all those Gram-negative bacteria in which presence of a respiration-driven primary sodium pump was demonstrated (Halomonas halophila, Halomonas variabilis, Chromohalobacter canadensis, Salinivibrio costicola, Pseudomonas beijerinckii, and "Pseudomonas halosaccharolytica"). Two Grampositive bacteria that lack a respiration-driven pump (Marinococcus halophilns, "Micrococcus varians subsp. halophilus") do not require for AIB uptake (Unemoto et al., 1993). NaCl causes a 2-3 fold stimulation of the transport activity of proline in Chromohalobacter israelensis. As in the case of AIB transport in Salinivibrio costicola and Halomonas elongata, the maximum uptake rate of proline was observed at salt concentrations similar to those in which the cells were grown (Peleg et al., 1980). A membrane-bound 5'-nucleotidase of Salinivibrio costicola is optimally active at 2 M NaCl or KCl or higher. This enzyme splits both ATP, ADP and AMP; these are dephosphorylated to adenosine, which is subsequently transported into the cells. Its activity is distinct from that of the ATPase also present in the membrane, and can be differentiated from that of the proton channel ATPase by its higher magnesium

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requirement and its insensitivity to dicyclohexylcarbodiimidc (DCCD) (Bengis-Garber and Kushner, 1981, 1982). Also the DCCD-inhibited ATPase of Salinivibrio costicola requires high salt for both activity and stability. Optimal activity was reported in 0.5 M KCl. KCl concentrations of up to 3 M caused relatively little inhibition, but 75% inhibition was found by 3 M NaCl (Cazzulo, 1978; Higa and Cazzulo, 1978). Also the NADH:quinone oxidoreductase of Chromohalobacter israelensis depends on sodium for activity. Sodium ions increase the rate of quinone reduction several-fold, but oxidation of the quinol with oxygen is not affected by sodium (Ken-Dror et al., 1986). Extracellular enzymes of halophilic Bacteria, even those that maintain very low intracellular salt concentrations, may be expected to be halophilic proteins, adapted to function at the high salt concentrations prevailing outside the cell. As Table 7.5 shows, this assumption is true in most cases. Such extracellular enzymes also show an excess of acidic amino acids characteristic for salt-adapted proteins. Thus, the amylase of Halomonas meridiana has 12.4% acidic residues and only 5.5% basic residues (Coronado et al., 2000b). This excess negative charge is much higher than that of non-halophilic but still less than that of the archaeal exoenzyme excreted by Natronococcus (Table 7.6). An intriguing case of salt adaptation of medium-exposed enzymes in halophilic Bacteria is that of the phosphatases of Halomonas elongata. This organism has both alkaline and acid phosphatases located on the cell envelope. In cells grown at low salt (50 mM), very little phosphatase activity was detected. In cultures grown at higher salinities (1.4 and 3.4 M), the activities of both enzymes were found optimal at the same salt concentration in which the cells had grown. These enzymes thus appear to "adapt" to the NaCl concentration in the medium. The mechanism of such an adaptation was never further elucidated. The effect was probably not due to the presence of isozymes, as only one band of each enzyme was detected on electrophoresis gels (Bylund et al., 1990).

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An exceptional case among the aerobic halophilic Bacteria is presented by the recently discovered Salinibacter ruber. The cytoplasm of this red, extremely halophilic organism has a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein shows a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine (Oren, and Mana, 2002). The salt dependence of four cytoplasmic enzymatic activities has been examined (Table 7.7). The NAD-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl activity was low. The organism also contained NADPdependent isocitrate dehydrogenase activity. Its activity was about constant over the whole range from 0 to 3.2 M NaCl, with a slight increase with increasing KCl. NADdependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NADdependent glutamate dehydrogenase, assayed by the reductive amination of 2oxoglutarate, showed activity in the absence of salt. The rate decreased with increasing KCl concentration, and no activity was found above 2.5 M KCl. However, specific stimulation occurred by high NaCl concentrations, optimum activity being found at 3-3.5 M. Salinibacter enzymes thus appear to be adapted to function in the presence of high salt concentrations, similar to the proteins of the Halobacteriaceae (Oren and Mana, 2002).

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7.6. HALOPHILIC BEHAVIOR OF ENZYMES FROM THE ANAEROBIC HALOPHILIC BACTERIA The fermentative anaerobic Halanaerobiales occupy a special position within the halophilic Bacteria: high intracellular ionic concentrations occur in the representatives of this order, and organic osmotic solutes have never been detected within the cells in significant concentrations. Accordingly, presence of salt-adapted enzymes is to be expected in this group. This is indeed the case, as shown by the examples given in Table 7.8. The enzymes tested generally function better in the presence of molar concentrations of salts than in salt-free medium, and they can be expected to be fully active at the actual salt concentrations present in the cytoplasm (Oren and Gurevich, 1993; Rengpipat et al., 1988; Zavarzin et al., 1994). The bulk cellular protein of those members of the Halanaerobiales tested is highly acidic, almost as much as the proteins of the aerobic halophilic Archaea (Oren, 1986). However, no especially high content of acidic amino acids was found in the ribosomal A-protein of Halanaerobium praevalens (Matheson et al., 1987). Analysis of a partial genome library of Halothermothrix orenii, a thermophilic representative of the group, failed to show high levels of acidic amino acids in the putative proteins coded by the 85 kb fragment analyzed. It was argued that this may be related to the thermophilic nature of the organism and the intermediate salt concentration at which it lives (Mijts and Patel, 2001).

7.7. HALOPHILIC BEHAVIOR OF ENZYMES FROM THE HALOPHILIC EUCARYA Most cytoplasmic and chloroplast-located enzymes of Dunaliella are salt-sensitive. Table 7.9 lists a number of enzymes whose relationship to salt has been tested. Dunaliella cells contain low intracellular ionic concentrations (see Section 6.6), and they accumulate glycerol as osmotic solute (see Section 8.4). Some Dunaliella enzymes (chloroplast ATPase, dihydroxyacetone kinase) are specifically stimulated and/or stabilized by glycerol. The toxic factor for many salt-sensitive enzymes of Dunaliella is the chloride anion, not the cations sodium or potassium. It was suggested that chloride competes with anionic substrates for active site of the enzymes. Saltsensitive enzymesof Dunaliella parva that are 50% inhibited by less than 0.5 M NaCl include dihydroxyacetone reductase, glutathione reductase, acid phosphatase, ATP synthase, glycerol-3-phosphate phosphatase, phosphoenolpyruvate carboxylase, and ribulose-l,5-bisphosphate carboxylase. Relatively salt-resistant enzymes are lactate dehydrogenase, invertase, and carbonic anydrase, as are uncoupled photosynthetic electron transport from water to ferricyanide and from ascorbate-dichlorophenol indophenol to diquat (Gimmler et al., 1984). The nitrate reductase of Dunaliella parva is more salt-tolerant than its counterparts from Chlorella and tobacco cells (Heimer, 1973).

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Some enzymes located in the cytoplasmic membrane of Dunaliella, that are exposed to the salt concentrations present in the outer medium, show marked halophilic properties, including an excess of acidic amino acids. The membrane-bound carbonic anhydrase that is induced at elevated salt concentrations is an example of such a halophilic enzyme (Fischer et al., 1996).

7.8. REFERENCES Ahonkhai, I., Kamekura, M., and Kushner, D.J. 1989. Effects of salts on the aspartate transcarbamylase of a halophilic eubacterium, Vibrio costicola. Biochem. Cell Biol. 67: 666-669. Aitken, D.M., Wicken, A.J., and Brown, A.D. 1970. Properties of a halophil nicotinamide-adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme. Biochem. J. 116: 125-134. Altekar, W., and Rajagopalan, R. 1990. Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria. Arch. Microbiol. 153: 169-174. Alvarez-Ossorio, M., Muriana, F.J.G., de la Rossa, F.F., and Relimpio, A.M. 1992. Purification and characterization of nitrate reductase from the halophile archaebacterium Haloferax mediterranei. Z. Naturforsch. 47c: 670-676. Arndt, E., Breithaupt, G., and Kimura, M. 1986. The complete amino acid sequence of ribosomal protein H-S11 from the archaebacterium Halobacterium marismortui. FEBS Lett. 194: 227-234. Ban, N., Nissen, P., Hansen, J., Moore, P., and Steitz, T.A. 2000. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905-934. Bandyopadhyay, A.K., and Sonawat, H.M. 2000. Salt dependent stability and unfolding of [Fe2-S2] ferredoxin of Halobacterium salinarum: spectroscopic investigations. Biophys. J. 79: 801-810. Bandyopadhyay, A.K., Krishnamoorthy, G., and Sonawat, H.M. 2001. Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum. Biochemistry 40: 1284-1292. Baxter, R.M., and Gibbons, N.E. 1954. The glycerol dehydrogenases of Pseudomonas salinaria, Vibrio costicolus. and Escherichia coli in relation to bacterial halophilism. Can. J. Biochem. Physiol. 32: 206-217. Baxter, R.M., and Gibbons, N.E. 1956. Effects of sodium and potassium chloride on certain enzymes of Micrococcus halodenitrificans and Pseudomonas salinaria. Can. J. Microbiol. 2: 599-606. Bayley, S.T., and Griffiths, E. 1968. A cell-free amino acid incorporating system from an extremely halophilic bacterium. Biochemistry 7: 2249-2256. Belmans, D., and van Laere, A. 1988. Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella. Arch. Microbiol. 150: 109-112. Bengis-Garber, C., and Kushner, D.J. 1981. Purification and properties of 5'-nuclcotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium. J. Bacteriol. 146: 24-32. Bengis-Garber, C., and Kushner, D.J. 1982. Role of membrane-bound 5'-nucleotidase in nucleotide uptake by the halophile Vibrio costicola. J. Bacteriol. 149: 808-815. Bickel-Sandkötter, S., and Ufer, M. 1995. Properties of a dissimilatory nitrate reductase from the halophilic archaeon Haloferax volcanii. Z. Naturforsch. 50c: 365-372. Bishoff, K., and Rodwell, V.W. 1996. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii, purification, characterization and expression in Escherichia coli. J. Bacteriol. 178: 19-25. Blecher, O., Goldman, S., and Mevarech, M. 1993. High expression in Escherichia coli of the gene encoding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii. Reconstitution of the active enzyme and mutation studies. Eur. J. Biochem. 216: 199-203. Böhm, G., and Jaenicke, R. 1994. A structure-based model for the halophilic adaptation of dihydrofolate reductase from Halobacterium volcanii. Protein Engin. 7: 213-220. Bolobova, A. V., Siman'kova, M.V., and Markovich, N.A. 1993. Cellulase complex of a new halophilic bacterium Halocella cellulolytica. Mikrobiologiya 61: 804-811 (Microbiology 61: 557-562, 1993). Bonet, M.L., Llorca, F.I., and Cadenas, E. 1992. Alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium. Selective activation by manganese and effect of other divalent cations. Int. J. Biochem. 24: 839-845. Bonet, M.L., Llorca, F.I., and Cadenas, E. 1993. Evidence that the alkaline p-nitrophenylphosphate phosphatase from Halobacterium halobium is a manganese-containing enzyme. Int. J. Biochem. 25: 7-12.

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Bonete, M.J., Camacho, M.L., and Cadenas, E. 1987. A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity. Int. J. Biochem. 19: 1149-1155. Bonete, M.-J., Pire, C., Llorca, F.I., and Camacho, M.L. 1996. Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: enzyme purification, characterisation, and N-terminal sequence. FEBS Lett. 383: 227-229. Bonete, M.J., Ferrer, J., Pire, C., Penades, M., and Ruiz, J.L. 2000. 2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family. Biochimie 82: 1143-1150. Bonneté, F., Ebel, C., Eisenberg, H., and Zaccai, G. 1993. A biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions of native and recombinant enzyme. J. Chem. Soc. Faraday Trans. 89: 2659-2666. Bonneté, F., Madern, D., and Zaccai, G. 1994. Stability against denaturation mechanisms in halophilic malate dehydrogenase "adapt" to solvent conditions. J. Mol. Biol. 244: 436-447. Britton, K.L., Stillman, T.J., Yip, K.S.P., Forterre, P., Engel, P.C., and Rice, D.W. 1998. Insights into the molecular basis of salt tolerance from the study of glutamate dehydrogenase from Halobacterium salinarum. J. Biol. Chem. 273: 9023-9030. Brown, A.D. 1990. Microbial water stress physiology. Principles and perspectives. John Wiley & Sons, Chichester. Brown-Peterson, N.J., and Salin, M.L. 1993. Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme. J. Bacteriol. 175: 41974202. Brown-Peterson, N.J., and Salin, M.L. 1995. Purification and characterization of a mesohaline catalase from the halophilic bacterium Halobacterium halobium. J. Bacteriol. 177: 378-384. Bylund, J.E., Dyer, J.K., Fecly, D.E., and Martin, E.L. 1990. Alkaline and acid phosphatases from the extensively halotolerant bacterium Halomonas elongata. Curr. Microbiol. 20: 125-131. Bylund, J.E., Dyer, J.K., Thompson, T.L., and Martin, E.L. 1991. Alanine dehydrogenase activity of the extensively halotolerant eubacterium Halomonas elongata. Microbios 66: 45-53. Cadenas, Q., and Engel, P.C. 1994. Activity staining of halophilic enzymes: substitution of salt with a zwitterion in non-denaturing electrophoresis. Biochem. Mol. Biol. Int. 33: 785-792. Camacho, M.L., Brown, R.A., Bonete, M.J., Danson. M.J., and Hough, D.W. 1995. Isocitrate dehydrogenasc from Haloferax volcanii and Sulfolobus solfataricus, enzyme purification, characterisation, and N-temiinal sequence. FEMS Microbiol. Lett. 134: 85-90. Cazzulo, J.J. 1978. The adenosine triphosphatase from the moderate halophile, Vibrio costicola, pp. 447-451 In: Caplan, S.R., and Ginzburg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam. Cazzulo, J.J. 1979. Las bacterias halofilas moderadas. Revistas Argentina de Microbiologia 11: 118-128. Cazzulo, J.J., and Vidal, M.C. 1972. Effect of monovalent cations on the malic enzyme from the extreme halophile, Halobacterium cutirubrum. J. Bacteriol. 109: 437-439. Cendrin, F., Chroboczek, J., Zaccai, G., Eisenberg, H., and Mevarech, M. 1993. Cloning, sequencing, and expression in Eschenchia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui. Biochemistry 32: 4308-4313. Cendrin, F., Jouve, H.M., Gaillard, J., Thibault, P., and Zaccai, G. 1994. Purification and properties of a halophilic catalase-peroxidase from Haloarcula marismortui. Biochim. Biophys. Acta 1209: 1-9. Chaga, G., Porath, J., and Illéni, T. 1993. Isolation and purification of amyloglucosidase from Halobacterium sodomense. Biomed. Chromatogr. 7: 256-261. Choquet, C.G., Kamekura, M., and Kushner, D.J. 1989. In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of ions. J. Bacteriol. 171: 880-886. Connaris, H., Chaudhuri, J.B, Danson, M.J., and Hough, D.W. 1999. Expression, reactivation, and purification of enzymes from Haloferax volcanii in Eschenchia coli. Biotechnol. Bioengin. 64: 38-45. Coronado, M.J., Vargas, C., Hofemeister, J., Ventosa, A., and Nieto, J.J. 2000a. Production and biochemical characterization of an from the moderate halophile Halomonas elongata. FEMS Microbiol. Lett. 183: 67-71. Coronado, M.J., Vargas, C., Mellado, E., Tegos, G., Drainas, C., Nieto, J.J., and Ventosa, A. 2000b. The amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization. Microbiology UK 146: 861-868. Daniel, E., Azem, A., Shaked, I., and Mevarech, M. 1993. Subunit structure of malate dehydrogenase from Haloarcula marismortui. Comp. Biochem. Physiol. 106B: 401-405.

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CHAPTER 8 ORGANIC COMPATIBLE SOLUTES

Adaptation at the cellular level, in a mechanism that is probably energy-dependent that maintains the intracellular salt concentration at a level considerably below that of the environment. (Baxter and Gibbons, 1954) Glycerol may be regarded as God's gift to solute-stressed eukaryotes. (Brown, 1990)

8.1. ORGANIC OSMOTIC SOLUTES AND THEIR DISTRIBUTION Microorganisms that live at high salt concentrations are exposed to media of a low water activity, and they need special adaptations to their high-salt, low-water environment. At least some level of turgor pressure has to be maintained to allow expansion and growth of the cells. Regulation of the intracellular turgor pressure is therefore required, using solutes that are fully compatible with all essential cell functions (Brown, 1978, 1990). A minority of the known halophiles use salt (mainly KCl) to provide the osmotic balance with the outside medium. This strategy is used by the aerobic halophilic Archaea of the order Halobacteriales, the anaerobic halophilic Bacteria of the order Halanaerobiales, and the recently discovered red halophilic bacterium Salinibacter ruber. The mode of osmotic adaptation of these organisms has been discussed in Chapter 6. The second option, which is realized in most halophilic and halotolerant representatives of the Bacteria, in the halophilic methanogenic Archaea, and in eucaryal halophilic microorganisms, involves the maintenance of a cytoplasm much lower in salt than the outside medium. Low molecular weight organic compounds are then used to provide osmotic balance and turgor. The term "compatible solute" was originally coined by Brown and Simpson (1972) to describe a low molecular weight solute that accumulates to a high intracellular concentration and that, by virtue of being a very poor enzyme inhibitor, protects enzymes against the inhibition which would otherwise occur in solutions of low water availability. While inorganic ions were at the time also included in this definition, for example in the case of the Halobacteriaceae that accumulate KCl, the term nowadays relates to organic solutes only. The compatible nature of solutes is expressed by their low inhibitory action on metabolic processes as compared to other solutes. Compatibility includes protection against inactivation, inhibition and denaturation of

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enzymes and macromolecular structures under conditions of low water activity (Brown, 1976). Accumulation of such compatible organic osmotic solutes may provide osmotic equilibrium while still enabling activity of "conventional", non-salt adapted enzymes (Da Costa et al., 1998; Galinski, 1993, 1995). The concentrations of such osmotic solutes are regulated according to the salt concentration in which the cells live (Galinski and Louis, 1999), and they can often be rapidly adjusted as required when the outside salinity is changed. Osmoadaptation, i.e. the physiological and genetic alterations that take place in the cell as the level of environmental water changes (Reed, 1984), may involve de novo synthesis and/or uptake of suitable compounds from the medium upon salt upshock, and degradation, transformation into osmotically inactive forms, or excretion to the outer medium following dilution stress (Trüper and Galinski, 1990). As the intracellular enzymes are able to function over a wide range of concentrations of compatible solutes, the use of such solutes as osmotic stabilizers may bestow a high degree of flexibility and adaptability to changing salt concentrations. The use of organic solutes as osmotic stabilizers was first recognized in the early 1970s for the eukaryotic alga Dunaliella, which accumulates glycerol for the purpose (Ben-Amotz and Avron, 1973). The first report of the occurrence of such solutes in the Bacteria appeared in the early 1980s when glycine betaine was found to be accumulated by Halorhodospira halochloris (Galinski and Trüper, 1982). However, the possible role of glycine betaine in the osmotic adaptation of bacteria had already been recognized in the late 1960s. Respiratory activity in Chromohalobacter israelensis at high salt concentrations was found to be stimulated by glycine betaine (Rafaeli-Eshkol and Avi-Dor, 1968). Moreover, it was shown that growth inhibition of several Bacteria at elevated solute concentrations could be relieved by addition of yeast extract, and the active compound was identified as glycine betaine (Dulaney et al., 1968). Compatible solutes are polar, highly soluble molecules, most of them being either uncharged or zwitterionic at the physiological pH (Reed, 1986). The list of compounds known to be synthesized as compatible solutes by halophilic microorganisms is steadily growing (Galinski, 1993, 1995; Galinski and Trüper, 1994; Imhoff, 1986, 1993; Oren, 1999a, 1999b; Reed, 1986; Trüper et al., 1991; Ventosa et al., 1998; Wohlfarth et al., 1990). Figure 8.1 shows the most important known osmotic solutes. They belong to several categories: Polyols (glycerol, arabitol, mannitol, erythritol), sugars (sucrose, trehalose) and heterosides (glucosylglycerol) Betaines (trimethylammonium compounds) and thetines (dimethylsulfonium compounds) Amino acids (proline, glutamate, glutamine, and derivatives) Glutamine amide derivatives amide; glutaminylglutamine amide N-acetylated diamino acids Ectoines (ectoine, Figure 8.1 shows the chemical structures of the most common organic compatible solutes.

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Although there is a great structural diversity among the known compatible solutes, a number of generalizations can be made relating to their structure and their use (Galinski, 1993, 1995): Disaccharides such as sucrose and trehalose have only a limited potential as compatible solutes. However, they are often found as minor components together with other osmotic solutes in halophilic organisms. Trehalose and sucrose are also known to stabilize membranes. De novo biosynthesis of polyols such as glycerol, arabitol and inositol is often found in halophilic and/or osmophilic fungi, algae and salt-tolerant plants, but is seldom detected in halophilic prokaryotes. An exceptional case is the production of mannitol (in addition to amide and glutamate) in Pseudomonas putida S12, an organism that grows up to about 0.6 M salt (Kets et al., 1996). Apart from glycine betaine, all nitrogen-containing compatible solutes are derived from the glutamate or the aspartate branch of amino acid biosynthesis. Charged amino acids such as glutamate, glutamate betaine, and others are never accumulated to very high concentrations (up to approximately 0.4 M only). All compatible solutes that are employed at concentrations of 0.5 M and above are polar, highly soluble molecules which carry no net charge. Table 8.1 provides a (not necessarily exhaustive) overview of the presence of the different osmotic solutes in different taxonomic groups of halophilic microorganisms.

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Many microorganisms are able to take up and accumulate compatible solutes when these are present in the medium. Transport systems for the active uptake of glycine betaine, ectoine, proline, and other compounds that may be used as osmotic stabilizers, are commonly found in the membranes of halophilic (and also in less halophilic)

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microorganisms. Uptake of such solutes from the medium appears to have preference over de novo synthesis (Galinski and Trüper, 1994). Uptake, also sometimes termed "sponging" (Da Costa et al., 1998), is advantageous as it enables the cell to economize its biosynthetic expenditure. Compatible solutes such as glycine betaine released from senescent or dying cells, or excess solutes excreted from the cells following dilution stress, may thus become available to other members of the microbial community, either to be taken up and to be further used as osmotic stabilizers, or to be degraded and to serve as carbon and energy source. As a result, the metabolism of compatible solutes may have a significant impact on the functioning of hypersaline ecosystems. This was clearly shown in the case of the Dead Sea and in saltern crystallizer ponds. Glycerol, the osmotic solute produced in large concentrations by Dunaliella, the sole primary producer in these environments, may well be the principal carbon and energy source that supports the massive blooms of halophilic Archaea of the family Halobacteriaceae that accompany the algae (Oren, 1995). Methanogenesis in anaerobic hypersaline sediments is based not on reduction of with hydrogen or on the degradation of acetate, but on the use of trimethylamine and other methylated amines and/or dimethylsulfide, all compounds which are derived from the degradation of osmotic solutes such as glycine betaine and dimethylsulfoniopropionate (Oremland and King, 1989; Oren, 1990) (Figure 8.2). The impact of these osmotic solutes may extend far beyond the local ecosystem where they are produced and consumed; dimethylsulfide released during breakdown of dimethylsulfoniopropionate can escape to the atmosphere where it influences the global climate (Welsh, 2000). Some compatible solutes produced by halophilic Bacteria have found interesting biotechnological applications. These will be discussed in further detail in Section 11.3. 8.2. COMPATIBLE SOLUTES IN THE DOMAIN ARCHAEA The aerobic halophilic Archaea of the family Halobacteriaceae use inorganic ions, mainly KCl, for osmotic stabilization (see Section 6.3.1). Their intracellular enzymatic machinery is adapted to the presence of high ionic concentrations, and such high salt concentrations are even required to stabilize the cells' proteins (Chapter 7). Such organisms do therefore not depend on organic osmotic solutes. There has been a report on the occurrence of glycine betaine in different representatives of the group. The compound was found in low concentrations only, with intracellular concentrations estimated between 1 and 20 mM. It was mostly associated with the membrane fraction and not with the cytoplasm (Nicolaus et al., 1989). The function of glycine betaine in the Halobacteriaceae is still unknown. In view of the fact that the Halobacteriaceae depend on inorganic ions for osmotic stabilization, the finding of substantial concentrations of an organic solute, 2sulfotrehalose in certain alkaliphilic halophilic Archaea (Natronomonas pharaonis, Natrialba magadii, Natronobacterium gregoryi) came as a surprise. The compound behaves as a true osmotic solute: its intracellular content increases with the salinity of the medium, and

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its concentration can reach substantial levels: cells of Natronococcus occultus grown in medium with 3.4 M NaCl were found to contain about 1 M 2-sulfotrehalose (Desmarais et al., 1997).

Not all halophilic Archaea rely on salt for osmotic stabilization. This is clearly demonstrated by the halophilic representatives of the methanogens. A high content of potassium ions is characteristic of many methanogenic Archaea, even those that do not grow at high salt concentrations. Most of this is probably used as counterion for intracellular reserve materials and/or thermostabilizers such as cyclic 2,3diphosphoglycerate (Jarrell et al., 1984). Truly halophilic methanogens, and methanogens adapted to life in the marine environment as well, rely on organic solutes for osmotic adaptation. Compounds used for this purpose include glycine betaine, glutamine, and others (Lai et al., 1999, 2000; Lai and Gunsalus, 1992; Menaia et al., 1993; Robertson et al., 1990a, 1990b). Glycine betaine can both be accumulated from the medium by halophilic methanogens (Lai et al., 2000; Robertson et al., 1990a, 1992) or be synthesized de novo. Methanohalophilus portucalensis synthesizes glycine betaine by stepwise

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methylation of glycine via sarcosine and N,N-dimethylglycine. S-adenosyl-methionine is used as donor of the methyl groups (Lai et al., 1999, 2000; Roberts et al., 1992). The intermediate dimethylglycine can also sometimes be detected intracellularly at significant concentrations (Menaia et al., 1993). Biosynthesis of probably starts from (Roberts et al., 1992). The synthesis of this compound is repressed by addition of glycine betaine (Sowers et al., 1990). In Methanohalophilus portucalensis grown at NaCl concentrations between 1.2 and 2.9 M, the ratio between intracellular glycine betaine, and primarily depends on the methanogenic substrate used, and less on the total osmolarity (Robertson et al., 1992). Zwitterionic solutes such as glycine betaine, and lysine were the predominant solutes in two moderately halotolerant strains (tolerating up to 1.5 M NaCl), when grown at the higher concentrations, while in cells grown at lower salinities the anionic and were found in a higher proportion (Lai et al., 1991).

8.3. COMPATIBLE SOLUTES IN THE DOMAIN BACTERIA 8.3.1. Compatible solutes in the oxygenic photosynthetic bacteria The cyanobacteria can be divided into three groups according to their salt tolerance and the types of compatible solutes they synthesize as a reaction to salt stress. The less salt tolerant types generally produce disaccharides (sucrose and/or trehalose). These sugars are not very effective as osmotic solutes, and they provide osmotic protection only up to relatively low salinities (Blumwald and Tel-Or, 1982; Blumwald et al., 1983; Gabbay-Azaria and Tel-Or, 1993). The most salt-tolerant and halophilic types produce glycine betaine (Mackay et al., 1984; Oren, 2000; Reed et al., 1984, 1986). Many species with an intermediate salt tolerance make the heteroside glucosylglycerol. Additional osmotic solutes are sometimes found, such as L-glutamate betaine (Ntrimethyl-L-glutamate), which was detected in combination with sucrose and/or trehalose in halophilic Calothrix isolates (Mackay et al., 1984). Glucosylglycerol has been detected in a many salt-tolerant cyanobacteria, including Microcoleus chthonoplastes (Kevbrin et al., 1991), different Synechococcus and Synechocystis isolates (Borowitzka et al., 1980; Mackay and Norton, 1987; Richardson et al., 1983), Spirulina platensis (Warr et al., 1985), Agmenellum quadruplicatum (Tel-Or et al., 1986), and Microcystis firma (Erdmann et al., 1992). Large concentrations of glucosylglycerol were found in communities of Microcoleus chthonoplastes in Lake Sivash, East Crimea (Zavarzin et al., 1993) and in a Microcoleus-dominated hypersaline microbial mat derived from Solar Lake, Sinai (Oren et al., 1994). In Microcoleus grown in salt, glucosylglycerol accounted for 30% of the cell dry weight (Zavarzin et al., 1993). Trehalose is often present as a minor component in Microcoleus chthonoplastes besides glucosylglycerol. The cells' content of glucosylglycerol increased with increasing salinity, while the concentration of trehalose decreased (Karsten, 1996). Upon salt downshock, Synechocystis PCC

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6714 releases part of the excess glucosylglycerol to the medium (Fulda et al., 1990). Transient changes in nonspecific membrane permeability may be responsible for this excretion of osmotic solute (Reed et al., 1986). However, in the marine Agmenellum quadruplicatum salt downshock induces increased carbohydrate turnover, and excess glucosylglycerol is probably converted intracellularly to glycogen (Tel-Or et al., 1986). Synechocystis strain PCC 6803 has become the model organism for the study of the processes regulating the production and degradation of glucosylglycerol (Hagemann and Zuther, 1992; Hagemann et al., 1994, 1996, 1999). This organism was isolated from a freshwater habitat, but it can grow up to about NaCl (Erdmann et al., 1992; Richardson et al., 1983). Glucosylglycerol is synthesized from ADP-glucose and glycerol-3-phosphate in a two step reaction in which glucosylglycerol-phosphate is an intermediate. The glucosylglycerol forming enzyme system is activated by salt; organic osmolytes are inactive as activators (Hagemann and Erdmann, 1994). Activation of glucosylglycerol biosynthesis does not require synthesis of new proteins (Hagemann et al., 1990). Mutants of Synechocystis PCC 6803 defective in glucosylglycerol synthesis are unable to grow at elevated salt concentrations (Hagemann and Zuther, 1992). One such salt-sensitive mutant was found to be defective in glucosylglycerol-phosphate phosphatase. It accumulated glucosylglycerolphosphate intracellularly, a compound that is not effective as an osmoprotectant (Hagemann et al., 1996). Exogenously added glucosylglycerol can be taken up from the medium, and restores salt tolerance of this mutant (Mikkat et al., 1996). The activity of the glucosylglycerol transport system is enhanced in cells grown at high NaCl concentrations. The affinity of the uptake system is relatively low, with a of about Sucrose and trehalose were found to compete for the same transport system. This transport system probably serves to prevent loss of glucosylglycerol from salt-stressed cells (Mikkat et al., 1996). The most salt tolerant among the cyanobacteria produce glycine betaine as osmotic solute. These include Aphanothece halophytica (Reed et al., 1984a), Spirulina subsalsa (renamed as Halospirulina tapeticola) (Gabbay-Azaria et al., 1988), Dactylococcopsis salina (Moore et al., 1987), and Synechocystis strain DUN52, an isolate from calcareous stromatolites of intertidal flats in Kuwait (Reed et al., 1984a). Glycine betaine can be accumulated to very high concentrations: values of 1.18, 2.43, and 2.98 M were measured in cells grown at 1, 1.7, and 3.4 M salt, respectively (Mohammad et al., 1983). Glycine betaine was detected in massive amounts in the Oscillatoria mat that covered the bottom of the hypersaline sulfur spring of Hamei Mazor (Oren et al., 1994) (sec Section 17.6). In a Gloeocapsa strain glycine betaine occurs together with trehalose (Mackay et al., 1984), and minor amounts of glucosylglycerol were found together with glycine betaine in Synechocystis DUN52 (Mohammad et al., 1983). Cyanobacteria synthesize glycine betaine by the oxidation of choline, not by the stepwise methylation of glycine such as occurs in the halophilic methanogens and in the anoxygenic photosynthetic bacteria (see Sections 8.2 and 8.3.2). Glycine betaine can be degraded by stepwise demethylation. A betainehomocysteine methyltransferase has been purified from Aphanothece halophytica and characterized. It converts glycine betaine + homocysteine to N,N-dimethylglycine +

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methionine. Its activity was increased upon salt downshock and during starvation (Waditee and Incharoensakdi, 2001). Glycine betaine protects the structural integrity and activity of Aphanothece halophytica ribulose-l,5-bisphosphate carboxylase against the damaging influence of salts (Incharoensakdi and Takabe, 1988; Incharoensakdi et al., 1986). It also restores the loss of activity of glucose-6-phosphate dehydrogenase in the same organism (Hawkins et al., 1987). The glucose-6-phosphate dehydrogenase ofSpirulina subsalsa (Halospirulina tapeticola) is only little sensitive to salt (50% inhibition at 1.25 M NaCl), but in the presence of glycine betaine full activity was obtained at NaCl concentrations as high as 1.5 M (Gabbay-Azaria et al., 1988). In Synechocystis DUN52 concentrations of glycine betaine between 0.8 and 2 M alleviated the inhibition of glutamine synthetase by 1.7-5 M NaCl (Warr et al., 1984). When exposed to a salt upshock from 0.5 to 1.5-2 M NaCl, photosynthesis in Aphanothece halophytica is strongly inhibited, and returns to normal levels only after the synthesis of sufficient quantities of glycine betaine had been completed. The adaptation process is lightdependent (Ishitani et al., 1993). Glycine betaine producing cyanobacteria can accumulate glycine betaine present in their environment by active transport. Transport activity of glycine betaine was detected in Aphanothece halophytica, Dactylococcopsis salina, Synechococcus PCC 7418, and Synechocystis DUN 52. The scavenging of glycine betaine available in the medium may be an effective strategy in environments of fluctuating salinity (Moore et al., 1987). Glycine betaine transport activity was not detected in halophilic cyanobacteria that accumulate sucrose or glucosylglycerol as compatible solutes. Aphanothece accumulated 120 mM glycine betaine intracellularly within 30 minutes when the compound was supplied at a concentration of 1 mM. After one hour of incubation with 1 mM glycine betaine, an intracellular concentration of 250 mM was measured in Synechocystis DUN52. The of the transport system for glycine betaine is about and optimal activity was found at pH 8-8.5. NaCl concentrations above 80 mM are required to stimulate the transport activity (Moore et al., 1987). Upon salt downshock excess glycine betaine is excreted from the cells and released into the surrounding medium (Moore et al., 1987), possibly as a result of transient permeability changes of the cell membrane (Reed et al., 1986). However, when a suspension of Aphanothece halophytica was diluted from 1.5 to 0.5 M NaCl, no release of glycine betaine to the outside medium could be detected; the intracellular concentration decreased slowly, to reach a new equilibrium value within 10 hours (Ishitani et al., 1993). Mycosporine-like amino acids may under certain conditions provide at least part of the osmotically active pool of small intracellular compounds in unicellular, Aphanothece (Cyanothece)-like cyanobacteria. Mycosporine-like amino acids act as sunscreen compounds, protecting the cells against solar ultraviolet radiation (GarciaPichel and Castenholz, 1993). Large concentrations of mycosporine-like amino acids were found in a community of unicellular cyanobacteria within a gypsum crust that develops on the bottom of a hypersaline saltern pond in Eilat, Israel (Oren et al., 1995) (see also Section 14.2). Two such compounds were present, one with an absorption

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maximum at 332 nm, and one at 365 nm. The intracellular concentrations of mycosporine-like amino acids in the cyanobacterial community were estimated to be at least 100 mM. Evidence for a possible osmotic function was obtained from salt downshock experiments. When material from the upper layer of the gypsum crust was subjected to slow dilution with distilled water, mycosporine-like amino acids rapidly appeared in the outer medium, the extent of loss of the intracellular pool being approximately proportional to the extent of the dilution stress applied (Oren, 1997). The mechanism of release may again be related to transient permeability changes of the cell membrane.

8.3.2. Compatible solutes in the anoxygenic photosynthetic bacteria Halophilic phototrophic sulfur bacteria of the genera Halorhodospira and Ectothiorhodospira have become useful model systems for the study of the production and functioning of compatible solutes. The finding of glycine betaine in Halorhodospira halochloris was probably the first reported case of the occurrence of organic compatible solutes in the bacterial world (Galinski and Trüper, 1982). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was also first discovered in this organism, the name ectoine being derived from Ectothiorhodospira, the basonym of the genus Halorhodospira (Galinski et al., 1985). Halorhodospira halochloris uses cocktails of three compatible solutes to provide osmotic balance: glycine betaine, ectoine, and trehalose. Under carbon limitation conditions, both trehalose and ectoine are metabolized, and glycine betaine remains as the sole compatible solute in the stationary phase cells (Trüper and Galinski, 1989). When nitrogen is in short supply, trehalose (a compound that does not contain nitrogen) is preferentially used to replace ectoine (a molecule with two nitrogen atoms). Glycine betaine is not degraded as nitrogen source, not even when nitrogen is depleted (Galinski and Herzog, 1990; Trüper and Galinski, 1990). Glycine betaine is synthesized in Halorhodospira by stepwise methylation of glycine (Nyyssölä et al., 2000; Trüper and Galinski, 1990). The glycine sarcosine Nmethyltransferase and the sarcosine dimethylglycine N-methyltransferase of Halorhodospira halochloris have been purified and characterized following expression in Escherichia coli. S-adenosylmethionine donates the methyl groups (Nyyssölä et al., 2001). Halorhodospira can also take up glycine betaine from the medium, using a transport system that is probably driven by the proton electrochemical gradient (Peters et al., 1992). The biosynthesis of ectoine shares the first enzymatic steps (aspartokinase and Ldehydrogenase) with the biosynthesis of amino acids of aspartate family (lysine, threonine and methionine) (Peters et al., 1990). The enzymology and the molecular biology of the biosynthesis of ectoine are discussed in further detail in Section 8.2.3. Halorhodospira halochloris synthesizes trehalose by reaction of uridinediphosphate-glucose and glucose-6-phosphate to form trehalose-6phosphate, which is then dephosphorylated (Lippert et al., 1993).

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When subjected to dilution stress, Halorhodospira releases excess glycine betaine and ectoine to the outer medium, and these can later again be taken up by the cells. An "overshoot" phenomenon was observed, in which the amount of glycine betaine released exceeded the amount needed to balance the intracellular environment with the new salinity to which the cells had been exposed. The missing amount of glycine betaine was subsequently taken up from the medium (Tschichholz and Trüper, 1990). Excess trehalose is degraded inside the cell following a salinity downshock. The trehalase responsible for the first step in the trehalose degradation in Halorhodospira halochloris has been characterized. Its affinity for its substrate trehalose is very low (a of 0.5 M, which is lowered to 0.16 M in the presence of glycine betaine). Such a low affinity is not surprising in view of the high concentrations at which the substrate may be present within the cell (Herzog et al., 1990). Less halotolerant anoxygenic photosynthetic bacteria use different cocktails of osmotic solutes. Halochromatium salexigens accumulates glycine betaine, sucrose, and N-acetyl-glutaminylglutamine amide (Welsh and Herbert, 1993; Galinski, 1995). Glycine betaine and sucrose are also used by Thiohalocapsa halophila, while Rhodovibrio salinarum produces glycine betaine and some ectoine. Rhodovulum sulfidophilum (which is not a truly halophilic organism) accumulates glucosylglycerol (Galinski, 1995). The compound amide has thus far been detected only in Ectothiothiorhodospira marismortui, where it occurs together with glycine betaine and sucrose (Galinski, 1995; Galinski and Oren, 1991; Oren et al., 1991). Upon downshock the compound is degraded intracellularly (Fischel and Oren, 1993).

8.3.3. Compatible solutes in aerobic heterotrophic bacteria A wide variety of organic osmotic solutes have been detected in the halophilic heterotrophic aerobic Bacteria (Table 8.1). Additional information on the distribution of these solutes can be found in review articles (e.g. Galinski, 1995; Ventosa et al., 1998). The stimulatory action of glycine betaine on salt-stressed heterotrophic Bacteria has been known for many years. Already in 1968 it was shown that respiratory activity in Chromohalobacter israelensis at high salt concentrations is stimulated by glycine betaine (Rafaeli-Eshkol and Avi-Dor, 1968). The compound was found to be accumulated by the cells in increasing amounts as the osmolarity of the medium was raised. However, part of the stimulating effect was claimed to be due to the presence of glycine betaine at the outer side of the cytoplasmic membrane (Shkedy-Vinkler and Avi-Dor, 1975). Glycine betaine is the main compatible solute accumulated by different species of heterotrophic halophilic Bacteria when grown in rich media containing yeast extract (Imhoff and Rodriguez-Valera, 1984). Yeast extract contains 1 to 3% glycine betaine (Galinski, 1993), and most heterotrophic Bacteria accumulate the compound from the medium. Under these conditions the Bacteria do not need to spend energy for de novo

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synthesis of other osmotic solutes. However, very few aerobic heterotrophs have the ability to synthesize glycine betaine themselves. High-affinity transport systems for glycine betaine and for choline were characterized in Chromohalobacter salexigens (Cánovas et al., 1996) and in Tetragenococcus halophilus (Robert et al., 2000). Conversion of choline to glycine betaine is also commonly found in the halophilic Bacteria. Salinivibrio costicola has a membrane-bound choline dehydrogenase and a soluble betaine aldehyde dehydrogenase (Choquet et al., 1991). Similar enzymes have been found in Chromohalobacter salexigens (Cánovas et al., 1996). Chromohalobacter salexigens converts choline to betaine in a salt-dependent reaction, and NaCl concentrations as high as were found highly stimulatory (Cánovas et al., 1998a). The genes betB (coding for soluble betaine aldehyde dehydrogenase) and betA (coding for the membrane-bound choline dehydrogenase) have been characterized by functional complementation in an Escherichia coli strain defective in glycine betaine synthesis (Cánovas et al., 2000). Halomonas elongata can grow on glycine betaine as carbon and energy source. Under these conditions its tolerance to NaCl is lower than when grown on glucose (120 versus This effect has been attributed to the inhibition of the enzymes responsible for glycine betaine catabolism by salt, so that at high salt concentrations the compound serves as an osmotic solute only (Cummings and Gilmour, 1995). The only halophilic aerobic bacterium known to be able of de novo synthesis of glycine betaine is the actinomycete Actinopolyspora halophila (Severin et al., 1992). In cells grown in NaCl glycine betaine made up 33% of the cellular dry weight. In addition, trehalose was found at concentrations up to 9.7% of the dry weight. The concentration of glycine betaine increased with increasing medium NaCl, while the cellular content of trehalose was highest at the lowest NaCl concentrations tested (Nyyssölä and Leisola, 2001). The biosynthesis of glycine betaine was first shown to proceed by stepwise methylation of glycine, using glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase (Nyyssölä et al., 2000). However, it recently appeared that the enzymes required for the synthesis of glycine betaine via choline and betaine aldehyde are present as well. Actinopolyspora halophila is therewith the first organism known to use both pathways (Nyyssölä and Leisola, 2001). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and hydroxyectoine are the most widespread compatible solutes synthesized de novo by aerobic halophilic Bacteria (see also Table 8.1). Ectoine is readily assayed both by natural abundance nuclear magnetic resonance and by high performance liquid chromatography. A sensitive HPLC assay has been developed based on derivation with 9-fluorenylmethyl chloroformate (FMOC) following hydrolysis to yield N-acetylated aminobutyric acid derivatives and use of a fluorescence detector (Kunte et al., 1993). Studies using natural abundance nuclear magnetic resonance showed ectoine and hydroxyectoine to be present in different members of the Halomonadaceae, in Salinivibrio costicola, in Marinobacter hydrocarbonoclasticus, and in many other halophilic heterotrophs when grown in defined media that did not contain glycine

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betaine or its precursor choline (Fernandez-Linares et al., 1996; Ono et al., 1998; Regev et al., 1990; Severin et al., 1992; Wohlfarth et al., 1990). The intracellular concentration found was often calculated to be sufficient to provide osmotic balance with the salt concentration present in the medium (Wohlfarth et al., 1990). Calorimetric measurements have shown that the conversion of glucose to ectoinc by Halomonas elongata is energetically highly efficient (Maskow and Babel, 2001). Many halophilic Bacteria can take up ectoinc from their growth medium. Halomonas elongata possesses an osmoregulated transport system for ectoine (TeaABC) (Grammann et al., 2002; Tetsch and Kunte, 2002). All the genes teaA, teaB, and teaC are mandatory for ectoine uptake. The transport system belongs to the tripartite ATP-independent periplasmic transport family (TRAP-T). Affinity for ectoine is high Deletion of teaC or teaBC in the wild type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. The physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane (Grammann et al., 2002). TeaA, the putative periplasmic substrate binding protein, was characterized following overexpression in Escherichia coli (Tetsch and Kunte, 2002). Addition of ectoine to the medium highly stimulated growth of a Brevibacterium sp. isolated from the marine environment at salt concentrations as high as salt (Nagata and Wang, 2001). This organism can grow up to about NaCl by producing ectoine, growth at higher salt concentrations being dependent on uptake of compatible solutes such as glycine betaine or its precursor choline (Bernard et al., 1993). When Halomonas elongata is subjected to sudden dilution from 150 to NaCl, the cells rapidly extrude excess ectoine to the medium. The viability of the cells is not affected in the process. This selective extrusion is probably mediated by stretch-activated or mechanosensitive channels which are closed at normal turgor and open above a critical turgor pressure (see also Section 6.3.7). Ectoine is synthesized from aspartate semialdehyde. Three enzymes are involved: L-2,4-diaminobutyric acid transaminase (EctB), L-2,4-diaminobutyric acid acetyltransferase (EctA), and L-ectoine synthase (EctC) (Figure 8.3) (Cánovas et al., 1998b; Louis and Galinski, 1997; Peters and Galinski, 1990). The three enzymes from Halomonas elongata have been purified and characterized. The donor of the amino group for the 2,4-diaminobutyrate aminotransferase is Lglutamate. The enzyme requires pyridoxal 5'-phosphate and ions for activity and stability. The L-2,4-diaminobutyric acid acetyltransferase binds acetyl coenzyme-A to the 2,4-diaminobutyrate. It is optimally active in the presence of 0.4 M NaCl. Ectoine synthase catalyzes a cyclic condensation reaction to form ectoine, functions optimally at 0.5 M NaCl (Ono et al., 1999). The genes involved have been isolated from Halomonas elongata (Goller et al., 1998), from Halomonas sp. KS-3 (Min-Yu et al., 1993), from Chromohalobacter salexigens (Cánovas et al., 1997), and from Marinococcus halophilus (Louis and Galinski, 1997). The ectoine synthase gene (ectC) from Halomonas sp. KS-3 was amplified and cloned using primers designed on the basis of the N-terminal amino acid sequence of the isolated protein (Min-Yu et al., 1993). Genetic analysis of the regulation of ectoine

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production became possible after the ectoine synthesis genes of Marinococcus halophilus had been cloned in Escherichia coli to obtain a recombinant strain that produces increasing cytoplasmic ectoine concentration in response to salinity (Louis and Galinski, 1997; Galinski and Louis, 1999). The genes are arranged in a cluster in the order ectABC (Louis and Galinski, 1997). The Marinococcus halophilus ectC gene shares 47% identity with the corresponding gene of Halomonas sp. KS-3. Analysis of a NaCl-sensitive mutant of Halomonas elongata that is impaired in ectoine biosynthesis and accumulates L-2,4-diaminobutyric acid showed an identical arrangement of the genes (Göller et al., 1998). A number of salt-sensitive mutants of Chromohalobacter salexigens have been generated by transposon mutagenesis. Some of these mutants accumulated intermediates of the ectoine biosynthesis pathway. One such mutant was found to produce diaminobutyrate, another mutant, affected in the ectoine synthase gene ectC, accumulated The latter compound proved as effective as an osmotic solute as ectoine itself (Cánovas et al., 1997). NMR analysis showed the presence of hydroxyectoine, ectoine, and glucosylglycerate in the cytoplasm of this mutant. The concentration of the last-named compound did not depend on the salt concentration at which the cells were grown (Cánovas et al., 1999). analysis of the solutes present in the wild type strain and its mutants indicated that Chromohalobacter salexigens can synthesize hydroxyectoine by two different pathways, one that uses ectoine as substrate and a second alternative pathway that converts to hydroxyectoine without ectoine as an intermediate (Cánovas et al.. 1999).

Many Gram-positive Bacteria accumulate proline, and/or acetyllysine (Del Moral et al., 1994; Galinski, 1995; Severin et al., 1992; Wohlfarth et al., 1993). The heteroside glucosylglycerol, found in many cyanobacteria (see Section

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8.3.1) also occurs in Pseudomonas mendocina and in Pseudomonas pseudoalkaligenes, two organisms with a low salt tolerance (Galinski, 1995). Moderately halophilic methanotrophic Bacteria were found to accumulate sucrose and 5-oxo-1-proline when grown in the lower salt concentration range at more elevated salinities (30NaCl) ectoine is made in addition (Khmelenina et al., 1999, 2000). Little is known about the mode and regulation of the biosynthesis of the above compounds. The use of the green fluorescent protein gene as a reporter gene linked to a 480 kbp Marinococcus halophilus DNA fragment upstream of the ectoine genes ectABC in recombinant Escherichia coli has recently yielded some information on the signals that lead to formation of ectoine (Bestvater and Galinski, 2002). More extensive information, including genetic data, has been obtained on osmotic adaptation, uptake of osmotic solutes, and turgor regulation in Gram-negative and Gram-positive Bacteria with low salt tolerance such as Escherichia coli and Bacillus subtilis. The review article by Kempf and Bremer (1998) provides an excellent overview of the insights obtained from these studies. As these organisms cannot be considered halophilic according to the definition used here, the many data collected will not be discussed here. However, it should be realized that many of the findings reported may be extrapolated to extend our understanding of the more halophilic representatives within the bacterial domain.

8.4. COMPATIBLE SOLUTES IN THE DOMAIN EUCARYA Unicellular algae of the genus Dunaliella are the only truly halophilic Eucarya in which the synthesis and regulation of organic osmotic solutes has been investigated indepth. Dunaliella (Chlorophyceae) and the less-well studied Asteromonas gracilis (Prasinophyceae) use glycerol as compatible solute when growing at high salt concentrations, while maintaining their intracellular ionic concentrations at very low levels (see Section 6.6) (Ben-Amotz and Avron, 1973, 1980; Ben-Amotz and Grunwald, 1981; Borowitzka and Brown, 1974). Dunaliella can maintain extremely high intracellular glycerol concentrations: Dunaliella salina grown at 1.5 M NaCl showed an intracellular glycerol concentration of about 1.9 M, which is osmotically equivalent to 1.25 M NaCl (Degani et al., 1985). Cells grown in 4 M NaCl contain approximately 7.8 M glycerol inside, equivalent to a solution of glycerol in water (Brown, 1990). Glycerol is probably present in all cell compartments, including the chloroplast and the cell cytoplasm (Maeda and Thompson, 1986). Glycerol in high concentrations stimulates cyclic photophosphorylation in Dunaliella bardawil thylacoid membranes, showing that it is truly "compatible" with the intracellular enzymatic machinery. Such high glycerol concentrations are inhibitory to cyclic photophosphorylation in spinach thylacoid membranes (Finel et al., 1984). While most biological membranes are freely permeable to glycerol, the membranes of Dunaliella and Asteromonas are virtually impermeable to the compound (Brown, F.F. et al., 1982; Gimmler and Hartung, 1988). Only at elevated temperatures (above 50 °C) do substantial amounts of glycerol leak out of the cells (Wegmann et al., 1980).

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The passive permeability of the cytoplasmic membrane of Dunaliella to glycerol was estimated at (at 17 °C), to be compared for example with the value of measured in pig erythrocytes (at 25 °C) (Brown et al., 1982). The mechanism that causes the glycerol to be retained by the Dunaliella while it readily passes other eukaryotic membranes has never been fully clarified. The carbon atoms required for glycerol biosynthesis are derived from the Calvin cycle or from degradation of intracellular storage starch-like polysaccharides. Dihydroxyacetone phosphate, an intermediate of both the autotrophic Calvin cycle and the glycolytic pathway, is the central intermediate that provides the starting point for glycerol biosynthesis. In the so-called "glycerol cycle" (Figure 8.4), dihydroxyacetone phosphate is reduced to glycerol-3-phosphate by glycerol-3-phosphate dehydrogenase using NADH as electron donor, whereafter the phosphate is split off by a glycerol-1phosphatase. When Dunaliella tertiolecta is exposed to a hyperosmotic shock, the intracellular concentration of glycerol-3-phosphate transiently increases. This effect is probably due to the increased activity of glycerol-3-P dehydrogenase in vivo, caused by changes in the concentrations of effectors such as ATP (Belmans and van Laere, 1987). Excess glycerol can also be returned to the central metabolic pathways by action of an

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NADP-dependent glycerol dehydrogenase (dihydroxyacetone reductase) that oxidizes glycerol to dihydroxyacetone. The dihydroxyacetone is then phosphorylated by dihydroxyacetone kinase, the phosphate group being derived from ATP. Excess dihydroxyacetone phosphate can subsequently be converted to osmotically inactive starch (Ben-Amotz and Avron, 1978; Borowitzka et al., 1977; Brown, A.D. et al., 1982). The dihydroxyacetone reductase has a very low affinity for glycerol about 1.5 M), as may be expected for an enzyme whose activity has to be regulated in an environment that contains extremely high concentration of its substrate. The two reversible steps in the glycerol cycle are spatially separated: the NAD-specific glycerol3-phosphate dehydrogenase is located in the chloroplast, and so is probably the glycerol-1-phosphatase. However, the NADP-specific glycerol dehydrogenase is found in the cytosol. The intracellular distribution of the dihydroxyacetone kinase is yet uncertain (Brown, A.D. et al., 1982; Gimmler and Lotter, 1982). Breakdown of starch to glycerol is suppressed under isoosmotic conditions in high light. Salt upshock triggers enhanced breakdown of starch by a phosphorylytic mechanism, which is stimulated by the presence of salt and by phosphate (Goyal et al., 1987). Dunaliella cells lack a rigid cell wall. As a result they shrink and swell following sudden changes in the osmotic conditions (Ben-Amotz and Avron, 1981). Dunaliella salina was observed to increase in volume by 76% within 2 to 4 minutes when rapidly diluted from 1.71 M to 0.86 M NaCl (Maeda and Thompson, 1986). A new level of intracellular glycerol may be achieved within 30 minutes to a few hours, both in the light and in the dark, using the enzymatic mechanisms described above ((Kessly and Brown, 1981). In Dunaliella tertiolecta (a species adapted to relatively low salt concentrations) dilution stress generally does not cause leakage of glycerol out of the cell; and leakage and possible burst of the cell membrane are only observed following a very drastic sudden dilution (Goyal, 1989). However, another study with the same organism showed a substantial loss of intracellular glycerol upon salt downshock. Up to 75% of the observed decrease in intracellular glycerol was accounted for by loss to the medium, the remainder had been converted to osmotically inactive compounds (Zidan et al., 1987). Addition of the uncoupler CCCP (carbonyl cyanide mchlorophenylhydrazone) inhibited extrusion of glycerol out of the cells, suggesting that the process may be ATP-dependent, and does not represent a simple passive leakage through the membrane. and nuclear magnetic resonance studies with Dunaliella salina showed a temporal decrease in glycerol with an increase in content following hypo-osmotic shock. The opposite trend was found when the cells were subjected to a sudden increase in salt concentration. No release of glycerol to the medium was observed during these treatments (Degani et al., 1985). The halophilic unicellular green alga Picocystis from Mono Lake, California, contains glycine betaine and dimethylsulfoniopropionate. When the medium salinity was increased from 19 to the intracellular content of glycine betaine increased, while the concentration of dimethylsulfoniopropionate did not change significantly. These two compatible solutes together are present in sufficiently high concentrations to omotically balance the extracellular salt concentration (Roesler et al., 2002).

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Although many fungi tolerate moderately high salt concentrations, there are only few true halophilic species among the fungi. However, life at low water activity is very common in fungi, as many species are well adapted to survive drought stress and others thrive in concentrated sugar solutions. Halophilic and osmophilic fungi and yeasts generally use polyols such as glycerol, arabitol and sorbitol to provide osmotic balance of their cytoplasm with the extracellular environment (Brown, 1990). Glycerol is the main organic osmotic solute detected in cells of Saccharomyces cerevisiae, Zymomonas rouxii and Debaryomyces hansenii growing at NaCl. Stationary phase Debaryomyces cells contain arabitol as well, and the disaccharide trehalose has been detected in Saccharomyces cerevisiae and in many other yeasts. 8.5. THE MODE OF ACTION OF COMPATIBLE SOLUTES

As documented above, compatible solutes belong to different chemical classes with little structural similarity. The question should therefore be asked what common principles underlie the action of compatible solutes and what makes these solutes compatible? Based on the information available, the following generalization can be made: compatible solutes are very soluble, they have no (net) charge, and they show only limited interaction with proteins. Galinski (1995) states that it is this lack of interaction with proteins that is most characteristic for compatible solutes. Compatible solutes are strong water structure formers and as such they are to a large extent excluded from the hydration shell of proteins. This "preferential exclusion" probably defines their function as effective stabilizers of the hydration shell of proteins and other cytoplasmic structural elements, and the low level of interaction with the protein itself explains their compatibility, not only in vitro but also within the living cell (Arakawa and Timasheff, 1985; Timasheff, 1992). The phenomenon of preferential hydration of proteins favors a more compact protein conformation, opposes an increase in surface area, and, since unfolding usually results in a surface area increase, favors the native state (Figure 8.5). It is thus primarily the free water (as opposed to bound water) which responds to osmotic changes of the environment, and the "compatibility" of osmotic solutes is based on the fact that they specifically adjust the osmotic equilibrium of the free water fraction. Exclusion of compatible solutes from the hydration sphere of proteins is consistent with a decrease in entropy of the system (higher ordening). This entropically unfavorable situation in turn causes minimization of the excluded volume and subsequently stabilizes the conformation of a protein (Galinski, 1993). The operation of the preferential exclusion principle was nicely illustrated in a recent study of the stabilizing effects of and ectoine on bovine ribonuclease A as model protein, using differential scanning calorimetry (Knapp et al., 1999). at a concentration of 3 M increased the melting temperature of the protein by more that 12 °K and caused a stability increase of at room temperature. Exclusion of the osmolytes from the protein surface was claimed to be the main factor responsible for the stabilization of the native protein.

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A model proposed by Wiggins (1990) explains the behavior of osmotic solutes on the basis that water near interfaces is structurally different (more dense), allowing compatible solutes to preferentially move towards the less dense water fractions. Compatible solutes, due to their strongly bonded hydration shell, fit much better in the less dense water fraction. Detailed calculations have recently been made of the hydration structure of ectoine (Suenobu and Nagaoka, 1998). According to the model obtained, ectoine is hydrogen-bonded to one water molecule both at the oxygen atom of the -COO group and at the hydrogen atom of the -NH group. Other structures are possible with four water molecules, and still other solutions show that more than four water molecules could solvate to ectoine.

The mechanism of non-specific exclusion of compatible solutes has also been explained in terms of increased surface tension of water, the presence of solutes effecting forces of cohesion between water molecules, and/or in terms of enhancement of water structure by solutes, enforcing the formation of large hydration clusters (Gekko and Timasheff, 198la, 1981b). As a thermodynamic consequence (minimization of entropy, reinforcement of the hydrophobic effect), compatible solutes display a general stabilizing effect that opposes the unfolding/denaturation of proteins

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and other labile macromolecular structures (Galinski, 1993). Their protective function against a range of stress factors is a direct consequence of this behavior. Compatible solutes thus provide a general stabilizing effect on proteins by limiting the extent of their unfolding and denaturation during heating, freezing, and drying (Galinski, 1993, 1995; see also Section 11.3.3). They may also affect protein-nucleic acid interactions. Thus, ectoine and were found to inhibit DNA cleavage by the endonuclease EcoR1 (Malin et al., 1999). Bolen and coworkers have refined the preferential exclusion model. According to their calculations, reduced exposure of the peptide backbone towards the solvent in the presence of compatible solutes specifically stabilizes the protein structure. In natural selection of organic osmolytes as protein stabilizers, it appears that the osmotic property selected for is the unfavorable interaction between the osmolyte and the peptide backbone ("osmophobic effect") (Bolen and Baskakov, 2001; Liu and Bolen, 1995). In spite of our increased insight in the modes of osmotic adaptation in halophilic microorganisms, our understanding of the molecular principles that govern the functioning of organic compatible solutes remains rather limited. 8.6. REFERENCES Arakawa, T., and Timasheff, S.N. 1985. The stabilization of proteins by osmolytes. Biophys. J. 47: 411-414. Baxter, R.M., and Gibbons, N.E. 1954. The glycerol dehydrogenases of Pseudomonas salinaria, Vibrio costicola, and Escherichia coli in relation to bacterial halophilism. Can. J. Biochem. Physiol. 32: 206-217. Belmans, D., and van Laere, A. 1987. Glycerol cycle enzymes and intermediates during adaptation of Dunaliella tertiolecta cells to hyperosmotic stress. Plant Cell Environ. 10: 185-190. Ben-Amotz, A. 1978. Adaptation of the unicellular alga Dunaliella parva to a saline environment. J. Phycol. 11: 50-54. Ben-Amotz, A., and Avron, M. 1973. The role of glycerol in the osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiol. 51: 875-878. Ben-Amotz, A., and Avron, M. 1980. Osmoregulation in the halophilic algae Dunaliella and Asteromonas, pp. 91-99 In: Rains, D.W., Valentine, R.C., and Hollaender, A. (Eds.), Genetic engineering of osmoregulation. Plenum Publishing Co., New York. Ben-Amotz, A., and Avron, M. 1981. Glycerol and metabolism in the halotolerant alga Dunaliella: a model system for biosolar energy conversion. Trends Biochem. Sci. 6: 297-299. Ben-Amotz, A., and Grunwald, T. 1981. Osmoregulation in the halotolerant alga Asteromonas gracilis. Plant Physiol. 67: 613-616. Bernard, T., Jebbar, M., Rassouli, Y., Himdi-Kabbab, S., Hamelin, J., and Blanco, J. 1993. Ectoine accumulation and osmotic regulation in Brevibacterium linens. J. Gen. Microbiol. 139: 129-136. Bestvater, T., and Galinski, E.A. 2002. Investigation into a stress-inducible promoter region from Marinococcus halophilus using green fluorescent protein. Extremophiles 6: 15-20. Blumwald, E., and Tel-Or, E. 1982. Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum. Arch. Microbiol. 132: 168-172. Blumwald, E., Mehlhorn, R.J., and Packer, L. 1983. Studies of osmoregulation in salt adaptation with ESR spinprobe techniques. Proc. Natl. Acad. Sci. USA 80: 2599-2602. Bolen, D.W., and Baskakov, I.V. 2001. The osmophobic effect: natural selection of a thermodynamic force on protein folding. Mol. Biol. 310: 955-963. Borowitzka, L.J., and Brown, A.D. 1974. The salt relations of marine and halophilic species of the unicellular green alga, Dunaliella. The role of glycerol as a compatible solute. Arch. Microbiol. 96: 37-52. Borowitzka, L.J., Kessly, D.S., and Brown, A.D. 1977. The salt relations of Dunaliella. Further observations on glycerol production and its regulation. Arch. Microbiol. 113: 131-138.

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Louis, P., and Galinski, E.A. 1997. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology UK 143: 1141-1149. Mackay, M.A., and Norton, R.S. 1987. nuclear magnetic resonance study of biosynthesis of glucosylglycerol by a cyanobacterium under osmotic stress. J. Gen. Microbiol. 133: 1535-1542. Mackay, M.A., Norton, R.S., and Borowitzka, L.J. 1984. Organic osmoregulatory solutes in cyanobacteria. J. Gen. Microbiol. 130: 2177-2191. Maeda, M., and Thompson, G.A., Jr. 1986. On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock. J. Cell Biol. 102: 289-297. Malin, G., Iakobbashvili, R., and Lapidot, A. 1999. Effects of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. J. Biol. Chem. 274: 6920-6929. Maskow, T., and Babel, W. 2001. Calorimetrically obtained information about the efficiency of ectoine synthesis from glucose in Halomonas elongata. Biochim. Biophys. Acta 1527: 4-10. Menaia, J.A.G.F., Duarte, J.C., and Boone, D.R. 1993. Osmotic adaptation of moderately halophilic methanogenic archaeobacteria, and detection of cytosolic N,N-dimethylglycine. Experientia 49: 1047-1054. Mikkatt, S., Hagemann, M., and Schoor, A. 1996. Active transport of glucosylglycerol is involved in salt adaptation of the cyanobacterium Synechocystis sp. strain PCC 6803. Microbiology UK 142: 1725-1732. Min-Yu, L., Ono, H., and Takano, M. 1993. Gene cloning of ectoine synthase from Halomonas sp. Annu. Rep. Int. Cent. Coop. Res. Biotechnol. Japan 16: 193-200. Mohammad, F.A.A., Reed, R.H., and Stewart, W.D.P. 1983. The halophilic cyanobacterium Synechocystis DUN52 and its osmotic responses. FEMS Microbiol. Lett. 16: 287-290. Moore, D.J., Reed, R.H., and Stewart, W.D.P. 1987. A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria. Arch. Microbiol. 147: 399-405. Nagata, S., and Wang, Y.B. 2001. Interrelation between synthesis and uptake of ectoine for the growth of he halotolerant Brevibacterium species JCM 6894 at high osmolarity. Microbios 104: 7-15. Nicolaus, B., Lanzotti, V., Trincone, A., De Rosa, M., Grant, W.D., and Gambacorta, A. 1989. Glycine betaine and polar lipid composition in halophilic archaebacteria in response to growth in different salt concentrations. FEMS Microbiol. Lett. 39: 157-160. Nyyssölä, A., and Leisola, M. 2001. Actinopolyspora halophila has two separate pathways for betaine synthesis. Arch. Microbiol. 176: 294-300. Nyyssölä, A., Keruvuo, J., Kaukinen, P., von Weymarn, N., and Reinikainen, T. 2000. Extreme halophiles synthesize betaine from glycine by methylation. J. Biol. Chem. 275: 22196-22201. Nyyssölä, A., Reinikainen, T., and Leisola, M. 2001. Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase. Appl. Environ. Microbiol. 67: 2044-2050. Ono, H., Okuda, M., Tongpim, S., Shinmyo, A., Sakuda, S., Kaneko, Y., Murooka, Y., and Takano, M. 1998. Accumulation of compatible solutes, ectoine and hydroxyectoine, in a moderate halophile, Halomonas elongata KS3 isolated from dry salty land in Thailand. J. Ferment. Bioengin. 85: 362-368. Ono, H., Sawada, K., Khunajakr, N., Tao, T., Yamamoto, M., Hiramoto, M., Shinmyo, A., Takano, M., and Murooka, Y. 1999. Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J. Bacteriol. 181: 91-99. Oremland, R.S., and King, G.M. 1989. Methanogenesis in hypersaline environments, pp. 180-190 In: Cohen, Y., and Rosenberg, E. (Eds.), Microbial mats. Physiological ecology of benthic microbial communities. American Society for Microbiology, Washington, DC. Oren, A. 1995. The role of glycerol in the nutrition of halophilic archaeal communities: a study of respiratory electron transport. FEMS Microbiol. Ecol. 16: 281-290. Oren, A. 1997. Mycosporine-like amino acids as osmotic solutes in a community of halophilic cyanobacteria. Geomicrobiol. J. 14: 233-242. Oren, A. 1990. Formation and breakdown of glycine betaine and trimethylamine in hypersaline environments. Antonie van Leeuwenhoek 5: 291-298. Oren, A. 1999a. Life at high salt concentrations, In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.-H. and Stackebrandt, E. (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. 3rd. Ed. Springer-Verlag, New York (Electronic publication). Oren, A. 1999b. Bioenergetic aspects of halophilism. Microbiol. Mol. Biol. Rev. 63: 334-348. Oren, A. 2000. Salts and brines, pp. 281-306 In: Whitton, B.A., and Potts, M. (eds.), Ecology of cyanobacteria: Their diversity in time and space. Kluwer Academic Publishers, Dordrecht.

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Oren, A., Simon, G., and Galinski, E.A. 1991. Intracellular salt and solute concentrations in Ectothiorhodospira marismortui: glycine betaine and glutamineamide as osmotic solutes. Arch. Microbiol. 156: 350-355. Oren, A., Fischel, U., Aizenshtat, Z., Krein, E.B., and Reed, R.H. 1994. Osmotic adaptation of microbial communities in hypersaline mats, pp. 125-130 In: Stal, L.J., and Caumette, P. (Eds.), Microbial mats. Structure, development and environmental significance. Springer Verlag, Berlin. Oren, A., Kühl, M., and Karsten, U. 1995. An endevaporitic microbial mat within a gypsum crust: zonation of phototrophs, photopigments, and light penetration. Mar. Ecol. Prog. Ser. 128: 151-159. Peters, P., Galinski, E.A., and Trüper, H.G. 1990. The biosynthesis of ectoine. FEMS Microbiol. Lett. 71: 157162. Peters, P., Tel-Or, E., and Trüper, H.G. 1992. Transport of glycine betaine in the extremely haloalkaliphilic sulphur bacterium Ectothiorhodospira halochloris. J. Gen. Microbiol. 138: 1993-1998. Rafaeli-Eshkol, D., and Avi-Dor, Y. 1968. Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system. Biochem. J. 109: 687-691. Reed, R.H. 1984. Use and abuse of osmo-terminology. Plant, Cell and Environment 7: 165-170. Reed, R.H. 1986. Halotolerant and halophilic microbes, pp. 55-81 In: Herbert, R.A., and Codd, G.A. (Eds.), Microbes in extreme environments. Academic Press, London. Reed, R.H., Chudek, J.A., Foster, R., and Stewart, W.D.P. 1984. Osmotic adjustment in cyanobacteria from hypersaline environments. Arch. Microbiol. 138: 333-337. Reed, R.H., Borowitzka, L.J., MacKay, M.A., Chudek, J.A., Foster, R., Warr, S.R.C., Moore, D.J., and Stewart, W.D.P. 1986. Organic solute accumulation in osmotically stressed cyanobacteria. FEMS Microbiol. Rev. 39: 51-56. Regev, R., Peri, I., Gilboa, H., and Avi-Dor, Y. 1990. NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Arch. Biochem. Biophys. 278: 106112. Richardson, R.L., Reed, R.H., and Stewart, W.D.P. 1983. Synechocystis PCC6803, a euryhaline cyanobacterium. FEMS Microbiol. Lett. 18: 99-102. Robert, H., Le Marrec, C., Blanco, C., and Jebbar, M. 2000. Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila. Appl. Environ. Microbiol. 66: 509-517. Roberts, M.F., Lai, M.-C., and Gunsalus. R.P. 1992. Biosynthetic pathways of the osmolytes and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analysis. J. Bacteriol. 174: 6688-6693. Robertson, D.E., Noll, D., Roberts, M.F., Menaia, J.A.G.F., and Boone, D.R. 1990a. Detection of the osmoregulator betaine in methanogens. Appl. Environ. Microbiol. 56: 563-565. Robertson, D.E., Roberts, M.F., Belay, N., Stetter, K.O., and Boone, D.R. 1990b. Occurrence of a novel osmolyte, in marine methanogenic bacteria. Appl. Environ. Microbiol. 56: 1504-1508. Robertson, D.E., Lai, M.-C., Gunsalus, R.F., and Roberts, M.F. 1992. Composition, variation, and dynamics of major osmotic solutes in Methanohalophilus strain FDF1. Appl. Environ. Microbiol. 58: 2438-2443. Roesler, C.S., Culbertson, C.W., Etheridge, S.M., Goericke, R., Kiene, R.P., Miller, L.G., and Oremland, R.S. 2002. Distribution, production, and ecophysiology of Picocystis strain ML in Mono Lake, California. Limnol. Oceanogr. 47: 440-452. Severin, J., Wohlfarth, A., and Galinski, E.A. 1992. The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol. 138: 1629-1638. Shkedy-Vinkler, H., and Avi-Dor, Y. 1975. Betaine-induced stimulation of respiration at high osmolarities in a halotolerant bacterium. Biochem. J. 150: 219-226. Sowers, K.R., Robertson, D.E., Noll, D., Gunsalus, R.P., and Roberts, M.F. 1990. an osmolyte synthesized by methanogenic archaebacteria. Proc. Natl. Acad. Sci. USA 87: 9083-9087. Suenobu, K., and Nagaoka, M. 1998. Ab initio molecular orbital study on molecular and hydration structures of ectoine. J. Phys. Chem. A 102: 7505-7511. Tel-Or, E., Spath, S., Packer, L., and Mehlhorn, R.J. 1986. Carbon-13 NMR studies of salt shock influenced carbohydrate turnover in the marine cyanobacterium Agmenellum quadruplicatum. Plant Physiol. 82: 646652. Tetsch, L., and Kunte, H.J. 2002. The substrate-binding protein TeaA of the osmoregulated ectoine transporter TeaABC from Halomonas elongata: purification and characterization of recombinant TeaA. FEMS Microbiol. Lett., in press.

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Timasheff, S.N. 1992. A physicochemical basis for the selection of osmolytes by nature, pp. 70-86 In: Somero, G.N., Osmond, C.B., and Bolis, C.L. (Eds.), Water and life. Springer-Verlag, New York. Trotsenko, Y.A., and Khmelenina, V.N. 2002. The biology of osmoadaptation of halophilic methanotrophs. Mikrobiologiya 71: 149-159 (Microbiology 71: 123-132). Trüper, H.G., and Galinski, E.A. 1989. Compatible solutes in halophilic phototrophic procaryotes, pp. 342-348 In: Cohen, Y., and Rosenberg, E. (Eds.), Microbial mats. Physiological ecology of benthic microbial communities. ASM Press, Washington, DC. Trüper, H.G., and Galinski, E.A. 1990. Biosynthesis and fate of compatible solutes in extremely halophilic phototrophic eubacteria. FEMS Microbiol. Rev. 75: 247-254. Trüper, H.G., Severin, J., Wohlfarth, A., Müller, E., and Galinski, E.A. 1991. Halophily, taxonomy, phylogeny and nomenclature, pp. 3-7 In: Rodriguez-Valera, F. (Ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York. Tschichholz, I., and Trüper, H.G. 1990. Fate of compatible solutes during dilution stress in Ectothiorhodospira halochloris. FEMS Microbiol. Ecol. 73: 181-186. Ventosa, A., Nieto, J.J., and Oren, A. 1998. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biot. Rev. 62: 504-544. Waditee, R., and Incharoensakdi, A. 2001. Purification and kinetic properties of betaine-homocysteine methyltransferase from Aphanothece halophytica. Curr. Microbiol. 44: 107-111. Warr, S.R.C., Reed, R.H., and Stewart, W.D.P. 1984. Osmotic adjustment of cyanobacteria: the effects of NaCl, KCl, sucrose and glycine betaine on glutamine synthetase activity in a marine and a halotolerant strain. J Gen. Microbiol. 130: 2169-2175. Warr, S.R.C., Reed, R.H., Chudek, J.A., Foster, R., and Stewart, W.D.P. (1985) Osmotic adjustment in Spirulina platensis. Planta 163: 424-429. Wegmann, K., Ben-Amotz, A., and Avron, M. 1980. Effect of temperature on glycerol retention in the halotolerant algae Dunaliella and Asteromonas. Plant Physiol. 66: 1196-1197. Welsh, D.T. 2000. Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate. FEMS Microbiol. Rev. 24: 263-290. Welsh, D.T., and Herbert, R.A. 1993. Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance nuclear magnetic resonance spectroscopy. FEMS Microbiol. Ecol. 13: 143-150. Welsh, D.T., Lindsay, Y.E., Caumette, P., Herbert, R.A., and Hannan, J. 1996. Identification of trehalose and glycine betaine as compatible solutes in the moderately halophilic sulfate reducing bacterium Desulfovibrio halophilus. FEMS Microbiol. Lett. 140: 203-207. Wiggins, P.M. 1990. Role of water in some biological processes. Microbiol. Rev. 54: 432-449. Wohlfarth, A., Severin, J., and Galinski, E.A. 1990. The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae. J. Gen. Microbiol. 136: 705-712. Wohlfarth, A., Severin, J., and Galinski, E.A. 1993. Identification of as a novel osmolyte in some Gram-positive eubacteria. Appl. Microbiol. Biotechnol. 39: 568-573. Zavarzin, G.A., Gerasimenko, L.M., and Zhilina, T.N. 1993. Cyanobacterial communities in hypersaline lagoons of Lake Sivash. Mikrobiologiya 62: 1113-1126 (Microbiology 62: 645-652). Zidan, M.A., Hipkins, M.F., and Boney, A.D. 1987. Loss of intracellular glycerol from Dunaliella tertiolecta after decreasing the external salinity. J. Plant Physiol. 127: 461-469.

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CHAPTER 9 HALOPHILIC BACTERIOPHAGES AND HALOCINS

The existence of bacteriophage for Halobacterium opens the existing possibilities of further exploration of the genetic basis of extreme halophilism. (Torsvik and Dundas, 1974) The isolation of phage for Halobacterium allows a study of genetic adaptation in the extremes of ionic strength. (Wais et al., 1975)

9.1. BACTERIOPHAGES OF HALOPHILIC MICROORGANISMS The first bacteriophages that attack halophilic Archaea ("halophages" or "haloviruses") were discovered in the early 1970s (Torsvik and Dundas, 1974; Wais et al., 1975). Such bacteriophages require high concentrations of salt for structural stability, just as their hosts. Both lytic and temperate halophages occur. Halophilic archaeal bacteriophages have since been isolated from a variety of environments inhabited by Halobacteriaceae, including the Great Salt Lake (Post, 1981), saltern crystallizer ponds, and fermented fish sauce. In addition, some cultures of halophilic Archaea maintained in laboratories for many years were found to harbor temperate bacteriophages (Pfeifer, 1988; Torsvik and Dundas, 1980). In spite of the promising statements on the future use of halophages in genetic studies made in the first publications on halophages, as quoted above, this potential has hardly been realized thus far. Halophilic Bacteria too have their bacteriophages. The first report suggesting that halophilic Bacteria may be subject to lysis by phages is probably the observation by Gochnauer et al. (1975) that cultures of Actinopolyspora halophila grown on agar plates containing media of relatively low salinity (100 to 120 being near the lower limit required for growth of this organism) showed holes resembling viral plaques. A few bacteriophages from different halophilic Bacteria have since been characterized.

9.1.1. Bacteriophages of halophilic Archaea Halophages have been found that lyse Halobacterium salinarum (Daniels and Wais, 1990; Torsvik and Dundas, 1974; Wais et al., 1975), Haloarcula hispanica (Bath and Dyall-Smith, 1998), Haloferax volcanii (Nuttall and Dyall-Smith, 1993), Natrialba

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magadii (Witte et al., 1997), and other species (Nuttall and Dyall-Smith, 1993). Table 9.1 summarizes the properties of a number of such bacteriophages that have been studied. Most halophages have a "head and tail" morphology, but there are also spindle-shaped or lemon-shaped phages such as His1 of Haloarcula hispanica (Bath and Dyall-Smith, 1998), and SH1 is spherical in shape. Most of these phages contain double-stranded DNA only, but phage of the alkaliphilic Natrialba magadii contains both DNA and RNA (Witte et al., 1997). Most of the phages that have been characterized attack Halobacterium salinarum strains. Burst sizes between 140 and 1,300 have been reported for lytic halophages. The largest value reported was for phage S45 (Daniels and Wais, 1984). A large burst size (1,100) was also observed during lysis of Halobacterium salinarum by phage Hh-1 (Pauling, 1982). There exist many strains of Halobacterium salinarum (a species that now includes those designated in the past Halobacterium halobium or Halobacterium cutirubrum), and their susceptibility to lysis by the different halophages differs greatly. Lytic activity of different phages may therefore be used to discriminate between strains within the genus Halobacterium. Several phages can enter a carrier state inside their host, and revert to a lytic cycle when conditions become suitable. Examples of such temperate phages are Halobacterium salinarum phages Hh1, Hh3 (Pauling, 1982), and Hs1 (Torsvik and Dundas. 1980). In the case of phage Hs1 the transition between the temperate and the virulent state is induced by a decrease in salt concentration from 300 to , being near the lower salinity level in which the host can grow (Torsvik and Dundas, 1980). Similarly, an increased salinity reduces the burst size and prolongs the latent period of infection of bacteriophage S5100 (Daniels and Wais, 1990). A temperate phage that has been studied in depth is of Halobacterium salinarum (Schnabel et al., 1982a, 1982b; Schnabel and Zillig, 1984). Phage production is spontaneously induced at a rate of about Cells lysogenic for the phage are immune to infection by the phage. The prophage state consists of a covalently closed circle of DNA of 57 kbp, terminally redundant and partially circularly permuted (Schnabel, 1984). The structure of this prophage genome varies with a high frequency. Six phage variants have been described that differ by several insertions, a deletion, and an inversion (Schnabel et al., 1982b). Two of these variants both have two copies of the 1.8-kbp insertion element ISH1.8 in inverted orientation, whereas the other variants have only one copy. This same IS element also occurs in the genome of Halobacterium strain NRC-1 (see Chapter 10). As in the linear phage DNA, a segment flanked by two copies of the insertion element ISH1.8 is frequently inverted. This L segment can also circularize to a plasmid of 12 kpb with simultaneous loss of the remaining phage DNA. A strain of Halobacterium which contains this plasmid is immune to infection by A variant of the phage, which carries an insertion of 1 kbp in its L segment, is able to grow within L, but not within normal lysogens, showing that the plasmid confers to only part of the immunity of normal lysogens.

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Phages HF1 and HF2 were isolated from salt lakes, are both virulent, and have large genomes (78 kbp). No less than 121 putative open reading frames have been identified in the genome of HF2, many of which (37) show small overlaps. Five tRNA genes were present as well within the phage genome. Many restriction enzymes do not cut this long stretch of DNA, as it contains very few palindromic sequences (Nuttall and Dyall-Smith, 1993). Restriction and modification have been shown to occur in the DNA of phage S45 in Halobacterium salinarum. The host-controlled restriction and modification involves strain-specific endonuclease activities (Daniels and Wais, 1984). Phage a temperate phage of the alkaliphilic Natrialba magadii, was isolated from a laboratory culture that had lysed spontaneously. Besides being the first phage isolated from a haloalkaliphile, this phage is unique as it contains both DNA and RNA in the mature phage particles (Witte et al., 1997). The phage requires at least 120 salt for structural stability. The prophage DNA is integrated in the host chromosome. Part of the phage DNA population is modified; restriction analysis revealed evidence for adenine methylation within 5'-GATC-3' and related sequences. A adenine methyltransferase (DAM methylase) was identified in the viral genome. Heterologous expression of this enzyme in Escherichia coli showed it to be active also in the absence of salt (Baranyi et al., 2000). The importance that bacteriophages may play in controlling the community sizes of the Halobacteriaceae in hypersaline water bodies appears from the high number of virus-like particles - one to two orders higher than the number of bacteria present observed during electron microscopical examination of brines from saltern crystallizer ponds (Guixa–Boixareu et al., 1996) and the Dead Sea (Oren et al., 1997). Virus-like particles were abundantly found in the crystallizer ponds of salterns in Spain. Numbers of presumed viruses as high as were observed in NaCl-saturated brines. In the lower salinity range (up to about 150 around prokaryote cells and virus-like particles, most of them with icosahedral heads, were counted per ml of brine. Most viruses were untailed, but tailed and spindle-shaped viruses were observed as well (Figure 9.1). Between one and ten percent of the flat square Archaea in the crystallizer ponds had visible phages inside, often in high numbers; the estimated burst size was more than 200 viruses per cell. However, it was concluded that viruses, although present in high numbers, did not exert a strong control over the prokaryotic abundance and growth rate. At the highest salinities the percentage of cells lost daily by viral lysis was calculated to be lower than 5%. No virus-infected cells were observed in the lower salinity evaporation ponds (Guixa-Boixareu et al., 1996). In an attempt to obtain more information on the types of bacteriophages present in Spanish saltern ponds, viral-sized particles were concentrated from the brines (134-350 salt) by tangential flow filtration and ultracentrifugation. The nucleic acids present in the viral assemblage were then analyzed by pulsed-field gel electrophoresis. Between one and eight DNA bands were obtained. No correlation was found between the number of bands and the brine salinity. The apparent sizes of the viral DNA bands was between 25 and 300 kbp. Whether these bands consisted of circular or of linear DNA was not determined. For each salinity a different pattern was obtained. Viral

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diversity in the saltern brines was much lower than that observed in the marine environment using similar techniques (Diez et al., 2000). The Dead Sea also showed high concentrations virus-like particles (head and tail or spindle-shaped). In view of the dominance of halophilic Archaea in this lake, these virus-like particles were most probably derived from lysis of Archaea. Lysis by halophilic phages may be the cause of sudden drops in the community size of halophilic Archaea observed in the Dead Sea (Oren et al., 1997, see also Section 13.3). Halophages may become active in natural communities of halophilic Archaea when these are exposed to suboptimal salt concentrations. In a study of a brine pool in Jamaica, Wais and Daniels (1985) observed an increase in phage numbers following a lowering of the salinity. Phage enrichments were performed with Halobacterium salinarum (cutirubrum) as a host, using different volumes of brine as inoculum. When using saturated brines, the minimal volumes resulting in positive enrichments were 1, 5, or 50 ml. However, after the destruction of the halobacterial population in the pond by dilution following rainfall, phages were much more abundant: positive results were already obtained using a sample size of 0.1-0.2 ml. This observation is consistent with laboratory results, such as the finding that the transition between the lysogenic and the virulent state of phage Hs1 is induced by a decrease in salt concentration to a level close to the lower salinity level in which Halobacterium salinarum can grow (Torsvik and Dundas, 1980). The burst size of bacteriophage S5100 is reduced and the latent period of infection prolonged at increased salinity (Daniels and Wais, 1990). Bacteriophage isolates from the Jamaica salt ponds active against Halobacterium salinarum were found to be of low virulence, probably due to the slow adsorption of the virus particles to the host (Daniels and Wais, 1998). Dilution of brines by rain is a gradual process, and intermediate salinity levels may thus have existed long enough to enable multiplication of the phages. Subsequently the host population was destroyed by further dilution. The phage thus appears to have grown at the expense of a host doomed to destruction by factors other than phage infection. The phage did thus not exert a selective disadvantage on its host (Wais and Daniels, 1985). The salinitydependent shift from lytic to persistent infection may be of ecological importance to halobacteria and their phage coexisting in their natural environment. In saltern ponds that approach the lower limit for the existence of halobacteria, production of phage particles is favored, thus maximizing the chance of phage survival. At saturating salt concentrations the bacteria reach their optimal growth conditions. A carrier state is established, in which the bacteria are protected from extensive phage-induced lysis, and the phage is perpetuated at the same time. The Halobacterium – halophage system is therefore well adapted to the variations in salinity occurring in the salterns (Torsvik and Dundas, 1980). Halophilic Archaea may possess restriction-modification systems that can protect them against halophage infection. Two such systems occur in Halobacterium salinarum (cutirubrum) NRC 34001. as appeared from experiments in which the plating efficiencies of halophage Hh-3 were investigated (Patterson and Pauling, 1985).

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9.1.2. Bacteriophages of halophilic Bacteria Halophilic Bacteria also have their bacteriophages. The list of such phages known is quite short (Table 9.2), and this probably due to the fact that relatively few attempts have been made toward the isolation and characterization of such bacteriophages. We know little about the occurrence of phages in hypersaline environments inhabited by halophilic Bacteria and on their importance in the regulation of the community density of their host organisms. Studies in which these bacteriophages are exploited to investigate the genetics of their hosts are lacking altogether. All bacteriophages isolated thus far from the moderate halophiles are doublestranded DNA phages with heads and tails. Most are almost equally stable in the presence and the absence of salt. They may retain infectivity for weeks in dilute solutions (Kauri et al., 1991). In contrast, the halophilic archaeal phages, like their hosts, are inactivated in the absence of salt. The bacterial phages are "halophilic" only to the extent that their hosts are: the phages multiply only when the halophilic host is growing. In view of the relatively low salt concentrations within the cytoplasm of halophilic bacteria compared to the outside medium, the process of attachment and infection of the host cell may occur at high salt concentrations, while intracellular multiplication occurs in a low-salt medium (Kauri et al., 1991). A phage designated UTAK, isolated from an organism resembling Salinivibrio costicola isolated from the salterns of Alicante, Spain, has been studied in some detail. The phage particles are stable for a long time in distilled water. Burst size was maximal in cells grown at 60 to 120 NaC1 (80 to 105 phages per cell), and decreased to an average of only 12.5 phages per cell at 30 . Intracellular phage replication may thus be controlled by the salinity of the medium. Additional bacteriophages were isolated from the saltern brine in the course of this study, some of which attacked morphologically different hosts (Goel et al., 1996). Most of these phages were not studied further. A water sample from lake Chaplin, Canada, was the source of isolation of a bacteriophage that lysed a bacterium designated Pseudomonas strain G3, which is able to grow from 15 to over 175 NaCl. The phage also infects Salinivibrio costicola and two unidentified halophilic bacteria, and is stable in the absence of salt (Kauri et al., 1991). Induction of lysogenic phages by mitomycin C treatment led to the isolation of phage F9-11 from a Halomonas halophila strain obtained from soil. This phage replicates over a wide range of salinities (25 to 150 (Calvo et al., 1988; Garcia de la Paz et al., 1989). Two bacteriophages lysing Tetragenococcus halophilus, a bacterium involved in soy sauce fermentation, have been characterized. Phage has an isometeric head and a contractile tail, and consists of an isometric head and a noncontractile tail. Both phages could propagate at all salinities in which their host could grow.

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Phage

was stable at all salinities between 1.8 and 150 However, phage was speficically unstable between 2 and 10 while being stable in both the lower(0.5to2 and the higher (10 to 150 salt range.This effect is probably due to the salting-in effect of ions which cause destruction of phage protein and DNA-protein complexes (Uchida and Kanbe, 1993).

9.2. HALOCINS The formation of halocins (halophilic "bacteriocins" or better: archaeocins), protein antibiotics excreted by halophilic Archaea, was first described in 1982, when it was discovered that colonies of Haloferax mediterranei on agar plates inhibit growth of Halobacterium salinarum and other members of the Halobacteriaceae (RodriguezValera et al., 1982). Since then it has become clear that halocin production is an almost universal feature among the halophilic Archaea (Meseguer and RodriguezValera, 1986; Meseguer et al., 1986; O’Connor and Shand, 2002; Shand et al., 1999; Torreblanca et al., 1994). Comparative studies with large numbers of isolates have shown that there are many types of halocins with different activity spectra (Torreblanca et al., 1994). Several halocins have been characterized in depth. These include halocin H4 of Haloferax mediterranei, halocin H1 of Haloferax mediterranei X1a.3, halocin H6 of Haloferax gibbonsii, halocin Hal R1 of Halobacterium sp. GN 101, halocin A4 produced by a halophilic archaeon obtained from Tunisia (Rdest and Sturm, 1987), and halocin S8 of a yet uncharacterized rod-shaped halobacterium isolated from Great Salt Lake, Utah. Their properties are listed in Table 9.3. A general overview of the halocins was given by Shand et al. (1999). Halocin H4 is a heat-sensitive and salt-dependent protein. Its size was originally described as 28 kDa (Meseguer and Rodriguez-Valera, 1985), but analysis of the cloned gene has shown it to be a 34.9 kDa protein, made from a pre-protein of 39.6 kDa. It contains a signal sequence of 46 amino acids assisting the transport of the protein through the cell membrane (Cheung et al., 1997). Halocin H4 interacts with the membrane of the target cells, where it probably causes permeability changes that result in an ionic imbalance, leading to death and cell lysis (Meseguer and RodriguezValera, 1985, 1986; Meseguer et al., 1991; Rodriguez-Valera et al., 1982). Sensitive cells become swollen and assume a spherical shape (Meseguer and Rodriguez-Valera, 1986). Halocin H6 has a size of 32 kDa, is heat-resistant, and does not depend on salt for stability and activity (Torreblanca et al., 1989). It is the only halocin whose specific target of action is known: it inhibits the antiporter activity of sensitive cells, thus targeting the central device used by the halobacteria to adapt to highly saline environments (Meseguer et al., 1995). Halocin Hal R1 is a small protein of 3.8 kDa (to be classified as a "microcin"). It is thermostable, does not depend on salt, and shows bacteriostatic effect on a wide variety of halophilic Archaea (Rdest and Sturm, 1987).

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Halocin S8 is another microcin with a very narrow specificity: the only strains known to be inhibited are Halobacterium salinarum NRC 817, Halobacterium sp. strain GRB, and Haloferax gibbonsii. The 36 amino acids, 3,580 Da protein is produced by processing of a much larger, 33,962 Da protein coded by a 933 bp open reading frame located on a 200 kbp megaplasmid. The halocin is thermostable and does not depend on salt for stability (O'Connor and Shand, 2002; Shand et al., 1999). Halocin H1, an 31 kDa protein, is produced by a strain of Haloferax mediterranei (Platas et al., 1996). Halocin A4, excreted by halophilic archaeon TuA4 isolated from a salt lake in Tunisia, is of special interest as it is the first halocin known to be inhibitory not only toward members of the Halobacteriaceae (the haloalkaliphiles Natronobacterium gregoryi and Natrialba magadii), but also against phylogenetically much more remote Sulfolobus species (Haseltine et al., 2001).

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Halocins are typically formed when the cultures of the producing strains enter the stationary growth phase (Shand et al.,1999). In Haloferax mediterranei the transcript for halocin H4 is present at a low level during exponential growth, but increases dramatically at the end of the exponential growth phase when resources become exhausted. Activity first appears as the culture enters the stationary phase. As halocin activity increases, so do transcript levels, but then activity levels decrease sharply while transcript levels remain elevated (Figure 9.2). This suggests that the synthesis of the halocin may be regulated post-transcriptionally (Cheung et al., 1997). Transcription of the gene for halocin S8 production likewise increases sharply during the transition to the stationary phase. The activities of halocins H4, H6 and S8 decrease during the stationary phase, but Hal R1 maintains a high activity for prolonged times (Shand et al., 1999). Halocin production may be expected to be of considerable ecological advantage, as the ability to compete for nutrients and other resources may be enhanced by excreting archaeocins. However, it has never yet been proven that halocins are indeed excreted by natural communities of halophilic Archaea in concentrations sufficient to inhibit the development of competing strains, thus substantiating their ecological role. A study aimed toward the quantitation of the presence of halocins in saltern crystallizer ponds at different locations worldwide failed to detect halocin activity, even not after 53-fold concentration of the protein fraction in the brines by ultrafiltration (Kis-Papo and Oren, 2000). 9.3. REFERENCES Baranyi, U., Klein, R., Lubitz, W., Krüger, D.H., and Witte, A. 2000. The archaeal halophilic virus-encoded Dam-like methyltransferase M. methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli. Mol. Microbiol. 38: 1168-1179. Bath, C., and Dyall-Smith, M.L. 1998. His1, an archaeal vims of the Fuselloviridae family that infects Haloarcula hispanica. J. Virol. 72: 9392-9395. Calvo, C., Garcia de la Paz, A., Bejar, V., Quesada, E., and Ramos-Cormenzana, A. 1988. Isolation and characterization of phage F9-11 from a lysogenic Deleya halophila strain. Curr. Microbiol. 17: 49-53. Cheung, J., Danna, K.J., O'Connor, E.M., Price, L.B., and Shand, R.F. 1997. Isolation, sequence, and expression of the gene encoding halocin H4, a bacteriocin from the halophilic archaeon Haloferax mediterranei R4. J. Bacteriol. 179: 548-551. Daniels, L.L., and Wais, A.C. 1984. Restriction and modification of halophage S45 in Halobacterium. Curr. Microbiol. 10: 133-136. Daniels, L.L., and Wais, A.C. 1990. Ecophysiology of bacteriophage S5100 infecting Halobacterium cutirubrum. Appl. Environ. Microbiol. 56: 3605-3608. Daniels, L.L., and Wais, A.C. 1998. Virulence in phage populations infecting Halobacterium cutirubrum. FEMS Microbiol. Ecol. 25: 129-134. Diez, B., Antón, J., Guixa-Boixereu, N., Pedrós-Alió, C., and Rodríguez-Valera, F. 2000. Pulsed-field gel electrophoresis analysis of virus assemblages present in a hypersaline environment. Int. Microbiol. 3: 159164. Dyall-Smith, M.L. 2001. The halohandbook: protocols for halobacterial genetics (http://www.microbiol. unimelb.edu.au/micro/staff/mds/HaloHandbook/Index.html, updated December 2001; accessed January 4, 2002). Garcia de la Paz, A.M., Perez Martinez, A., Calvo Sainz, C., and Ramos Cormenzana, A. 1989. Isolation and characterisation of bacteriophages active on moderately halophilic microorganisms, p. 425 In: Da Costa,

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M.S., Duarte, J.C., and Williams, R.A.D. (Eds.), Microbiology of extreme environments and its potential for biotechnology. Elsevier Applied Science, London. Gochnauer, M.B., Leppard, G.G., Komaratat, P., Kates, M., Novitsky, T., and Kushner, D.J. 1975. Isolation and characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can. J. Microbiol. 21: 1500-1511. Goel, U., Kauri, T., Ackermann, H.-W., and Kushner, D.J. 1996. A moderately halophilic Vibrio from a Spanish saltern and its lytic bacteriophage. Can. J. Microbiol. 42: 1015-1023. Guixa-Boixareu, N., Calderón-Paz, J.I., Heldal, M., Bratbak, G., and Pedrós-Alió, C. 1996. Viral lysis and bacterivory as prokaryotic loss factors along a salinity gradient. Aquat. Microb. Ecol. 11: 215-227. Haseltine, C., Hill, T., Montalvo-Rodviguez, R., Kemper, S.K., Shand, R.F., and Blum, P. 2001. Secreted euryarchaeal microhalocins kill hyperthermophilic crenarchaea. J. Bacteriol. 183: 287-291. Kauri, T., Ackermann, H.-W., Goel, U., and Kushner, D.J. 1991. A bacteriophage of a moderately halophilic bacterium. Arch. Microbiol. 156: 435-438. Kis-Papo, T., and Oren, A. 2000. Halocins: are they involved in the competition between halobacteria in saltern ponds? Extremophiles 4: 35-41. Meseguer, I., and Rodriguez-Valera, F. 1985. Production and purification of nhalocin H4. FEMS Microbiol. Lett. 28: 177-182. Meseguer, I., and Rodriguez-Valera, F. 1986. Effect of halocin H4 on cells of Halobacterium halobium. J. Gen. Microbiol. 132: 3061-3068. Meseguer, I., Rodríguez-Valera, F., and Ventosa, A. 1986. Antagonistic interactions among halobacteria due to halocin production. FEMS Microbiol. Lett. 36: 177-182. Meseguer, I., Torreblanca, M., and Rodriguez-Valera, F. 1991. Mode of action of halocins H4 and H6: are they effective against the adaptation to high salt environments?, pp. 157-164 In: Rodriguez-Valera, F. (Ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York. Meseguer, I., Torreblanca, M., and Konishi, T. 1995. Specific inhibition of the halobacterial antiporter by halocin H6. J. Biol. Chem. 270: 6450-6455. Nuttall, S.D., and Dyall-Smith, M.L. 1993. HF1 and HF2: novel bacteriophages of halophilic archaea. Virology 197: 678-684. O'Connor, E.M., and Shand, R.F. 2002. Halocins and sulfolobicins: The emerging story of archaeal protein and peptide antibiotics. J. Int. Microbiol. Biotechnol. 28: 23-31. Oren, A., Bratbak, G., and Heldal, M. 1997. Occurrence of virus-like particles in the Dead Sea. Extremophiles 1: 143-149. Patterson, N.H., and Pauling, C. 1985. Evidence for two restriction-modification systems in Halobacterium cutirubrum NRC 34001. J. Bacteriol. 163: 783-784. Pauling, C. 1982. Bacteriophages of Halobacterium halobium: isolation from fermented fish and primary characterization. Can. J. Microbiol. 28: 916-921. Pfeifer, F. 1988. Genetics of halobacteria, pp. 105-133 In: Rodriguez-Valera, F. (Ed.), Halophilic bacteria, Vol. II. CRC Press, Boca Raton. Platas, G., Meseguer, I., and Amils, R. 1996. Optimization of the production of a bacteriocin from Haloferax mediterranei Xia3. Microbiología SEM 12: 75-84. Post, F.J. 1981. Microbiology of the Great Salt Lake north arm. Hydrobiologia 81: 59-69. Price, L.B., and Shand, R.F. 2000. Halocin S8: a 36-amino-acid microhalocin from the haloarchaeal strain S8A. J. Bacteriol. 182: 4951-4958. Rdest, U., and Sturm, M. 1987. Bacteriocins from halobacteria, pp. 271-278 In: Burgess, R. (Ed.), Protein purification: micro to macro. Alan R. Liss, New York. Rodriguez-Valera, F., Juez, G., and Kushner, D.J. 1982. Halocins: salt dependent bacteriocins produced by extremely halophilic rods. Can. J. Microbiol. 28: 151-154. Rohrmann, G.F., Cheney, R., and Pauling, C. 1983. Bacteriophages of Halobacterium halobium: virion DNAs and proteins. Can. J. Microbiol. 29: 627-629. Schabel, H. 1984. An immune strain of Halobacterium halobium carries the invertible L segment of phage as a plasmid. Proc. Natl. Acad. Sci. USA 81: 1017-1020. Schnabel, H., and Zillig, W. 1984. Circular structure of the genome of phage in a lysogenic Halobacterium halobium. Mol. Gen. Genet. 193: 422-426. Schnabel, H., Zillig, W., Pfäffle, M., Schnabel, R., Michel, H., and Delius, H. 1982a. Halobacterium halobium phage EMBO J. 1:87-92. Schnabel, H., Schramm, E., Schnabel, R., and Zillig, W. 1982b. Structural variability of the genome of phage ΦH of Halobacterium halobium. Mol. Gen. Genet. 188: 370
377.

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Shand, R.F., Price, L.B., and O'Connor, E.M. 1999. Halocins: protein antibiotics from hypersaline environments, pp. 295-306 In: Oren, A. (Ed.), Microbiology and biogeochemistry of hypersaline environments. CRC Press, Boca Raton. Soppa, J., and Oesterhelt, D. 1989b. Halobacterium sp. GRB – a species to work with? Can J. Microbiol. 35: 205-209. Torreblanca, M., Meseguer, I., and Rodríguez-Valera, F. 1989. Halocin H6, a bacteriocin from Haloferax gibbonsii. J. Gen. Microbiol. 135: 2655-2661. Torreblanca, M., Meseguer, I., and Ventosa, A. 1994. Production of halocin is a practically universal feature of archaeal halophilic rods. Lett. Appl. Microbiol. 19: 201-205. Torsvik T., and Dundas, I.D. 1974. Bacteriophage of Halobacterium salinarium. Nature 248: 680-681. Torsvik, T., and Dundas, I.D. 1978. Halophilic phage specific for Halolobacterium salinarium str. 1., pp. 609-614 In: Caplan, S.R., and Ginzburg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam. Torsvik, T., and Dundas, I.D. 1980. Persisting phage infection in Halobacterium salinarium str. 1. J. Gen. Virol. 47: 29-36. Uchida, K., and Kanbe, C. 1993. Occurrence of bacteriophages lytic for Pediococcus halophilus, a halophilic lactic-acid bacterium, in soy sauce fermentation. J. Gen. Appl. Microbiol. 39. 429-437. Wais, A.C., and Daniels, L.L. 1985. Populations of bacteriophage infecting Halobacterium in a transient brine pool. FEMS Microbiol. Ecol. 31: 323-326. Wais, A.C., Kon, M., MacDonald, R.E., and Stollar, B.D. 1975. Salt-dependent bacteriophage infecting Halobacterium cutirubrum and Halobacterium halobium. Nature 256: 314-315. Witte, A., Baranyi, U., Klein, R., Sulzner, M., Luo, C., Wanner, G., Krüger, D.H., and Lubitz, W. 1997. Characterization of Natronobacterium magadii phage ΦCh1, a un ique archaeal phage containing D N A and RN A. Mol. Microbiol. 23: 603
616.

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CHAPTER 10 GENETICS AND GENOMICS OF HALOPHILIC ARCHAEA AND BACTERIA

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related microchromosomes. (Ng et al., 2000) The relatively simple growth requirements and sensitivity of halophilic eubacteria to antibiotics should make them attractive for both physiological and genetic studies. They have been used for physiological studies...., but practically nothing is known about their genetics. There seems to be a silent agreement among molecular biologists, who think of halophilic eubacteria at all, that these will be very close to the non-halophilic eubacteria in gene organization and expression. This implies that such attributes will not be affected by the high salt concentration around, and sometimes in, the cells. If true, this would be so strange to be worth establishing. I suspect, however, that the halophilic eubacteria will eventually prove to have their own fascinating genetic, as well as physiological, properties. (Kushner, 1993)

10.1. GENETICS OF HALOPHILIC MICROORGANISMS AN HISTORICAL SURVEY In 1963 Joshi et al. analyzed DNA of different halophilic Archaea in cesium chloride density gradients in the ultracentrifuge. They observed that some of these extreme halophiles gave two rather than one band of DNA. The discovery of a minor fraction of "satellite DNA" or "FII DNA" with a lower G+C content than the main DNA band was probably the first evidence that the halophilic Archaea have special properties also on the level of the genome structure. Thirty seven years later, a group of scientists led by Shiladitya DasSarma published the complete genome sequence of Halobacterium strain NRC-1 (Ng et al., 2000). The genome structure has fully clarified the nature of the FII DNA, and our insight in the properties of Halobacterium has greatly deepened. The wealth of information to be found in the 2,571,010 base pairs of this genome is now being intensively exploited to obtain answers to many open questions. Complete sequences of other halophilic

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Archaea (Halobacterium salinarum strain R1, Natronomonas pharaonis, Haloferax volcanii) will probably become available soon. A number of techniques have been developed during the past two decades for the genetic manipulation of Halobacterium salinarum, Haloferax volcanii, and other halophilic Archaea. Transformation with foreign DNA is possible, plasmid vectors with selectable markers have been designed for the introduction of genetic material into the cells, and techniques for the knock-out of specific genes are available as well. An unusual mating process involving formation of cytoplasmic bridges has been identified in Haloferax volcanii (Mevarech and Werczberger, 1985), and this process has also opened new possibilities for genetic manipulation. Reviews of the older studies on the genetics of the halophilic Archaea have been presented by Pfeifer (1988), Charlebois et al. (1989), Doolittle et al. (1992), and Schalkwyk (1993), An overview of the genetic information available in the pregenomics era was given more recently (Oren, 2001). A wealth of technical information, including detailed protocols for performing genetic experiments with the halophilic Archaea, is available online (Dyall-Smith, 2001). The halophilic Bacteria have for long been neglected as objects of genetic studies. No complete genome sequences of such organisms have yet been determined. However, the genetic analysis of certain groups, especially of genera belonging to the Halomonadaceae (Halomonas, Chromohalobacter) has made tremendous progress in recent years. Also here have naturally occurring plasmids been converted into suitable shuttle vectors. The use of these genetic techniques has already deepened our understanding of the regulation of the biosynthesis of compatible solutes such as ectoine and glycine betaine (see Section 8.3.3), thus enabling an insight in some of the fundamental properties that enable these microorganisms to adapt to life at high salt concentrations. The following sections discuss the present state of our understanding of the structure of the genomes of halophilic Archaea and Bacteria, the techniques that have been developed to study and manipulate the genetic system, and the results that have been obtained while using these techniques.

10.2. GENETICS OF HALOPHILIC ARCHAEA 10.2.1. Organization of the genome of the halophilic Archaea In the 1960s it was discovered that buoyant density centrifugation in CsCI gradients resolves the DNA of Halobacterium salinarum and a few other species of the Halobacteriaceae into a major ("FI DNA"; 66-68 mol% G+C) and a minor ("FII DNA" or "satellite DNA") band of lower G+C content (57-60 mol%) (Joshi et al., 1963; Moore and McCarthy, 1969). The FII fraction accounts for 11-36% of the total DNA. The A+T-rich fractions contain the large cccDNA (covalently closed circular extrachromosmal DNA), as well as some "islands" of low G+C DNA located within the chromosome. Such a 70 kbp island of A+T-rich DNA within the Halobacterium

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chromosome was characterized by Pfeifer and Betlach (1985). This fraction contains many transposable insertion sequence elements (Pfeifer, 1986, 1988). The frequent occurrence of insertion sequences within the genome of Halobacterium strain NRC-1, the strain most frequently used in genetic and genomic studies, has been recognized a long time ago. The resulting instability of the genome, which undergoes spontaneous rearrangements at a high frequency, highly complicates genetic analysis (Pfeifer, 1988; Pfeifer et al., 1981b). We now know that the Halobacterium NRC-1 genome contains no less than 91 insertion sequences representing 12 families (Charlebois and DasSarma, 1995; Ng et al., 2000). These insertion sequences are responsible for the high frequency of spontaneous mutations in this strain (Charlebois, 1999; DasSarma, 1993; DasSarma et al., 1983; Doolittle and Sapienza, 1980; Ng et al., 1998; Pfeifer et al., 1997). They are highly mobile, and are arranged in both clustered and dispersed fashions within the genome (Sapienza and Doolittle, 1982). As a result, gas vesicle defective mutants may occur at a frequency of and production of the bacteriorhodopsin components retinal and bacterio-opsin is lost at a frequency of This high frequency of mutation could be tracked down to alterations (insertions, deletions, rearrangements) in one of the two large plasmids (megaplasmids or minichromosomes) present in Halobacterium (Pfeifer et al., 198la). Megaplasmids or minichromosomes are found in many representatives of the Halobacteriaceae (Gutiérrez et al., 1986; Ng et al., 1998). Many isolates have between two and six large plasmids with sizes between 150 and 450 kbp. Out of the 65 isolates tested, including both culture collection strains and new isolates, 75% harbored at least one such plasmid. Most contained three or four megaplasmids of less than 100 to up to 300 MDa. Haloferax gibbonsii does not appear to possess any plasmids. Two Haloarcula strains (the species incertae sedis "Haloarcula sinaiiensis" and a strain designated as Haloarcula sp. WS-1) harbor five and eight plasmids, respectively, four and six of which, respectively, were larger than 100 MDa (Gutiérrez et al., 1986). Two megaplasmids are found in Halobacterium NRC-1: pNRC100 and pNRC200 (Ng et al., 1998, 2000). These large plasmids may be present in several copies per cell (Sapienza and Doolittle, 1982). In addition, minor cccDNAs of variable sizes and quantities are present in most strains (Pfeifer, 1988). Information on the structure of a number of plasmids from halophilic Archaea that have been characterized was supplied by DasSarma (1995). The genes for the production of gas vesicles in Halobacterium have for long been known to be plasmid-located (Simon, 1978; Weidinger et al., 1979), although chromosomal genes for gas vesicle formation are present as well (see Section 3.1.7). The complete sequence of pNRC100 was released in 1998 (Ng et al., 1998). It contains 27 insertion sequence elements representing 8 families. A total of 176 open reading frames or likely genes were originally identified in the plasmid, 39 of which were repeated within large inverted repeats. Further analysis increased the number of putative genes to 197 (Ng et al., 2000). These genes include those coding for a restriction-modification system and for gas vesicle production (DasSarma, 1993; DasSarma and Arora, 1997). Typical chromosomal genes are found as well, such as the electron transport cytochrome d oxidase, enzymes for the biosynthesis of

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nucleotides, eukaryotic-type TATA-binding protein transcription factors, and a chromosomal replication initiator protein. It was suggested that pNRC100 has evolved from several smaller plasmids through insertion element-mediated fusions. The finding of essential genes on pNCR100 and its resistance to curing suggest that this replicon may be evolving into a new chromosome. Acquisition of chromosomal genes was suggested to be insertion sequence mediated (Ng et al., 1998). The replication origin of the pNCRl100 has been subject to an in-depth analysis (Ng and DasSarma, 1993). The complete sequence of the Halobacterium NRC-1 genome was published in 2000. The total of 2,571,010 base pair genome is organized into a large chromosome (2,014,239 bp) and the two related minichromosomes pNRC100 and pNRC200 (191,346 and 365,425 bp, respectively) (Kennedy et al., 2001; Ng et al., 1998, 2000). Several smaller and variable minor circular DNAs may be present as well, most of which are deletion derivatives of pNRC100. The two minichromosomes contain a 145,428-bp region of identity, within which exist two copies of 33- to 39-kb inverted repeats that mediate inversion isomerization. The two smaller replicons arc less GC rich than the large chromosome (57.9 and 59.2 as compared to 67.9 mol% G+C). The genome contains 91 insertion sequences representing 12 families. Of these insertion sequences, 29 are located on pNRC100, 40 on pNRC200, and 22 on the large chromosome. Altogether, 14% of the genome, including the 91 IS elements, 33-37kbp inverted repeats, and 145-kb duplications in pNRC100 and pNRC200, is repeated DNA. The genome codes for 2,682 likely genes (including 52 RNA genes), of which 1,658 code for proteins with significant matches to the databases. Of these 1,067 were matches to proteins of known function. The large chromosome contains 2,111 putative genes, pNRC200 374, and pNRC100 197. About 40 genes located on the minichromosomes pNRC100 and pNRC200 code for proteins likely to be essential or important for cell viability such as a DNA polymerase, seven TBP and TFP transcription factors, and the arginyl-tRNA synthetase. The RNA genes in the genome include one copy each of 23S, 16S and 5S rRNA, 47 tRNAs, and RnaseP and the 7S signal recognition particle RNA (Mortiz et al., 1985). Figure 10.1 presents an overview of the biology of Halobacterium NRC-1 such as can be deduced from the analysis of its genome. Extensive information on the genome of strain NRC1 is found in the web site http://www./zdna2.umbi.edu/~haloweb (last accessed 15 November 2001). Whole proteome comparisons confirm the archaeal nature of Halobacterium NRC1. However, extensive similarities were noted to the Grain-positive bacterium Bacillus subtilis. A large number of unique homologs with the radiation-resistant Deinococcus radiodurans were found as well, suggesting the possibility that lateral gene transfer from the bacterial domain may have contributed many elements to the Halobacterium genome. As expected (see also Section 7.2), the whole proteome is highly acidic: the majority of the proteins have an isoelectric point of around 4.2, and the whole proteome has a median pI of 4.9. The 2 Mbp chromosome of Halobacterium as characterized in strain NRC-1 is highly conserved among different Halobacterium isolates. Thus, the chromosome of

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strain R1 (DSM 671) has 1,998,885 bp as compared to 2,014,239 in strain NRC-1. Comparison of the two genomes shows only 17 true sequence differences, 7 frameshifts and 5 jumps of 10-30 bp (D. Oesterhelt, personal communication). However, the plasmids of strain R1 differ greatly from those of strain NRC-1. More detailed information can be found in the web site http://www.halolex.mpg.de (last accessed: 20 January, 2002). Restriction mapping of a number of related isolates shows variations in the numbers and size of the plasmids, while the chromosome is well-conserved (Bobovnikova et al., 1994; Ebert et al., 1984; Hackett et al., 1994; Ng et al., 1991; Pfeifer et al., 1981a; St Jean et al., 1994). There are Halobacterium strains such as strain GRB, an isolate obtained from the saltern ponds of Gruissan at the Mediterranean coast of France, that lack multiple copy families of insertion sequences and are therefore genetically much more stable than strain NRC-1 (Ebert et al., 1984). Strain GRB also lacks a DNA restriction system, a property that facilitates the introduction of foreign DNA (Charlebois 1995b; Ebert et al., 1984, 1986; Soppa and Oesterhelt, 1989b). It contains five replicons: a 2,038 kbp chromosome and four plasmids of 305, 90, 37, and 1.8 kbp (St Jean et al., 1994). It is unknown whether the strain has escaped evasion by the more aggressive insertion elements or has been cured of them in the past. A physical map of the genome of Halobacterium sp. GRB has been prepared (Charlebois, 1995b). Haloferax volcanii and Haloferax mediterranei have much more stable genomes than Halobacterium NRC-1, and are therefore much better amenable to genetic analysis (López-García et al., 1995). The genome of Haloferax volcanii has been examined in considerable detail, and its complete sequence is expected to become available soon. The organism contains a 2,920 kbp chromosome and plasmids of 690, 442, 86, and 6.4 kbp (designated pHV4, pHV3, pHV1 and pHV2, respectively) (Charlebois, 1995a; Charlebois et al., 1987, 1989, 1991; Cohen et al., 1992; Doolittle et al., 1992). A large number of genes have been localized within this genome by means of hybridization experiments and by transformation with cosmids; these include the genes responsible for biosynthesis of uracil, adenine, guanine, and 14 amino acids (Cohen et al., 1992) (Figure 10.2). Also, there are transposable elements present. Haloferax volcanii possesses at least 49 copies of the ISH51 family, which are concentrated on two of the four plasmids (López-García et al., 1995). Analysis of large numbers of mutants has shown that all of the 29 tryptophan auxotrophic alleles characterized in Haloferax volcanii map in one of two positions in the genome. On the other hand, some other biosynthetic pathways controlled by operons in Escherichia coli are probably not organized in operons in Haloferax volcanii: 23 histidine auxotrophic mutants mapped in six unlinked positions (Doolittle et al., 1992). As in the case of Halobacterium, considerable variation can be found in the plasmid content of different Haloferax volcanii strains. Three isolates obtained from an archaeal bloom that developed in the Dead Sea in 1980, and identified as belonging to this species on the basis of a large number of taxonomic criteria, each contained different plasmids that showed no homology to each other or to plasmid pHV2 of the type strain of Haloferax volcanii (Rosenshine and Mevarech, 1989), which had been isolated earlier from the Dead Sea (Mullakhanbhai and Larsen, 1975).

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Some effort has also been devoted to the characterization of the genome of Haloferax mediterranei. Based on results of pulsed-field electrophoresis a physical map of the genome elements has been constructed. The species contains a circular chromosome of 2.9 Mbp and at least three extrachromosomal elements of 490,320 and 130 kbp (López-García et al., 1992). Comparison of the chromosome with that of Haloferax volcanii revealed only two inversions and a few translocations. In view of this striking similarity is was stated that "Forces known to promote genomic rearrangement, common in haloarchaea, have been inactive in changing global gene order throughout the nearly years of these species' divergent evolution" (LópezGarcia et al., 1995). A physical map has been prepared of a 257 kbp region from the FI fraction of the genome of Halococcus saccharolyticus. No long repeated sequences were detected. This genome was therefore suggested to be relatively genetically stable (Montero et al., 1991).

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10.2.2. Mutagenesis and DNA repair in halophilic Archaea The isolation of mutants, both spontaneous and induced, has opened the way toward the elucidation of the genomic organization in the Halobacteriaceae. In Halobacterium salinarum phenotypic markers such as changes in pigmentation and colony appearance have proven useful in genetic analyses. Properties such as the red pigmentation caused by presence of bacterioruberins, synthesis of purple membrane with bacteriorhodopsin, and content of gas vesicles were all shown to mutate spontaneously with a high frequency, as discussed above. Mutagenesis can also be induced chemically. Procedures such as mutagenesis by ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine have been proven effective for the isolation of auxotrophic mutants (Bonelo et al., 1984; Charlebois et al., 1989). After treatment with N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of between 1 and 10% of the Haloferax mediterranei cells survived, and among the survivors were mutants showing resistance to the antibiotic josamycin (Bonelo et al, 1984). A large number of ethyl methanesulfonate-induced auxotrophic mutants of Haloferax volcanii have been obtained (Cohen et al., 1992). Hydroxylamine is also an effective agent for isolation of auxotrophic mutants of Haloferax mediterranei and other halophilic Archaea (Fernández-Castillo et al., 1990). Mutants of Haloferax volcanii resistant to the dihydrofolate reductase inhibitors trimethoprim and methotrexate arise spontaneously at a frequency of to Resistance to these drugs correlates with the amplification of specific DNA sequences, and loss of these amplified sequences causes reversion to the wild-type phenotype. The resistant mutants overproduce a 20-kDa protein that corresponds in size to dihydrofolate reductase, the target of the drugs (Rosenshine et al., 1987).

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Soppa (1998) has designed an interesting protocol for the isolation of "loss of function" mutants, which has been applied for the isolation of nitrate respirationdeficient mutants of Haloferax volcanii. The procedure is based on 5'-bromo-2'deoxyuridine selection in a negative selection procedure analogous to the penicillin method used in the Bacteria. Cells are first grown in normal medium under conditions permissive for both the wild type and the mutant. Cultivation is then continued under selective conditions in the presence of 5'-bromo-2'-deoxyuridine. The mutants do not grow, while the wild-type cells incorporate the compound in their DNA instead of thymidine. Bromodeoxyuridine disintegrates upon illumination at 280 mm. A bromoradical is split off, and this reactive radical damages the DNA. Illumination of the cells with UV light of the proper wavelength therefore results in the selective killing of non-mutant cells. Using this protocol phototrophy-negative mutants of Halobacterium GRB have been isolated: each round of bromodeoxyuridine treatment resulted in an enrichment of two orders of magnitude in the frequency of mutants in the culture (Soppa and Oesterhelt, 1989a, 1989b). Transposon mutagenesis has been applied in Haloarcula hispanica, using a transposon based on the ISH28 insertion sequence from Halobacterium salinarum and the mevinolin resistance marker of Haloferax volcanii (Dyall-Smith and Doolittle, 1994). A novel procedure for homologous gene knockout has been devised for Halobacterium salinarum, using ura3 as a counterselectable marker. The gene ura3 encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. Halobacterium salinarum is sensitive to 5'-fluoroorotic acid, which can select for mutations in ura3. A spontaneous 5'-fluoroorotic acid-resistant mutant was found that contains an insertion in ura3 and behaves as an uracil auxotroph. Integration of ura3 at the bacterio-opsin (bop) locus of this mutant restored 5'fluoroorotic acid sensitivity and uracil prototrophy. The ura3 gene can thus be used in an efficient new genetic strategy towards the systematic knockout of genes in Halobacterium salinarum (Peck et al., 2000). Early studies have reported that the halophilic Archaea lack dark DNA repair (excision repair) of UV damage (Fitt and Sharma, 1987; Grey and Fitt, 1976). These organisms were thus believed to rely on their effective photoreactivation mechanism (Fitt and Sharma, 1987; Hescox and Carlberg, 1972; Sharma et al., 1984). This should imply that cyclobutane dimers are the only significant UV-induced lesions, and that these completely repaired by photoreactivation. However, in all organisms studied, pyrimidine 6-4 pyrimidone photoproducts are significant cytotoxic and mutagenic lesions, and these are generally constitute 10-30% of the UV photoproducts. It has now been demonstrated that both cyclobutane dimers and 6-4 photoproducts are induced in the DNA of Halobacterium salinarum and Haloferax volcanii at similar levels as in other organisms. A dark repair mechanism, presumably based on excision repair, exists in the halophiles as well. As in other organisms, 6-4 photoproducts are removed more efficiently in the dark than are cyclobutane dimers. In the light, cyclobutane dimers are repaired very rapidly, and there is also photoenhanced repair of 6-4 photoproducts (McCready, 1996). The possibility of dark repair in Halobacterium salinarum was also shown in "liquid holding recovery" experiments, in which the cells

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recover from UV irradiation damage in non-nutrient buffer in the dark (Fitt et al., 1983). Analysis of the Halobacterium NRC-1 genome has identified two of the three genes of the guanine oxidation pathway, mutT and mutY, which are involved in DNA repair. In addition, both the nucleotide and the base excision pathways appear to be complete as copies of the uvrABC nuclease and uvrD helicase, as well as endonucleases and glycosylase genes are present. Two of the three genes of methyl-directed mismatch repair were found, mutL and mutS (three copies), but the nuclease gene mutH is missing. Repair genes similar to those in yeast are also present in Halobacterium NRC-1, including rad2, rad3, rad24, and rad25. Several of these proteins appear to be active in the excision repair pathway. Products of rad3 and rad25 have been identified as repair helicases, and Rad2 is a single-stranded DNA endonuclease. This all suggests that Halobacterium NRC-1 has developed multiple pathways to repair UVinduced damage as a means for survival (DasSarma et al., 2001; Ng et al., 2000). Halobacterium salinarum is remarkably resistant to damage by mutagenic agents such as ultraviolet light, and mitomycin C. It was suggested that bacterioruberin carotenoids may be responsible for scavenging active oxygen species and/or act as UV absorbers (Shahmohammadi et al., 1997, 1998). A protective function has also been attributed to the high intracellular concentrations of KCl present in Halobacterium cells (Shahmohammadi et al., 1998). A carotenoid-less mutant proved more sensitive to UV and to ionizing radiation than the wild type (Shahmohammadi et al., 1998). The protective effect of bacterioruberin and KCl on damage to DNA caused by UV or was further investigated in vitro, using plasmid (pDEL19) DNA as a model. Formation of cyclobutane pyrimidine dimers in the DNA was slightly suppressed by 0.1 mM bacterioruberin (which was added together with a detergent to promote dispersal of the pigment in solution). Formation of DNA single strand breaks by ionizing radiation was efficiently suppressed by bacterioruberin. KCl at a concentration of 2 M was also effective in vitro in preventing the formation of DNA single breaks and formation of cyclobutane pyrimidine dimers by UV radiation, and was very efficient in protecting against damage by (Asgarani et al., 1999).

10.2.3. Genetic manipulation of halophilic Archaea A number of techniques have been developed for the genetic manipulation of different members of the Halobacteriaceae. These include conjugation, transformation, and transfection (Table 10.1). Together with the above-mentioned method of homologous gene knockout (Peck et al., 2000) these techniques have opened up many possibilities for the study of the halophilic Archaea. Transduction has not yet been described in the Halobacteriaceae. Studies with auxotrophic mutants of Haloferax volcanii have shown that cells can exchange genetic information by mating, following formation of cytoplasmic bridges. As a result, mixing of different auxotrophs may lead to the formation of prototrophic

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recombinants (Mevarech and Werczberger, 1985; Ortenberg et al., 1999; Rosenshine et al., 1989). Each parental type can serve both as a donor and as a recipient (Rosenshine et al., 1989). Direct contact of the cells is necessary for the process to occur, and this can be achieved e.g. by filtration on nitrocellulose filters or within cell pellets during centrifugation. Electron micrographs show that Haloferax volcanii cells grown in colonies are often connected by cytoplasmic bridges of up to long and in diameter (Rosenshine et al., 1989) (Figure 10.3). Similar cytoplasmic bridges can be seen in the micrographs and electron micrographs published in the original description of the species (Mullakhanbhai and Larsen, 1975). This phenomenon may be related to cell fusion during mating. Both plasmid and chromosomal DNA may cross in both directions. Plasmids and chromosomal markers are transferred in tight linkage and at similar frequencies (Ortenberg et al., 1999; Tchelet and Mevarech, 1994). The process is not inhibited by DNase (Rosenshine and Mevarech, 1991). Unidirectional interspecies gene transfer between Haloferax volcanii and Haloferax mediterranei has also been demonstrated, following fusion of cells and transfer of plasmids. In these experiments Haloferax volcanii WR1307, a histidine auxotroph which carries the 3-

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hydroxy-3-methylglutaryl-CoA reductase gene that bestows resistance to mevinolin, was mated with Haloferax volcanii WR1304, which is auxotrophic for arginine and contains the haloarchaeal shuttle vector pMDS1 that confers resistance to novobiocin. Haloferax volcanii WR1306, auxotrophic for histidine and containing the haloarchaeal shuttle vector pWL102 that confers resistance of mevinolin was mated with Haloferax mediterranei carrying plasmid pWL102 that bestows resistance to novobiocin. Colonies resistant to both mevinolin and novobiocin were obtained at a frequency of to The resistant cells had the morphology of Haloferax mediterranei and produced gas vesicles (Tchelet and Mevarech. 1994). Attempts to show a similar

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genetic transfer from Haloferax volcanii to Halobacterium salinarum or Haloarcula marismortui by selecting for novobiocin-rcsistant colonies failed. As these species are naturally resistant to the antifolate inhibitor trimethoprim, but are sensitive to novobiocin, the selection in this case was for colonies resistant to both trimethoprim and novobiocin (Tchelet and Mevarech, 1994). The development of a transformation protocol suitable for many different species of the Halobacteriaceae has enabled the use of shuttle vectors to transfer genetic information between different types of cells. These transformation protocols are based on the formation of spheroplasts following removal of divalent cations by EDTA, addition of foreign DNA together with low-molecular-weight polyethylene glycol, and subsequent regeneration of the cells on agar plates (Dyall-Smith, 2001). Formation and regeneration of spheroplasts aimed at genetic studies was pioneered in Halobacterium by Jarrell and Sprott (1984), who used a low salt (0.25 M NaCl), low magnesium (10 mM medium stabilized by 0.5 M sucrose. Spheroplast regeneration efficiency in the order of 5-20% can be achieved (Cline and Doolittle, 1992). A method based on freeze-thawing to introduce foreign DNA into Halobacterium salinarum was recently described (Zibat, 2001). The efficiency of the process was low. Transformation in the Halobacteriaceae was first shown in Haloferax volcanii using plasmid pHV2 (Charlebois et al., 1987). Polyethylene glycol-mediated transformation protocols have been optimized for efficient uptake of DNA for Haloferax volcanii, Halobacterium salinarum, Haloarcula hispanica, and Haloarcula vallismortis. Transformation of plasmid DNA as large as 20.4 kbp and linear phage and chromosomal DNA fragments of up to 25 kbp has been reported, and efficiencies of up to transformants per of DNA have been achieved in some cases (Cline et al., 1989a, 1989b, 1995; Doolittle et al., 1992; Holmes et al., 1991). However, in most cases transformation efficiencies are reduced to about transformants per DNA due to the action of restriction systems present in most halophile species (see e.g. Patterson and Pauling, 1985). Haloarcula hispanica is exceptional as it exhibits only low restriction activity (Cline and Doolittle, 1992). Restriction-negative mutants of Halobacterium salinarum have been described, and these may be used to enhance transformation efficiencies. Another strategy to circumvent restriction is the prior passage of the DNA through a dam strain of Escherichia coli such as strain JM110 to overcome the adenine methylation directed system in Haloferax volcanii (Holmes et al., 1991). Transformation has been achieved with native plasmids, with chromosomal DNA, with shuttle vectors grown in Haloferax volcanii or in Escherichia coli, with cosmid DNA from Escherichia coli, with restriction fragments of cosmid DNA, and with double- and single-stranded M13 sequencing templates (Doolittle et al., 1992). Transfection was shown already in 1987, when it was demonstrated that Halobacterium salinarum spheroplasts can be transfected with DNA from its phage at frequencies of to transfectants per DNA (Cline and Doolittle, 1987). Due to host restriction, transfection of Haloferax volcanii resulted in a to fold

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reduction in efficiency when DNA from Halobacterium salinarum was used (Charlebois et al., 1987). Different plasmid shuttle vectors carrying one or more selectable markers have been developed for genetic studies of halophilic Archaea, such as gene disruption experiments and construction of mutant strains (Cline and Doolittle, 1992; DasSarma, 1995; Doolittle et al., 1992; Dyall-Smith and Doolittle, 1994; Holmes and Dyall-Smith, 1990; Holmes et al., 1991, 1994; Lam and Doolittle, 1989). Table 10.2 summarizes the properties of some of these shuttle vectors.

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The vectors pMDS20 and pMLH3 (Holmes et al., 1994) provide good examples of the types of shuttle vectors used to transfer genes between halophilic Archaea and Escherichia coli. These vectors, being improved versions of the earlier developed shuttle vector pMDS1 (Holmes et al., 1991), contain the Escherichia coli E1 plasmid ori region, the ampicillin resistance-conferring bla gene, the Haloferax pKH2 replicon region, and the novobiocin-resistance-encoding gyrB gene, thus enabling maintenance and selection in both hosts. Plasmid pMLH3 has, in addition, a Haloferax volcanii mevinolin-resistance determinant and restriction sites allowing insertional inactivation of either marker, to facilitate the identification of Haloferax transformants harboring cloned sequences. Haloarcula vallismortis and Haloarcula hispanica can be transformed with the Halobacterium-Escherichia coli shuttle vector pWL102, which is was constructed on the basis of the Haloferax volcanii pHV2 replicon and of pUBP2, based on the Halobacterium salinarum (halobium) pHH1 replicon. The shuttle vector confers resistance to mevinolin (Cline and Doolittle, 1992). This type of vector is probably suitable for use in a wide range of phylogenetically diverse members of the Halobacteriaceae. Several of the shuttle vectors listed above have resistance to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, as selectable marker. Haloferax volcanii is resistant to the drug; its mevinolin resistance determinant can confer mevinolin resistance in Haloferax mediterranei, in Haloarcula species, and in Halobacterium salinarum (Holmes and Dyall-Smith, 1990; Sowers and Schreier, 1999). Other vectors bestow resistance to novobiocin, a DNA gyrase inhibitor. The determinant for novobiocin resistance was isolated from a spontaneous mutant of "Haloferax lucentensis". Several hybrid vectors have been constructed that use both markers for transformant selection and insertional inactivation (Table 10.2). Resistance to sparsomycin, a broad-spectrum antibiotic that acts at the peptidyltransferase center of the ribosome, has been reported in Halobacterium salinarum, but the altered 23 S rRNA gene responsible for the resistance has not yet been extensively exploited in genetic studies (Tan et al., 1996). Similarly, a Halobacterium salinarum mutant resistant to anisomycin and thiostrepton has been obtained through the use of a plasmid-borne, altered 23S rRNA gene (Mankin et al., 1992). A mevinolin-resistance marker has been isolated from Haloarcula hispanica and from Haloferax volcanii. The mevinolin-resistance determinant hmg encodes 3hydroxy-3-methylglutaryl CoA reductase that is involved in the biosynthesis of mevalonic acid, essential for the production of isoprenoid lipids, from acetyl-CoA. Plasmids with this marker are unstable in Haloferax volcanii because the resistance gene was derived from the genome of this species and is almost identical in sequence to the chromosomal copy. To reduce the level of homologous recombination between introduced plasmid vectors and the chromosome, a homologue of the hmg determinant was obtained from the distantly related Haloarcula hispanica. The nucleotide sequences of the wild type genes are only 78% identical. In comparison to the wild type hmgA gene, the resistance gene from a mutant resistant to simvastatin (an analogue of mevinolin) showed a single base substitution in the putative promoter.

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Plasmids constructed using the new resistance determinant were stably maintained under selection in Haloferax volcanii and showed very low recombination rates with the chromosome. Resistance to the drugs is based on overproduction of the enzyme (Wendoloski et al., 2001). The ShBle bleomycin resistance determinant from the actinomycete Streptoalloteichns hindustanus can be expressed in Haloferax volcanii. A gene cassette has been designed that confers resistance to bleomycin following expression of the bleomycin-resistance protein. Recombinant ShBle has been purified from Haloferax volcanii as correctly folded dimeric protein. This protein, which is the first non-halophilic and soluble heterologous protein to be expressed in halophilic Archaea, may prove a useful selective marker in genetic studies (Nuttall et al., 2000). Progress in developing phenotypic markers for genetic studies of the Halobacteriaceae has lagged behind that for selectable markers. A useful phenotypic trait that is underexploited in terms of use for genetic studies is the purple color associated with bacteriorhodopsin production in Halobacterium salinarum. When present on a plasmid, the bop gene coding for the protein moiety of bacteriorhodopsin can complement a insertion mutant, as detected by formation of purple-colored colonies (Krebs et al., 1991). Constructs containing functional bop genes are potentially useful not only for studies employing strains but also when propagated in naturally occurring bop-less halophiles such as Haloferax volcanii, provided that the organism is capable of synthesizing the retinal chromophore required for bacteriorhodopsin function or retinal is supplemented to the medium. The cloning and characterization of the gene bgaH of "Haloferax alicantei" (to be described as Haloferax lucentensis) (Holmes and Dyall-Smith, 1999; Holmes et al., 1997) has led to the development of a convenient reporter gene to be used in gene expression studies in halophilic Archaea, comparable to the use of the lacZ gene in Escherichia coli. Enzyme activity in cell lysates can be quantified using a simple colorimetric assay using the chromogenic substrate ONPG galactoside), and colonies can be screened on agar plates with X-Gal (5-bromo-4The gene can be transferred to Haloferax volcanii by means of shuttle plasmid pMDS20 (Holmes et al., 1994). The gene has been used as a reporter gene for promoter analyses in Halobacterium salinarum (Patenge et al., 2000). The genes for the production of gas vesicles from Haloferax mediterranei and Halobacterium salinarum were fused to bgaH and examined in Haloferax volcanii (Gregor and Pfeifer, 2001). The green fluorescent protein of the jellyfish Aequorea victoria has been functionally expressed as a reporter gene in Halobacterium salinarum. Recombinant green fluorescent protein has been fused with bacteriorhodopsin. Fusion proteins that had preserved the intrinsic functions of each protein were expressed in Halobacterium salinarum, and were properly inserted in the membrane. Green fluorescent protein was thus suggested as a useful reporter of gene expression of halophilic membrane proteins (Nomura and Harada, 1998), but the levels of fluorescence obtained were low. An overview of the techniques used in genetic manipulation of the halophilic Archaea, including detailed laboratory protocols, was given by Dyall-Smith (2001).

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10.2.4. DNA topology and the transcription machinery of the Halobacteriaceae Analysis of the Halobacterium NRC-1 genome has shown that its systems for DNA replication, transcription and translation resemble those of the more complex eukaryotic organisms in many aspects (Ng et al., 2000). Three types of DNA polymerase, a putative DNA ligase, primase, type I topoisomerase (TopA), and two type II topoisomerases (GyrA and B and Top6A and B) were identified. Nine copies of orc (the origin recognition complex) are present, three of which are scattered on the large chromosome, suggesting the possibility of multiple replication origins. The protein components of the translation apparatus also resemble more closely those of the Eucarya than those of Bacteria (Ng et al., 2000). One of the "eukaryotic" properties of the DNA replication system of the halophilic Archaea is its sensitivity to aphidicolin, an inhibitor of eukaryotic DNA polymerases. Aphidicolin inhibits DNA synthesis and interferes with cell division, causing the formation of long filaments in Halobacterium (Forterre et al., 1984, 1986; Schinzel and Burger, 1984). DNA replication of halophilic Archaea in vivo is inhibited by etoposide, a specific inhibitor of DNA topoisomerase II. DNA replication in halobacteria is also inhibited by coumarins at doses which specifically inhibit bacterial DNA gyrase. The putative archaeal type II DNA topoisomerase (DNA gyrase), the enzyme that introduces negative superturns in topologically closed DNA, thus exhibits both a eukaryotic feature (etoposide sensitivity) and a bacterial feature (sensitivity to coumarins) (Forterre et al., 1986). Additional evidence for the presence and functioning of a DNA gyrase-like enzyme in halophilic Archaea was derived from studies of the action of novobiocin and coumermycin (two coumarins that interact with the gyrB-encoded subunit of bacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone that interacts with the gyrAencoded subunit of DNA gyrase) (Sioud et al., 1988). The alkaliphilic Natronobacterium gregoryi is very sensitive to quinolones; the relatively low sensitivity of Halobacterium and other neutrophilic members of the Halobacteriaceae is probably related to the high magnesium content of the media used for their cultivation (Oren, 1996). Mutations in the DNA gyrase can result in novobiocin resistance in halophilic Archaea. Sequencing of the resistance determinant of cloning vector pMDS2 that confers novobiocin resistance as selectable marker revealed an open reading frame coding for 640 amino acid protein homologous to bacterial GyrB (Holmes and Dyall-Smith, 1991). Negative supercoiling is a characteristic feature of plasmids of halophilic Archaea. A 1.7 kbp plasmid of Halobacterium strain GRB was shown to be negatively supercoiled; novobiocin treatment caused relaxation of the plasmid DNA (Sioud et al., 1988). Negative supercoiling in vivo was also observed in several megaplasmids from different strains of Haloferax mediterranei, as deduced from the effect of intercalating agents affecting topology and electrophoretic mobility (López-García et al., 1994). The superhelical density of pHV11, a 3 kbp plasmid of Haloferax volcanii WR11, depends on the salt concentration and composition of the medium (Mojica et al., 1994).

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The DNA intercalating drugs actinomycin and 2-methyl-9-hydroxy-ellipticine inhibit positive supercoiling induced by novobiocin in halophilic Archaea. This observation was used as evidence that positive supercoiling in the Halobacteriaceae is due to transcription, as in the domain Bacteria (Gadelle and Forterre, 1994). It was suggested that type II DNA topoisomerase is essential for DNA replication, and that inhibition of this enzyme induces positive supercoiling of the plasmids. The topoisomerase is then required to relax positive superturns induced by transcription (Mojica et al., 1994). Transcriptional induction of purple membrane (expression of the bop gene) and gas vesicle synthesis (expression of gvpA) in Halobacterium salinarum is blocked by novobiocin (Yang and DasSarma, 1990). Although earlier studies failed to detect an eukaryotic type I DNA topoisomerase in members of the Halobactenaceae (Mojica et al., 1994), the gene for such an enzyme (topA) has been identified in the Halobacterium NRC-1 genome (Ng et al., 2000). The properties of the transcription apparatus are conserved throughout the domain Archaea. This became evident in a study of heterologous in vitro transcription from archaeal promoters. A cell-free extract of Sulfolobus shibatae can specifically initiate transcription in vitro at the promoter of the plasmid-encoded gene for the major gas vesicle protein of Halobacterium salinarum and at the promoter for the transcript T4 of the temperate Halobacterium salinarum phage (Hüdepohl et al., 1991). Eukaryotic-like TFIIB transcription initiation factors have been identified in Haloferax volcanii (Thompson et al., 1999), and multiple transcription factors have been detected in the genome of Halobacterium NRC-1 (Baliga et al., 2000). Introns have been found in several haloarchaeal genes. Thus, the trp tRNA gene of Haloferax volcanii has an intervening sequence of 105 bp (Daniels et al., 1985). The gene is interrupted at a position corresponding to two nucleotides 3' to the anticodon. The gene is transcribed in its entirety and the intervening sequence is excised posttranscriptionally. Another unusually structured gene is the 5S rRNA of Halococcus morrhuae, which is abnormally long (231 bp, containing a 108 bp insertion in the putative helix 5) (Luehrsen et al., 1981). The structure of the ribosomes of halophilic Archaea has been discussed earlier (see Section 3.1.5). The unusual structure of the S9 operon, one of the operons coding for ribosomal proteins in Haloarcula marismortui, may be mentioned here. Genes for three ribosomal proteins (HL29, HmaL13, HmaS9) are co-transcribed with a the glycolytic enzyme enolase, a putative membrane protein, and two unidentified open reading frames (Krömer and Arndt, 1991).

10.2.5. Codon use and proteomics of the Halobacteriaceae Examination of the codon usage of Halobacterium, based on examination of 130 protein coding genes, has shown a few general trends. For 12 out of the 18 amino acids which are coded for by more than one codon, the usage of codons with G or C at the third codon position is 90% or higher, as expected for an organism with a high G+C content in its DNA. Two of the remaining codon families prefer C-rich codons in

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more than 80% of the cases (alanine and arginine), three in more than 70% (glycine, glutamate and lysine). The only exception is the cysteine codon family where TGT and TGC are equally used. For the three amino acids that are coded for by six codons (arginine, leucine, serine), some of the possible codons are avoided. Examples are TTG/TTA (leucine), which are only used in 6% of the cases, and AGG/AGA only in 2%. The preference of GAC over GAT (aspartate) is 92%, whereas the preference of GAG over GAA (glutamate) is 74%. In the case of the stop codons, however, the ATrich codons TGA and TAA are preferentially used, and TAG is used only in 6% of the cases. The genes that form the gas vesicle gene clusters have a very different codon usage, as expected in view of the non-halophilic nature of the gas vesicle proteins (Soppa, 1994). This survey has been extended on the basis of the information that has become available with the completion of the sequencing of the genome of Halobacterium (Ng et al., 2000). Data can be found in the "Halohandbook" (DyallSmith, 2001) and in the web site http://www.kazusa.or.jp/codon (last accessed: 20 January 2002). Studies on the differential synthesis of proteins in cells grown under different conditions have been few in the Halobacteriaceae. Kamekura et al. (1986) examined the rate of protein turnover in Halobacterium salinarum by pulse-labeling with labeled amino acids followed by a chase with unlabeled amino acids. On the basis of measurements using non-essential amino acids such as proline, protein turnover was found to be slow. When the essential amino acid leucine was used in a low concentration, a great part of the label was first found in an unstable protein fraction, and only when sufficient unlabeled leucine was supplied did the label appear in the stable protein fraction. The bacteriorhodopsin fraction is very stable and does not appear to turn over at all. Exposure to different salt concentrations triggers the differential transcription of different sets of proteins. Different regions of the Haloferax volcanii genome are preferentially transcribed depending on medium salinity as observed by hybridization of cDNAs obtained at different salinities with a Haloferax volcanii cosmid library. A large domain within the largest megaplasmid (pHV4; 690 kbp) shows an especially strong response to a shift to low salinity (Ferrer et al., 1996). Differences were found in the restriction patterns obtained when genomic DNA of Haloferax mediterranei cells grown at low salt and high salt was digested with restriction enzymes. These differences were attributed to modification of specific sequences related to the reaction of the cells to salt (Juez et al., 1990). A number of general stress proteins that are also involved in the cells' reaction to heat shock are induced both following osmotic upshock and downshock (Mojica et al., 1997). Recently a chaperone-like protein complex has been characterized from Haloarcula marismortui that may protect cells under hyposaline conditions. This chaperonin needs only 0.5 M salt for structural stability, so it can work in relatively low salt concentrations (Franzetti et al., 2001). A chaperonin-like sequence has also been identified in the Halobacterium salinarum genome.

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10.3. GENETICS OF HALOPHILIC BACTERIA Because of a lack of suitable genetic tools, only little work has been done on the genetic aspects of the halophilic Bacteria until a few years ago. However, genetic systems have now been developed and optimized for a number of representatives of the group. The overview given below is to a large extent based on the information provided by Ventosa et al. (1998), supplemented by new data that have become available since. 10.3.1. Organization of the genome of halophilic Bacteria The genome sizes of a number of halophilic Bacteria have been determined by pulsedfield gel electrophoresis following digestion of the DNA with infrequently cutting restriction enzymes. Digestion of the genomes of fourteen isolates belonging to the closely related genera Halomonas and Chromohalobacter with SpeI and SwaI yielded characteristic fingerprints for each of the strains studied. The genomes of the eleven Halomonas strains tested ranged from 1,450 to 2,830 kbp, of the Chromohalobacter strains from 1,770 to 2,295 kbp. The macrorestriction fingerprint was suggested to be a useful tool to elucidate the taxonomic position of isolates to be classified within the Halomonas-Chromohalobacter complex (Mellado et al., 1998). A similar approach was used for the determination of the genome structure of Salinivibrio costicola strain E-367 following digestion with restriction enzymes SfiI and MboI. The genome size was estimated at 2,505 kbp (SfiI) or 2,259 (MboI) (Mellado et al., 1997). The apparent genome sizes of five other Salinivibrio costicola strains, as determined by SfiI digestion, ranged from 2,100 to 2,600 kbp. Analysis of the restriction profiles led to the conclusion that strain E-367 harbored three different plasmids, as well a megaplasmid. The DNA of Salinivibrio costicola is probably highly methylated because digestion with MboI (which recognizes the DNA sequence GATC, and hence the dam methylase site, but does not effectively cut the methylated site) yielded very few cleavage sites. Different MboI restriction patterns were observed when the strain was grown at different salinities, suggesting that the methylation system may be affected by the salinity of the growth medium. Plasmids have been found in many or most halophilic Bacteria examined thus far. The first plasmid characterized from a halophilic bacterium is pMH1, a 11.5 kbp plasmid isolated from a strain of Halomonas elongata. This plasmid mediates resistance to kanamycin, tetracycline and neomycin, is stable in Escherichia coli, and has three unique restriction sites, and has therefore proven suitable for the development of cloning vectors (see Section 10.3.3). The same plasmid was also found within the closely related Halomonas halmophila and Halomonas halophila, and also in Salinivibrio costicola. Another plasmid that has been extensively characterized is pHE1 of Halomonas elongata. This mobilizable 4.2 kbp plasmid contains a basic replicon of 1.7 kbp which contains the information for autonomous replication and stable maintenance. No homology with pMH1 was found. Two open reading frames were identified that appear to form one transcription unit, coding for the replication initiator proteins RepA and RepB, which have a high degree of similarity with

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replication initiator proteins from Gram-negative and Gram-positive non-halophilic bacteria. The mobilization (mob) region mobCABD contains genes which overlap considerably: mobB and mobD are entirely overlapped by mob A. The proteins show a high degree of homology to Mob proteins of ColE1 and related plasmids (Vargas et al., 1999a, 1999b; Ventosa et al., 1994). pCM1 is a narrow-host range 17.5 kpb plasmid of Chromohalobacter marismortui. Its minimal replicon is localized in a 1,600-bp region which includes the oriV region and the repA gene. The ori V region, located on a 700-bp fragment, contains four 20-bp direct repeats adjacent to a DnaA box that is indispensable for efficient replication. Besides, it requires trans-acting functions. The repA gene codes for a replication protein of 289 amino acids that is similar to the replication proteins of other Gramnegative bacteria (Mellado et al., 1995a; Ventosa et al., 1994). Other plasmids that have been isolated and characterized are pH11 from Chromohalobacter israelensis (48 kb) and pHS1 from Halomonas subglaciescola (about 70 kb) (Vargas et al., 1995). Salinivibrio costicola E-367 carries three plasmids designated pVC1, pVC2 and pVC3 (Mellado et al., 1997). Marinococcus halophilus harbors a 3,874 bp plasmid pPL1 that has been entirely sequenced. Four open reading frames were identified. Two of these had no sequence similarity to known proteins. The rep gene of this plasmid has high similarity to replication proteins of rolling circle plasmids, and it has therefore been suggested that the plasmid replicates according to the rolling circle mechanism (Louis and Galinski, 1997). The presence of a plasmid has previously been implicated in the high salt tolerance of "Spirillum lunatum" (possibly a strain of Oceanospirillum mans) (Morishita 1978a, 1978b). Such a plasmid-encoded halophilic behavior has never been confirmed, and its mechanism has not been clarified.

10.3.2. Mutagenesis and DNA repair in halophilic Bacteria Most attempts to obtain mutants of halophilic Bacteria have been aimed at the isolation of strains altered in their salt tolerance and requirement in an attempt to obtain information on their mode of osmotic adaptation. Following UV irradiation of Salinivibrio costicola at a dose that killed 95% of the cells and subsequent penicillin enrichment, several mutants were obtained that were unable to grow at high NaCl concentrations while all grew at NaCl. In some of these mutants was the salt range allowing growth temperature-dependent (Kogut et al., 1992). Hydroxylamine has proven a useful agent for the production of mutants of Chromohalobacter salexigens and Halomonas meridiana (Cánovas et al., 1997). Induced mutagenicity was assessed according to the frequency of appearance of mutants resistant to streptomycin (Nieto et al., 1993). A number of single and double auxotrophic mutants of Chromohalobacter salexigens were obtained, as well as saltsensitive mutants of both species. For the isolation of salt-sensitive mutants, an ampicillin enrichment was carried out and putative mutants were selected as those able

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to grow at 20 or NaCl but failed to grow at Some of the mutants were affected in the synthesis of ectoine or in the genes responsible for regulation of compatible solutes synthesis (Cánovas et al., 1997) (see also Section 8.3.3). Hydroxylamine mutagenesis has also been used for the isolation of exopolysaccharidedeficient variants of Halomonas eurihalina (Llamas et al., 1999). Transposon mutagenesis was introduced to the study of halophilic Bacteria for the isolation of Halomonas elongata mutants with altered salt tolerance. Suicide plasmids pSUP101 and pSUP102-Gm were used to introduce transposons Tn5 and Tn1732 into Halomonas elongata via Escherichia coli SM10 mediated conjugation (Kunte and Galinski, 1995). As Halomonas elongata is sensitive to aminoglycoside antibiotics at low salinities, transposons that mediate kanamycin resistance can be applied for the purpose. Use of Tn5 failed to yield auxotrophic or salt sensitive mutants. Southern hybridization showed that Tn5 and the vector DNA co-integrated in a non-random manner into the Halomonas elongata chromosome. However, Tn1732, a transposon of the Tn3 family, proved to be suitable for the isolation of mutants. Several auxotrophic mutants with a requirement for a single medium additive were generated, as well as a number of salt-sensitive mutants. Three of the 62 salt sensitive mutants isolated could grow in NaCl medium when supplemented with glycine betaine. These mutants are defective in the genes for the biosynthesis of the compatible solute ectoine (see Section 8.3.3 for additional details). Using a similar approach, Cánovas et al. (1997) isolated mutants of Chromohalobacter salexigens unable to grow at the highest salt concentrations. Hybridization analyses confirmed that the salt-sensitive phenotype was due to single insertions of the transposon. Also these mutants were impaired in ectoine biosynthesis. Isolation of exopolysaccharide-defective mutants of Halomonas eurihalina has also been achieved by transposon mutagenesis. Suicide plasmids pYT and pSUP102 were used to introduce the transposons mini-Tn5 and TN1732 into Halomonas eurihalina via Escherichia coli-mediated conjugation. Insertions of transposon mini-TN5 occurred randomly at single sites in the chromosome, whereas Tn1732 insertion also took place simultaneously at several sites (Llamas et al., 2000).

10.3.3. Genetic manipulation of halophilic Bacteria There are few genetic transfer procedures available for the halophilic Bacteria. Natural transformation has not been reported, and attempts to develop transformation protocols using electroporation or treatment have been either unsuccessful or non-reproducible (Ventosa et al., 1998). Although some bacteriophages have been described that attack halophilic Bacteria (see Section 9.1.2), transduction methods have not been developed thus far. Conjugation is therefore the only genetic transfer mechanism yet available. A major difficulty when using halophilic Bacteria for genetic transfer experiments is their generally high tolerance toward most antimicrobials used as selective markers in genetics when they are grown at their optimal salinity (Nieto et al., 1993). A study

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in which the response was tested of 13 strains of Chromohalobacter, Halomonas, and Salinivibrio to ten common antimicrobials showed a gradual increase of the toxicity of aminoglucosides gentamycin, kanamycin, neomycin and streptomycin at decreasing salinity. In the case of nalidixic acid, spectinomycin and tetracycline the effect of salinity was less pronounced. All strains tested showed a high sensitivity to rifampicin and trimethoprim regardless of the salt concentration (Coronado et al., 1995). In view of the increased antibiotic sensitivity at lowered medium salinity, the use of low salt media may be preferred in genetic studies of halophilic Bacteria that can grow over a broad salinity range when selection is based on antibiotic resistance (Kunte and Galinski, 1995; Vreeland, 1992). The ice-nucleation gene inaZ of Pseudomonas syringae has been employed as a reporter system for promoter activity and gene expression in halophilic Bacteria. The gene has been successfully expressed in Halomonas elongata, Halomonas eurihalina, Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Halomonas halophila. A promoterless version of inaZ was subcloned in vector pSH15. Constructed plasmids were mobilized from Escherichia coli to several halophilic Bacteria by triparental matings in which the RK2 tra genes were provided in trans by the helper plasmid pRK600. None of the strains used expressed native ice nucleation activity, but all halophilic transconjugants harboring a pHS15 derivative expressed activity. A significantly higher ice nucleation activity was found in a recombinant construct carrying a tandem duplication of inaZ in the same orientation, indicating that inaZ can be used for gene dosage studies. The gene was also transferred and expressed in Halomonas elongata and Halomonas eurihalina under the control of two heterologous promoters, (the promoter of the of Escherichia coli) and (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis), yielding an ice nucleation activity comparable to that obtained from the expression of the native putative promoters. It was thus demonstrated that foreign genes can be introduced independently and expressed in halophilic Bacteria, facilitating their genetic improvement and strain construction plans (Arvanitis et al., 1995; Tegos et al., 1997). Conjugation has been successfully used to transfer both native plasmid-derived vectors and broad-range plasmids of Gram-negative Bacteria from Escherichia coli to Halomonas, Chromohalobacter, and Salinivibrio (Kunte and Galinski, 1995; Mellado et al., 1995a, 1995b; Vargas et al., 1995, 1997). Different plasmids derived from the halophilic Bacteria have been engineered as shuttle vectors for the transfer of genetic material between different species. Plasmid pHE1 was selected for the construction of a shuttle vector for Halomonas. pHE1-derived constructs could be selected and maintained both in Escherichia coli and in Halomonas elongata. An improved shuttle vector, pHS15, has been constructed that contains an origin of replication from Escherichia coli as well as one from Halomonas elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2 (Vargas et al.. 1995). The constructed hybrid vector pHS134 contains the complete plasmid pHE1, the Escherichia coli vector pKS(-) and a

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streptomycin-resistance gene. pHS134 can be mobilized from Escherichia coli to Halomonas elongata assisted by pRK600 (Vargas et al., 1999b). Cloning vectors (pEE3 and pEE5) have also been constructed based on the minimal replicon of pCM1, a cryptic plasmid from Chromohalobacter marismortui, combined with the useful properties of pUC18 plasmid (small size, high copy number, multiple cloning sites, lacZ fragment) as well with the trimethoprim resistance as a selection marker for moderate halophiles. These vectors can be effectively transferred by PR4mediated conjugation from Escherichia coli to Chromohalobacter marismortui, Halomonas halophila, Halomonas elongata, Halomonas subglaciescola, and Halomonas eurihalina (Mellado et al., 1995b). Factors affecting the plasmid transfer frequency by conjugation (cell growth phase, mating time, donor: recipient ratio, composition and salinity of the mating medium) were evaluated by Vargas et al. (1997) to optimize the conditions for conjugation between Escherichia coli and halophilic Bacteria. Frequencies up to to transconjugants/recipient cell have been achieved. Intergeneric conjugation between Halomonas elongata and other moderate halophiles such as Halomonas eurihalina (transfer frequency of and Halomonas halophila was also demonstrated (Vargas et al., 1997). Plasmid pVC1 of Salinivibrio costicola E-367 was selected as a potential candidate for the construction of cloning vectors useful for Salinivibrio on the basis of its small size and the presence of four unique restriction sites. This plasmid was detected in strain 367 only, and no hybridization signals were detected against genomic DNA of 15 other Salinivibrio costicola strains. Southern hybridization experiments revealed no homology between pVC1 and the plasmids pMH1, pHE1, and pCM1 (Mellado et al. 1997). Gene transfer by means of plasmids has also been achieved in the halophilic anoxygenic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens. The Escherichia coli plasmids IncP and IncQ carrying antibiotic resistance markers were transferred into the halophilic phototrophs by triparental mating using plasmid pRK2013 as the helper. Resistance to kanamycin was well expressed in both phototrophs, while tetracycline resistance expression was poor. Chloramphenicol resistance was expressed at a low level in Rhodovibrio salinarum but not in Rhodothalassium salexigens. Streptomycin resistance was expressed in Rhodovibrio, but not in Rhodothalassium, while resistance to spectinomycin was expressed in both. These experiments provide the first step toward the development of a genetic system for these halophilic phototrophs (Borghese et al., 2001).

10.3.4. Genomics and proteomics of the halophilic Bacteria No complete genomes of halophilic Bacteria have been sequenced as yet. Probably the only halophilic Bacterium in which approaches derived from genomics have already been used to obtain information on the proteins potentially synthesized is the thermophilic anaerobic halophile Halothermothrix orenii. A pBluescriptSK+ vector

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library of 3,360 clones has been prepared with inserts of an average of 3.5 kbp of Halothermothrix orenii DNA. Seventy-seven of the clones were sequenced, representing approximately 85 kbp of the genome. Within the sequenced portions, 66 known proteins and another 15 conserved hypothetical proteins were identified (Mijts and Patel, 2001). The appearance and disappearance of specific proteins in response to changes in salinity has been studied in some Bacteria. When Halomonas halophila was transferred from to NaCl, the pattern of proteins labeled during pulse-labeling was changed. A salt downshock from 150 to NaCl also led to alterations in the protein labeling profile (Economou et al., 1989). In "Pseudomonas halosaccharolytica" a decrease in a major 43 kDa outer membrane protein was observed with an increase in a 50 kDa protein when the NaCl concentration of the medium was raised from 50 to . A shift from 120 to salt resulted in a decrease of the 50 kDa protein and the appearance of a 41 kDa protein (Hiramatsu et al., 1978, 1980a, 1980b). Also in Halomonas elongata specific sets of proteins were identified that were induced a high or at low salt. Some of these are general stress proteins also involved in reactions against heat shock (Mojica et al., 1997). The connection between salt stress proteins and heat shock protein was also shown in Chromobacterium marismortui. Heat shock proteins are maximally produced following 5 minutes exposure to 42 °C. High salt concentrations protect to some extent against thermal damage, and growth at elevated salt concentrations extends the upper temperature limit at which heat shock proteins can still be made (Katinakis, 1989). Heat shock proteins have also been identified in Halomonas halophila (Karamanou and Katinakis, 1988). 10.4. REFERENCES Arvanitis, N., Vargas, C., Tegos, G., Perysinakis. A., Nieto, J.J., Ventosa, A., and Drainas, C. 1995. Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae. Appl. Environ. Microbiol. 61: 3821-3825. Asgarani, E., Funamizu, H., Saito, T., Terato, H., Ohyama, Y., Yamamoto, O., and Ide, H. 1999. Mechanisms of DNA protection in Halobacterium salinarium, an extremely halophilic bacterium. Microhiol. Res. 154: 185190. Baliga, N.S., Goo, Y.A., Ng, W.V., Hood, L., Daniels, C.J., and DasSarma, S. 2000. Is gene expression I Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors? Mol. Microbiol. 36: 11841185. Blaseio, U., and Pfeifer, F. 1990. Transformation of Halobacterium halobium – development of vectors and investigation of gas vesicle synthesis. Proc. Natl. Acad. Sci. USA 87: 6772-6776. Bobovnikova, Y., Ng, W.-L., DasSarma, S., and Hackett, N.R. 1994. Restriction mapping the genome of Halobacterium halobium strain NRC-1. Syst. Appl. Microbiol. 16: 597-604. Bonelo, G., Megias, M., Ventosa, A., Nieto, J.J., and Ruiz-Berraquero, F. 1984. Lethality and mutagenicity in Halobacterium mediterranei caused by N-methyl-N'-nitro-N-nitrosoguanidine. Curr. Microbiol. 11: 165170. Borghese, R., Zagnoli, A., and Zannoni, D. 2001. Plasmid transfer and susceptibility to antibiotics in the halophilic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens. FEMS Microbiol. Lett. 197: 117-121. Cánovas, D., Vargas, C., Ventosa, A., and Nieto, J.J. 1997. Salt sensitive and auxotrophic mutants of Halomonas elongata and H. meridiana by use of hydroxylamine mutagenesis. Curr. Microbiol. 34: 85-90.

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Charlebois, R.L. 1995a. Appendix 3. Physical and genetic map of the genome of Halobacterium volcanii DS2, pp. 231-235 In: DasSarma, S., and Fleischmann, E.M. (Eds.), Archaea. A laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Charlebois, R.L. 1995b. Appendix 4. Physical and genetic map of the genome of Halobacterium sp. GRB, pp. 237-239 In: DasSarma, S., and Fleischmann, E.M. (Eds.), Archaea. A laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Charlebois, R.L. 1999. Evolutionary origins of the haloarchaeal genome, pp. 309-317 In: Oren, A. (Ed.), Microbiology and biogeochemistry of hypersaline environments. CRC Press, Boca Ralon. Charlebois, R.L., and DasSarma, S. 1995. Appendix 7. Insertion elements of halophiles, pp. 253-255 In: DasSarma, S., and Fleischmann, E.M. (Eds.), Archaea. A laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Charlebois, R.L., Lam, W.L., Cline, S.W., and Doolittle, W.F. 1987. Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium. Proc. Natl. Acad. Sci. USA 84: 8530-8534. Charlebois, R.L., Hofman, J.D., Schalkwyk, L.C., Lam, W.L., and Doolittle, W.F. 1989. Genome mapping in halobacteria. Can. J. Microbiol. 35: 21-29. Charlebois, R.L., Schalkwyk, L.C., Hofman, J.D., and Doolittle, W.F. 1991. Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2. J. Mol. Biol. 222: 509-524. Cline, S.W., and Doolittle, W.F. 1987. Efficient transfection of the archaebacterium Halobacterium halobium. J. Bacteriol. 169: 1341-1344. Cline, S.W., and Doolittle, W.F. 1992. Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. J. Bacteriol. 174: 1076-1080, Cline, S.W., Lam, W.L., Charlebois, R.L, Schalkwyk, L.C., and Doolittle, W.F. 1989a. Transformation methods for halophilic archaebacteria. Can. J. Microbiol. 35: 148-152. Cline, S.W., Schalkwyk, L.C., and Doolittle, W.F. 1989b. Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J. Bacteriol. 171: 4987-4991. Cline, S.W., Pfeifer, F., and Doolittle, W.F. 1995. Transformation of halophilic Archaea, pp. 197-204 In: DasSarma, S., and Fleischmann, E.M. (Eds.), Archaea. A laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Cohen, A, Lam, W.L., Charlebois, R.L., Doolittle, W.F., and Schalkwyk, L.C. 1992. Localizing genes on the map of the genome of Haloferax volcanii, one of the archaea. Proc. Natl. Acad. Sci. USA 89: 1602-1606. Coronado, M.-J., Vargas, C., Kunte, H.J., Galinski, E.A., Ventosa, A., and Nieto, J.J. 1995. Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies. Curr. Microbiol. 31: 365-371. Daniels, C.J., Gupta, R., and Doolittle, W.F. 1985. Transcription and excision of a large intron in the gene of an archaebacterium, Halobacterium volcanii. J. Biol. Chem. 260: 3132-3134. DasSarma, S. 1993. Identification and analysis of the gas vesicle cluster on an unstable plasmid of Halobacterium halobium. Experientia 49: 482-486. DasSarma, S. 1995. Natural plasmids and plasmid vectors of halophiles, pp. 241-250 In DasSarma, S., and Fleischmann, E.M. (Eds.), Archaea. A laboratory manual. Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. DasSarma, S., and Arora, P. 1997. Genetic analysis of the gas vesicle gene cluster in haloarchaea. FEMS Microbiol. Lett. 153: 1-10. DasSarma, S., RajBhandary, U.L., and Khorana, H.G. 1983. High-frequency spontaneous mutation in the bacterio-opsin gene in Halobacterium halobium is mediated by transposable elements. Proc. Natl. Acad. Sci. USA 80: 2201-2205. DasSarma, S., Kennedy, S.P., Berquist, B., Ng, W.N., Baliga, N.S., Spudich, J.L., Krebs, J.A., Johnson, C.H., and Hood, L. 2001. Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon. Photosynth. Res. 70: 3-17. Doolittle, W.F., and Sapienza, C. 1980. Selfish genes, the phenotype paradigm and genome evolution. Nature 284: 601-603. Doolittle, W.F., Lam, W.L., Schalkwyk, L.C., Charlebois, R.L., Cline, S.W., and Cohen, A. 1992. Progress in developing the genetics of the halobacteria, pp. 73-78 In: Danson, M.J., Hough, D.W., and Lunt, G.G. (Eds.), Archaebacteria: biochemistry and biotechnology. Biochemical Society Symposium no. 58. Biochemical Society, High Holburn, London.

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Dyall-Smith, M.L. 2001. The halohandbook: protocols for halobacterial genetics. Version 4.5. http://www.microbiol.unimelb.edu.au/micro/staff/mds/HaloHandbook/index.html (last accessed: December 23, 2001; last updated: December 2001). Dyall-Smith, M.L., and Doolittle, W.F. 1994. Construction of composite transposons for halophilic archaebacteria (Archaea). Can. J. Microbiol. 40: 922-929. Ebert, K., Goebel, W., and Pfeifer, F. 1984. Homologies between heterogeneous extrachromosomal DNA populations of Halobacterium halobium and four new halobacterial isolates. Mol. Gen. Genet. 194: 91 -97. Ebert, K., Goebel, W., Rdest, U., and Surek, B. 1986. Genome and gene structure in halobacteria. Syst. Appl. Microbiol. 7: 30-35. Economou, A., Roussis, A., Milioni, D., and Katinakis, P. 1989. Patterns of protein synthesis in the moderately halophilic bacterium Deleya halophila in response to sudden changes in external salinity. FEMS Microbiol. Ecol. 62: 103-110. Fernández-Castillo, R., Nieto, J.J., Megías, M., and Ruiz-Berraquero, F. 1990. Efficient hydroxylamine mutagenesis of Haloferax mediterranei and other extremely halophilic archaebacteria. Curr. Microbiol. 21: 83-89. Fernandez-Castillo, R., Vargas, C., Nieto, J.J., Ventosa, A., and Ruiz-Berraquero, F. 1992. Characterization of a plasmid from moderately halophilic eubacteria. J. Gen. Microbiol. 138: 1133-1137. Ferrer, C., Mojica, F.J.M., Juez, G., and Rodríguez-Valera, F. 1996. Differentially transcribed regions of Haloferax volcanii genome depending on the medium salinity. J. Bacteriol. 178: 309-313. Fitt, P.S., and Sharma, N. 1987. The fate of thymine-containing dimers in ultraviolet-irradiated Halobacterium cutirubrum. Biochim. Biophys. Acta 910: 103-110. Fitt, P.S., Sharma, N., and Castellanos, G. 1983. A comparison of liquid-holding recovery and photoreactivation in halophilic bacteria. Biochim. Biophys. Acta 739: 73-78. Forterre, P., Elie, C., and Kohiyama, M. 1984. Aphidicolin inhibits growth and DNA synthesis in halophilic archaebacteria. J. Bacteriol. 159: 800-802. Forterre, P., Nadal, M., Elie, C., Mirambeau, G., Jaxel, C., and Duguet, M. 1986. Mechanisms of DNA synthesis and topoisomerisation in archaebacteria – reverse gyration in vitro and in vivo. Syst. Appl. Microbiol. 7: 6771. Franzetti, B., Schoehn, G., Ebel, C., Gagnon, J., Ruigrok, R.W.H., and Zaccai, G. 2001. Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro. J. Biol. Chem. 276: 29906-29914. Gadelle, D., and Forterre, P. 1994. DNA intercalating drugs inhibit positive supercoiling induced by novobiocin in halophilic archaea. FEMS Microbiol. Lett. 123: 161-166. Gregor, D., and Pfeifer, F. 2001. Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea. Microbiology UK 147: 1745-1754. Grey, V.L., and Fitt, P.S. 1976. Evidence for lack of deoxyribonucleic acid dark-repair in Halobacterium cutirubrum. Biochem. J. 156: 569-575. Gutiérrez, M.C., García, M.T., Ventosa, A., Nieto, J.J., and Ruiz-Berraquero, F. 1986. Occurrence of megaplasmids in halobacteria. J. Appl. Bacteriol. 61: 67-71. Hackett, N.R., Bobovnikova, Y., and Heyrovska, N. 1994. Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium. J. Bacteriol. 176: 7711-7718. Hescox, M.A., and Carlberg, D.M. 1972. Photoreactivation in Halobacterium cutirubrum. Can. J. Microbiol 18: 981-985. Hiramatsu, T., Ohno, Y., Hara, H., Toriyama, S., Yano, I., and Masui, M. 1978. Salt-dependent change of cell envelope components of a moderately halophilic bacterium, Pseudomonas halosaccharolytica, pp. 515-520 In: Caplan, S.R., and Ginzburg, M. (Eds.), Energetics and structure of halophilic microorganisms. Elsevier/North Holland Biomedical Press, Amsterdam. Hiramatsu, T., Ohno, Y,, Hara, H., Yano, I., and Masui, M. 1980a. Effects of NaCl concentration on the envelope components in a moderately halophilic bacterium, Pseudomonas halosaccharolytica, pp. 189-200 In: Morishita, H., and Masui, M. (Eds.), Saline environments. Proceedings of the Japanese Conference on Halophilic Microbiology. Nakanishi Printing Co., Kyoto. Hiramatsu, T., Yano, I., and Masui, M. 1980b. Effect of NaCl concentration on the protein species and phospholipid composition of the outer membrane in a moderately halophilic bacterium. FEMS Microbiol. Lett. 7: 289-292. Holmes, M.L., and Dyall-Smith, M.L. 1990. A plasmid vector with a selectable marker for halophilic archaebacteria. J. Bacteriol. 172: 756-761.

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CHAPTER 11 BIOTECHNOLOGICAL APPLICATIONS AND POTENTIALS OF HALOPHILIC MICROORGANISMS

It is obvious ... that certain 'biotechnological' applications (production of fermented salted beans and fish) have been in use for centuries, while other ideas and concepts are being developed. However, it seems clear that a better understanding of the role played by halotolerant and halophilic micro-organisms may contribute to the future, whether it be in the Chinese restaurant, the biodegradable plastic carrier bag, or the use of enzymes stabilised by compatible solutes. (Galinski and Tindall, 1992)

11.1. INTRODUCTION The halophilic microorganisms have found a number of interesting applications in biotechnology. They may be less "fashionable" in biotechnology research than the thermophilic extremophiles, but the numbers of applications that are either already being exploited or are under development is impressive indeed. The uses that the halophiles have found in biotechnology can be divided into a number of categories. Firstly, the halotolerance of many of their enzymes can be exploited wherever enzymatic transformations are required to function at low water activities, such as found in the presence of high salt concentrations. Secondly, some organic osmotic stabilizers produced by halophiles have found interesting applications. Thirdly, a number of halophilic microorganisms produce valuable compounds. Some of these are unique and not found elsewhere in the living world. Halophiles may also present distinct advantages for the development of biotechnological production processes of certain products that can also be found in non-halophiles. Table 11.1 lists the most important applications of the halophilic Archaea, Bacteria and Eucarya, both those that are already commercially exploited and those that are in different stages of development. This list of possible biotechnological uses of halophilic microorganisms is by no means exhaustive. For a more complete account specialized review articles may be consulted (Galinski and Tindall, 1992; Margesin and Schinner, 2001; Oren, 2002; Rodriguez-Valera, 1992; Ventosa and Nieto, 1995). Generally the halophilic microorganisms have some distinct advantages over their non-halophilic counterparts. Possible contamination with undesired microorganisms is much less of a problem at high salt concentrations than in low salinity media. There are, however, also certain drawbacks associated with mass cultivation of halophiles.

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When dealing with aerobic, oxygen-requiring types, the low solubility of gases in concentrated brines may severely limit the supply of oxygen to the cultures (Shand and Perez, 1999). Cultivation in relatively large fermentors is possible (Kushner, 1966), but the aggressive nature of the salts should be taken into account when planning the construction of reactors with metal parts exposed to the medium. Special equipment may therefore be necessary. A corrosion-resistant bioreactor has been designed for the growth of halophiles, which is composed of polyetherether ketone. This bioreactor has been used for the optimization of the production of and polyby certain extremely halophilic Archaea (Hezayen et al., 2000). The tremendous diversity of halophilic microorganisms found in nature is still far from being fully exploited. Thanks to the new possibilities opened by the approaches derived from genomics and proteomics, as well as to the availability of genetic transfer systems in a variety of halophiles, novel applications may be developed in the future. 11.2. APPLICATIONS OF HALOPHILIC ARCHAEA

Much of the early research interest in the halophilic Archaea in the beginning of the century emerged from applied aspects: red halophiles caused significant damage to salted fish and salted hides (see also Section 1.2). The development of red microbial growth on hides ("red heat" or "the pink") has been known for long to be due to the development of Archaea such as Halobacterium or Halococcus (Shewan, 1971). Only in recent years, however, has the role of these Archaea as the causative agent of the deterioration of the hides been proven unequivocally. Treatment with commercial bactericides can prevent the damage (Bailey and Birbir, 1996; Birbir and Bailey, 1996; Vreeland et al., 1998). Different chemical treatments have been suggested to kill halophilic Archaea in crude solar salt (Tasch and Todd, 1973, 1974), but such procedures are not widely used. Degradative processes performed by halophilic Archaea also have positive aspects, as the preparation of certain traditionally fermented foods in the Far East depends on them. The considerable metabolic potential of halophilic Archaea can theoretically also be harnessed for the development of biodegradation processes that function at high salt concentrations. However, such applications are as yet in a developmental stage. Several species of halophilic Archaea synthesize potentially useful products such as exopolysaccharides, and others (Galinski and Tindall, 1992; Rodriguez-Valera, 1992; Ventosa and Nieto, 1995). An especially promising field of applied research is the development of different applications of bacteriorhodopsin, the retinal proton pump of Halobacterium salinarum. These uses range from holographic storage material, as building blocks for the construction of computer memories and processing units, as photoelectric converters, and others.

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11.2.1. Halophilic Archaea in the preparation of fermented foods The production certain traditional fermented foods in the Far East involves the activity of a variety of halophilic and/or highly halotolerant microorganisms, and in some cases these include Archaea of the family Halobacteriaceae. The microbiology of these processes is not well understood even today. One such preparation is "nam pla", a fermented fish sauce produced in Thailand. This product is traditionally made by adding two parts of fish and one part marine salts. The mixture is covered with concentrated brine and left to ferment for about a year. Red halophilic Archaea, identified as Halobacterium and Halococcus, reach their maximum density in the liquor after three weeks and persist throughout the fermentation period. The halobacterial proteases probably take part in the fermentation process (Thongthai and Siriwongpairat, 1990; Thongthai and Suntinalert, 1991; Thongthai et al., 1992). Products formed during the metabolism of the Archaea have been claimed to contribute to the aroma of the sauce (Saisithi et al., 1966; Lopetcharat et al., 2001) A more recent attempt to exploit the halophilic Archaea for human consumption has been the investigation on the suitability of the isoprenoid diether lipids of these organisms as a food additive, to serve as emulsifier and/or as a low-calorie fat substitute (Post and Collins, 1982). 11.2.2. The biodegradative potential of halophilic Archaea The Halobacteriaceae have always been considered to be a group of microorganisms with little metabolic versatility. For long it was believed that amino acids and a few organic acids were the only substrates degraded. Many species even do not use simple carbohydrates (see also Section 4.1.4). However, some isolates may have considerably more developed catabolic abilities. Thus, hydrocarbon-degrading halophilic Archaea have been described. A strain designated EH4, isolated by from a saltern pond in the south of France, can grow on saturated (tetradecane, hexadecane, eicosane, heneicosane, pristane) and aromatic hydrocarbons (acenaphtene, phenanthrene, anthracene, 9-methylanthracene). Degradation of between 48 and 88% of the straight-chain hydrocarbons or 19-24% of the aromatic hydrocarbons added at a concentration of was achieved after 30 days of incubation at 32 °C (Bertand et al., 1990). Hydrocarbon-degrading red Archaea were isolated as well from an oil deposit in Russia (Kulichevskaya et al., 1991). Haloferax strain D1227, isolated from petroleum-contaminated soil from a petroleum production well near Grand Rapids, Michigan (Oriel et al., 1997) can grow on aromatic compounds including benzoate, cinnamate, and 3-phenylpropionate (Emerson et al., 1994) (see also Section 4.1.4). Degradation of 3-phenylpropionate proceeds through the gentisate pathway (Fu and Oriel, 1998, 1999). Halophilic Archaea belonging to the genera Haloarcula, Halobacterium, and Haloferax have been subjected to a patented selection process, in the course of which they became adapted to the degradation of high concentrations (up to 1 mM) of

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halogenated hydrocarbons such as trichlorophenols or the insecticides lindane and DDT (Oesterhelt et al., 1998).

11.2.3. The role of halophilic Archaea in the production of solar salt The positive effect of the presence of dense communities of red halophilic Archaea in saltern crystallizer ponds has been recognized for a long time. The red coloration that develops in such ponds is mainly caused by Archaea, but strains of Dunaliella, and possibly even red halophilic Bacteria of the genus Salinibacter contribute as well toward the absorption of light energy. By trapping solar radiation these microorganisms raise the temperature of the brine and the rate of evaporation, thereby increasing salt production (Davis, 1974; Javor, 1989, 2002; Jones et al., 1981). To improve salt production in salterns that do not develop a sufficiently dense archaeal community, fertilization with organic nutrients has even been suggested (Davis, 1974). C50 bacterioruberin derivatives are the main carotenoids of the Halobacteriaceae (see Section 5.3). However, additional carotenoids may be present that have proven economic value. An isolate from a seawater evaporation pond near Alexandria, Egypt, produces considerable amounts of the ketocarotenoid canthaxanthin (Asker and Ohta, 1999). Exploitation of this organism for commercial canthaxanthin production has already been suggested (Asker and Ohta, 1999; Margesin and Schinner, 2001). There are even reports that the halophilic Archaea may be directly involved in the crystallization of halite in the crystallizer ponds (Castanier et al., 1999). This aspect will be discussed in further depth in the chapter on the microbiology of solar salterns (Section 14.8).

11.2.4. Biotechnological applications of bacteriorhodopsin The light-driven proton pump bacteriorhodopsin has many properties that make it an attractive material for a large number of possible applications (Hampp, 2000a, 2000b; Margesin and Schinner, 2001; Oesterhelt et al., 1991) (Table 11.2). Hampp (2000a) lists close to 100 patents that have been issued between 1983 and 1997 related to applications of bacteriorhodopsin. These applications are based on the protonmotive, photoelectric, and photochemical properties of the molecule. In some uses light energy is converted into chemical energy. Others exploit the properties of its photocycle. Upon excitation by light, the B state (absorbance maximum 570 nm) changes its conformation in a complex series of reactions. A key intermediate of this photocycle is the M state, in which the molecule is yellow and absorbs light of 400-450 nm. Bacteriorhodopsin can be used as an optoelectronic material for holographic image storage (Oesterhelt et al., 1991). The holographic interference patters are registered as purple or yellow areas. Two alternative recording systems have been proposed. One mechanism is based on the photoreactivity of bacteriorhodopsin in the ground state following illumination with 500-600 nm (see Section 5.4.1).

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Alternatively the transition can be recorded with light of 400-450 nm, using a film previously converted to the M state by illumination in the spectral range of the B state. Such M-type holograms offer certain advantages, especially when a mutant bacteriorhodopsin is used with a prolonged lifetime of the M-state. The exchange of a single amino acids (replacement of aspartic acid to asparagine in position 96) prolongs the lifetime of the M intermediate thousand-fold (Hampp, 2000b). As the transitions are reversible, a bacteriorhodopsin holographic matrix can be used repeatedly. Holographic bacteriorhodopsin films are suitable for the construction of computer memories enabling parallel processing. The developing technology may lead to a new generation of computers (Hong, 1986). The molecule is used here in "bioelectronic" elements of computer memories and information processing units, exploiting the properties of bacteriorhodopsin for information processing. It serves as an optical switching element, again based on the and transitions, analogous to the conducting/non-conducting stages in a semiconductor. A high density of information storage is possible, and all of the information can be processed simultaneously (parallel processing) (Birge, 1995; Birge et al., 1999; Oesterhelt et al., 1991). Bacterio-

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rhodopsin films show a high spatial resolution (> 35,000 lines and excellent reversibility cycles). Highly oriented films composed of purple membranes have been obtained by using two kinds of bispecific antibodies with different antigen binding sites, one binding to a specific side of bacteriorhodopsin and the other to a phospholipid haptene. Such films were used in the construction of a light-sensitive photoelectric device (Koyama et al., 1994). Patented new applications of the material include use as bioelement in a motion sensor (Ackley and Shieh, 1998), in an image sensor, or in a biocomputer (Margesin and Schinner, 2001). Other potential uses of bacteriorhodopsin include conversion of sunlight to electricity, ATP generation, desalination of seawater, use in chemo- and biosensors, and ultrafast light detection. To turn bacteriorhodopsin into a light sensor it is spread in a thin film sandwiched between an electrode and an electrically conductive gel. Changes in the shape of the molecule create a charge displacement generating an electrical signal (Vsevolodow and Dyukova, 1994). Bacteriorhodopsin was also suggested for use in a bio-photoelectrochemical reactor for photochemical hydrogen production. Here Halobacterium salinarum cells are immobilized in polyacrylamide gels or on cellulose acetate membranes. Presence of bacteriorhodopsin regulates the pH of the system, thereby increasing the hydrogen production and lowering the power requirement needed for electrochemical hydrogen production (Sediroglu et al., 1999). All of these applications are made possible by the excellent thermodynamic and photochemical stability of bacteriorhodopsin. In contrast to virtually all other proteins of Halobacterium it is not a truly "halophilic protein", as it is stable and active in the absence of salts as well. Bacteriorhodopsin is easy to immobilize on glass plates or to embed in polymers. It functions well between 0-45 °C and over the whole pH range from 1 to 11 (Chen and Birge, 1993). It is active both in fresh water and in highly saline solutions; it tolerates temperatures of over 80 °C in water and up to 140 °C in a dry state; it is stable to exposure to sunlight in air or oxygen for years, it resists digestion by most proteases; and, it produces very reproducible photoelectric signals. Bacteriorhodopsin is commercially available in the form of purple membrane patches isolated from Halobacterium salinarum strain S9. It is sold by MIB, Munich Innovative Biomaterials, Germany (http://www.mib-biotech.de) in lyophilized form. The same company also offers films and devices made of genetically modified bacteriorhodopsin for optical information storage and processing. The retinal-based light-driven chloride pump halorhodopsin (see Section 5.4.2) has also found potential biotechnological applications. A chloride-sensitive biosensor has been developed using an ion-sensitive field effect transistor on which membrane vesicles containing halorhodopsin had been immobilized. When illuminated, this sensor reacts to the concentration of chloride (Seki et al., 1994).

11.2.5. Production of biopolymers by halophilic Archaea Haloferax mediterranei cells may contain considerable amounts of hydroxyalkanoate, a copolymer of and

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(Fernandez-Castillo et al., 1986; Rodriguez Valera et al., 1991, see also Section 3.1.8). Its content of can reach values between 19 and 38 percent by dry weight, dependent on salinity. The highest concentrations were observed in cells grown in salt (Fernandez-Castillo et al., 1986). is used for the production of biodegradable plastics. Such thermoplastics ("biological polyesters") have excellent properties such as a high strength and a low melting point, similar to polypropylene. Although halophilic Archaea are not yet being used commercially for the production of hydroxyalkanoate, they have certain obvious advantages over an organism such as Ralstonia eutropha, which is already exploited for the production of biodegradable plastic ("Biopol" produced by ICI). Haloferax mediterranei can be grown on a cheap substrate such as starch. Moreover, downstream processing and purification of the product should be relatively simple as the cells are easily lysed in water (Ventosa and Nieto, 1995). Also the high genomic stability of the organism and the reduced danger of contamination are clear assets. production is maximal when grown on sugars (glucose or starch) and in the presence of low phosphate concentrations (Lillo and Rodriguez-Valera, 1990; Rodriguez-Valera and Lillo, 1992). formation has been demonstrated in certain other halophilic Archaea as well, such as Haloarcula marismortui (Kirk and Ginzburg, 1972) and Natrialba strain 56 (cited as DSM 13151, but not listed in the DSMZ online catalog in April 2002) (Hezayen et al., 2000). When grown in the presence of acetate and butyrate, about 0.43 g could be obtained per gram dried cells. Several species of halophilic Archaea, especially those of the genus Haloferax, produce copious amounts of extracellular polysaccharides (sec also Section 3.1.1). These polysaccharides may have considerable biotechnological potential (Antón et al., 1988; Rodriguez-Valera et al., 1991; Severina et al., 1989). Haloferax mediterranei produces up to of an acidic heteropolysaccharide with a high apparent viscosity at relatively low concentrations, which is resistant to extremes of salt concentration, temperature, and pH. The structure of this polymer was recently elucidated (Parolis et al., 1996). Haloferax volcanii and Haloferax gibbonsii produce exopolysaccharides as well (Paramonov et al., 1998; Severina et al., 1990). Such polymers may be used to modify rheological properties of aqueous systems, for viscosity stabilization as thickening agents, gelling agents and emulsifiers, and they may find applications in microbially enhanced oil recovery (Ventosa and Nieto, 1995). By decreasing surface tension they increase the solubility and thus the mobility of hydrophobic hydrocarbons. A salt-resistant surfactant is advantageous as high salinity brines are often encountered associated with oil deposits. Whole cell preparations may be used, and the membrane lipids liberated upon lysis of halophilic Archaea may also act as surfactants and improve the oil-carrying properties (Post and Al-Harjan, 1988). No large-scale production of the Haloferax mediterranei exopolysaccharide has yet been initiated. Another extracellular polymer that may find interesting biotechnological applications is excreted by Natrialba aegyptiaca (aegyptia)

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strain 40 (Hezayen et al., 2000). can be used as a biodegradable thickener, a humectant, or a drug carrier in the food or pharmaceutical industry.

11.2.6. Enzymes from halophilic Archaea Many halophilic Archaea excrete exoenzymes such as amylases, amyloglucosidases, proteases, and lipases that function at high salinity. Such enzymes may find applications in biotechnological processes requiring degradation of macromolecules in the presence of high salt concentrations (Chaga et al., 1993; Van Qua et al., 1981; Ventosa and Nieto, 1995). An overview of the biotechnological uses of archaeal exoenzymes, including those of the halophiles, was recently presented by Eichler (2001). In comparison to the thermophilic Archaea the haloarchaeal enzymes have received little attention in biotechnology as the demand for salt-tolerant enzymes in current manufacturing or related processes is relatively limited. Natronomonas pharaonis produces a chymotrypsinogen B-like protease that performs optimally at 61 °C and pH 10 (Stan-Lotter et al., 1999). Unlike the majority of other enzymes from halophiles, this alkaliphilic protease can function in salt concentrations as low as 3 mM. This suggests its usefulness as a detergent additive, which is currently a major market for alkaliphilic enzymes. The amyloglucosidase of Halorubrum sodomense has been purified and characterized in view of a possible biotechnological application of the enzyme (Chaga et al., 1993). production by immobilized cells of Halobacterium salinarum was optimized for a similar purpose (Bagai and Madamwar, 1997). For laboratory use more specialized enzymes may be derived from the halophilic Archaea. Thus, a patent has been filed for the production of a novel restriction enzyme of unusual specificity from a species of the genus Halococcus (Obayashi et al., 1988). The use of site-specific endonucleases of halophilic Archaea in molecular biological research has been suggested before (Schinzel and Burger, 1986). Peptidyl prolyl cistrans isomerase is a useful enzyme for the regeneration of denatured protein, for stabilization of proteins, and for production of recombinant proteins. Production of a novel cyclophilin type peptidyl prolyl cis-trans isomerase, using a gene amplified from the genome of Halobacterium salinarum, has been patented (lida et al., 1997).

11.2.7. Miscellaneous applications of halophilic Archaea There are additional potential applications for the halophilic Archaea of the family Halobacteriaceae; all those listed below are still in an experimental stage: A recombinant vector has been constructed capable of directing the synthesis of gas vesicles in nonfloating prokaryotic cells. This vector contains genes encoding those Halobacterium salinarum proteins required for the synthesis of gas vesicles. Cells transformed with this vector float, whereafter they can easily be separated from the medium by skimming or decanting, thus greatly facilitating cell harvesting (DasSarmna

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et al., 1999). These gas vesicles may also find interesting applications as an antigen epitope display and delivery system. Gas vesicles coupled to haptenes such as trinitrophenol or small peptides gave an excellent immunological response when injected in mice (Stuart et al., 2001). An archaeal 84-kDa protein from Halobacterium salinarum shares common epitopes with the human c-myc protein which is an oncogene product in the serum of certain cancer patients. This protein can therefore be used as an antigen to detect antibodies against the human c-myc protein in diagnostic procedures (Ben-Mahrez et al., 1988, 1991). Halophilic Archaea are inhibited by several drugs that interact with tubulin, actomyosin, and DNA topoisomerase II of Eucarya. They indeed contain proteins that may be the target of such drugs: a yeast actin probe hybridizes with DNA restriction digests of Halobacterium salinarum. Antibodies against tubulin and actine from chicken react in a crude extract of Halobacterium with polypeptides of 55 and 80 kDa. The epipodophyllotoxin VP16, a eukaryotic DNA topoisomerase II inhibitor, induces DNA strand breaks with DNA-protein covalent linkage in Halobacterium as well as in Eucarya. This suggests that halophilic Archaea can be used to prescreen antitumor drugs active on eukaryotic proteins. Plasmid pGRB-1 of Halobacterium strain GRB-1 presents a convenient model system to be used in the pre-screening of new antibiotics and anti-tumor drugs that affect eukaryotic-type II DNA topoisomerase (epipodophyllotoxins) and quinolone drugs that cause DNA relaxation. The single and double-stranded breaks in the DNA of the multicopy plasmid pGRB-1 can be monitored in vitro (Forterre, 1989; Sioud et al., 1987). Halophilic Archaea may prove suitable for the expression of certain human membrane proteins. The human a seven transmembrane helix protein, has been cloned in Halobacterium salinarum using an expression vector for bacterio-opsin modified to express the adenoreceptors under the control of bacterioopsin regulatory elements (Söhlemann et al., 1997). Archaeal ether lipid liposomes ("archaeosomes") have been tested as delivery systems for vaccines and drugs. However, the liposomes prepared from Halobacterium salinarum proved much more leaky than those made from methanogens or from Thermoplasma (Patel and Sprott, 1999). Halococcus morrhuae cells have a stimulatory effect on human lymphocytes as judged by (Montes et al., 1999). Whether useful applications will be found based on this property remains to be seen. In mammals, antiporter inhibitors protect the myocardium against ischemia and reperfusion injury. This led to the isolation of a Haloferax gibbonsii halocin H6 overproducing strain (Haloferax gibbonsii Alicante SPH7), the halocin of which has been renamed halocin H7. This haloarchaeal antiporter inhibitor was reported to decrease infarct size and ectopic beats after myocardial reperfusion in dogs, and decreased A-V intrinsic nodal conduction and heart rate in isolated rabbit hearts. This finding may lead to potential applications of the halocin to reduce injury during organ transplantation (Alberola et al., 1988; Such et al., 1988).

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11.3. APPLICATIONS OF HALOPHILIC BACTERIA Halophilic Bacteria possess a number of interesting applications as well (RamosCormenzana, 1989; Ventosa et al., 1998). Many produce compounds of industrial interest such as osmoprotectants, enzymes, polymers, and others. They are generally easy to grow, and their nutritional requirements are often simple. Many heterotrophic halophilic Bacteria can use a wide range of compounds as sole carbon and energy source (Kushner and Kamekura, 1988). Tools for their genetic manipulation are becoming increasingly available (see Section 10.3.3). The sections below discuss the biotechnological uses already made of the halophilic Bacteria and future uses that are being explored.

11.3.1. Halophilic Bacteria in the preparation of food products A characteristic flora of halophilic Bacteria is associated with cured salted fish. Examination of the microbial community of cured salted anchovies showed dominance of Pediococcus halophilus, with optimal growth between 60 and salt and it tolerated up to (Villar et al., 1985). Moderately halophilic Bacteria were also abundant on cured salted cod (bachalao). Viable counts up to were reported. These consisted of two types of cream to pinkish colonies that grew between 5 and 250 These colonies have not been characterized further (Vilhelmsson et al., 1996). Halophilic Bacteria are also involved in the production of traditionally fermented salted foods in the Far East. "Nukazuke", a paste of fermented fish in bran that contains between 10 and 15% salt, contains many halophilic cocci that are involved in the fermentation process. Lactic acid is the main product of their metabolism. Viable counts between and per gram have been obtained (Kuda et al., 2001). The microbial community involved in the production of fermented salted puffer fish ovaries in rice bran ("fugunoko nukazuke") in Japan has been investigated in more detail. The fermentation process here lasts up to 1-2 years, and salt concentrations from about 13% up to 30% are being used. Among the microorganisms abundantly found in the fermented material are Tetragenococcus halophilus and Tetragenococcus muraticus, Pseudomonas-like halophiles and also Archaea (Kobayashi et al., 1995, 2000). Halomonas alimentaria is an important component of the microbial community that develops during the preparation of "jeotgal", a traditional Korean fermented seafood (Yoon et al., 2002). Also related to the food industry is the commercial production of the flavoring agents 5'-guanylic acid (5'-GMP) and 5'-inosinic acid from RNA, using the halophilic nuclease H of "Micrococcus varians subsp. halophilus". This enzyme degrades RNA at 60 °C and NaCl. At these conditions an excellent yield is achieved, as there is little activity of contaminating 5'-nucleotidases (Kamekura et al., 1982). A bioreactor system of flocculated cells of "Micrococcus varians subsp. halophilus" has

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been developed for optimal production (Kamekura et al., 1982; Onishi et al., 1991; Yokoi and Onishi, 1990). Inclusion of at least 40 mM magnesium or more than 14 mM phosphate in the medium abolished extracellular nuclease activity and caused flocculation of the cells. Under these conditions the enzyme was found adsorbed to the surface of the floes (Kamekura and Onishi, 1978a). The enzyme is produced when the organism is grown in 1-4 M NaCl or KC1, with maximal production in media containing 2.5-3.5 M salt. The purified enzyme has both DNase and RNase activity. Activity is optimal in 2.9 M NaCl or 2.1 M KCl at 40 °C. Activity is lost by dialysis against water, but can be restored upon dialysis against buffer containing 3.4 M NaCl (Kamekura and Onishi, 1974, 1976). The protein has a large (21 mol%) excess of acidic over basic amino acids (Kamekura and Onishi, 1978b). To obtain a good yield of the desired product, the contaminating 5'-nucleotidase produced was further inactivated by desalting treatment of the flocculated cells in presence of 80 mM Inclusion of 0.05 mM also greatly improved the yield as it completely inhibited the 5'-nucleotidase, while more than 50% of the nuclease activity remained (Onishi et al., 1991; Yokoi and Onishi, 1989). The same "Micrococcus varians subsp. halophilus" also yields an interesting amylase with biotechnological potential (Kobayashi et al., 1986).

11.3.2. The biodegradative potential of halophilic Bacteria Industrial wastewaters often contain both high concentrations of toxic organic compounds and high salt concentrations, sometimes accompanied by high levels of toxic inorganic ions. Such hypersaline wastewalers are generated for example during the manufacture of pesticides and Pharmaceuticals. Wastewater brines are also produced during oil and gas recovery processes. Conventional biological waste treatment processes are generally unable to cope with such toxic brines. Processes based on the biodegradative potential of halophilic Bacteria may provide solutions to the problem. Special biological treatment systems have been developed for highly saline wastewaters, based on modifications of the activated sludge process, while employing aerated percolators or rotating discs to improve aeration and mixing of the sludge (Dinçer and Kargi, 2001; Kargi and Dinçer, 2000; Kargi and Uygur, 1996). Using a rotating disc system and synthetic model wastewaters containing molasses as organic substrate and salt at concentrations up a satisfactory rate of removal of the chemical oxygen demand of the wastes was achieved, although the efficiency of the process was lowered at the highest salinities tested. In these experiments a culture of the archaeon Halobacterium salinarum was added to the wastewater to improve biodegradative performance (Dinçer and Kargi, 2001). It is, however, improbable that this addition caused any stimulation of the process, as Halobacterium requires much higher salt concentrations for growth and activity than those present in the brines used. Therefore it may be assumed that the activity observed was due to Bacteria rather than to Archaea. The system was also applied to the purification of wastewater from the

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pickling industry (Kargi et al., 2000). The raw wastewater (130-150 salt) was adjusted to pH 6.5 with ammonium hydroxide and diluted to a final salt concentration between 30 and Also here a culture of Halobacterium was added in an attempt to speed up the degradation process. To what extent this addition indeed increased the biodegradation efficiency is unclear. A biological treatment system has been described that is used to clean up wastewater (salt concentration about 150 generated during the production of pickled plums in Japan. The system was reported to reduce the chemical oxygen demand of the wastewater by 70-90%. Two salt-tolerant bacteria were isolated from the system that grew optimally at salt concentrations between 0-100 but could tolerate up to 200 These isolates were tentatively identified as Staphylococcus sp. and Bacillus cereus (a species not otherwise known as highly halotolerant) (Kubo et al., 2001). It has been suggested that halophilic Bacteria might be utilized to remove phosphate from saline environments, as a cheaper alternative to chemical approaches (Ramos-Cormenzana, 1989). Among the toxic compounds shown to be broken down at high salt concentrations by halophilic Bacteria are formaldehyde (Azachi et al., 1995; Oren et al., 1992), phenol and other aromatic compounds, organophosphorus compounds, and others. Biodegradation of hypersaline wastewaters containing phenol was achieved by a biofilm of yet-to-be-identified halophilic bacteria isolated from a saltern at the Great Salt Lake, Utah. By using a biofilm reactor ("sequencing batch biofilm reactor") operated with periodical 12-hour cycles (1 hour filling-feeding with aeration, 8 hours reaction with aeration, 2.75 hours settling, and 0.25 hours drawing), more than 99% of the phenol from a waste containing salt was removed. The biofilm developed on the silicone tubing used for aerating the system (Woolard and Irvine, 1992, 1994, 1995). A Halomonas strain has been isolated that grows between 10 and salt with an optimum at and effectively degrades phenol (Hinteregger and Streichsbier, 1997). Benzoate and other aromatic compounds can be degraded by Halomonas halodurans by cleavage of the aromatic ring (Rosenberg, 1983). Bacteria belonging to the family Halomonadaceae have been isolated that can use chloroaromatic compounds such as the herbicide 2,4-dichlorophenoxyacetic acid (2,4D) as carbon and energy sources. They have been found in a highly saline site (Alkali Lake, Oregon) contaminated with 2,4-D. One isolate, designated strain I-18, showed high activities of catechol 1,2-dioxygenase, muconate cycloisomerase and dienelactone hydrolase at 1 M NaCl and pH 8.4-9.4. This strain could also use other aromatic compounds including benzoic acid, 3-chlorobenzoic acid, and 4-chlorophenol (Maltseva et al., 1996; Oriel et al., 1997). Halophilic Bacteria can degrade oil hydrocarbons up to quite high salt concentrations. A field study performed in the Great Salt Lake (see also Chapter 12) yielded microbial growth at salinities up to about after enrichment with mineral oil as substrate (Ward and Brock. 1978). Marinobacter hydrocarbonoclasticus is a specialized hydrocarbon degrader, able to function well at salt concentrations as high as (Gauthier et al., 1992). Several moderate halophiles

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that degrade hydrocarbons, including hexadecane and phenanthrene, have been isolated from Organic Lake, Antarctica (McMeekin et al., 1993). A halophilic Bacterium isolated from Grantsville Warm Springs, a hypersaline spring south of the Great Salt Lake, Utah, possesses high levels of enzymatic activity degrading highly toxic organophosphorus compounds. The organism (designated strain JD6.5, tentatively identified as an Alteromonas species) grew at salt (DeFrank and Cheng, 1991). The organophosphorus acid anhydrase of the organism was purified and characterized. Five other bacteria were isolated with properties similar to strain JD6.5, and showed hydrolytic activity against several organophosphorus compounds (DeFrank et al., 1993). Another organophosphonate degrading halophile was recently obtained from soil from underneath a road gritting salt pile in Northern Ireland (Hayes et al., 2000). It grows between 25 and salt with an optimum at and is phylogenctically most similar to Pseudomonas beijerinckii or Chromohalobacter marismortui (two organisms not known to be closely related). It uses phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutylmethyl- and ethyl-phosphonate as phosphorus source for growth. Organophosphorus anhydrases have considerable potential for the decontamination and mineralization of chemical warfare agents. Some halophilic Bacteria show a surprisingly high tolerance to heavy metal ions (Nieto, 1991; Nieto et al., 1989). Microorganisms that tolerate both high salt and high concentrations of heavy metals may be of much use in the biotransformation and bioremediation of heavy metals in hypersaline environments, including wastewater treatment plants. In a comprehensive study a large number of halophilic strains that show tolerance to mercury, cadmium, copper, chromium, and/or zinc have been isolated from different geographical sites in Spain. The majority of the isolates have been preliminary assigned to the genus Halomonas.. Approximately 30% of all strains displayed multiple resistances (Ventosa et al., 1998). Halophilic Bacteria have been identified that can perform biotransformations of selenium and uranium and aid in their bioremediation. From a selenium-contaminated hypersaline evaporation ponds in the San Joaquin Valley, California, built to reduce the volume of agricultural drainage water high in selenate, a number of Halomonaslike bacteria were isolated that tolerate up to 2 M selenate and NaCl. These organisms accumulated selenate and volatilized it to the relatively nontoxic dimethylselenide (D'Souza et al., 2001). Biotransformation of uranium compounds was observed in a halophilic denitrifying bacterium belonging to the genus Halomonas, isolated from the Waste Isolation Pilot Plant repository, Carlsbad, New Mexico. This organism caused dissolution of the insoluble uranyl hydroxide and uranylhydroxyphosphate species with the formation of soluble uranyl dicarbonate complexes as a result of production during metabolism of organic substrates such as succinate (Francis et al., 2000). All processes mentioned above are performed under aerobic conditions. Degradation of certain toxic compound can proceed anaerobically as well. Although many halophilic Bacteria are able to use nitrate as electron acceptor and grow as denitrifiers up to very high salt concentrations, little success has been achieved in the

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design of the anaerobic treatment of hypersaline wastewater based on denitrification. Already at salt denitrification was severely inhibited in model wastewaters (Dinçer and Kargi, 1999). Halomonas campisalis was recently suggested as a useful organism to remove nitrate at pH 9 in the presence of salt (Peyton et al., 2001). The moderately alkaliphilic denitrifying Halomonas desiderata, isolated from municipal sewage, may find use in the combined chemical and biological degradation of nitrocellulose (Berendes et al., 1996). Reductive dechlorination of chlorophenols and chlorophenoxyphenols has been observed under anaerobic conditions in hypersaline groundwaters near Alkali Lake, Oregon (Boone et al., 1989). The existence of the process was inferred from the nature of the shape of the concentration gradients of the chlorinated compounds from the source of pollution. No additional information is available on the nature of the organisms responsible for the dechlorination. Nitrosubstituted aromatic compounds (pnitrophenol, dinitrophenol, nitroanilines) were reduced to the corresponding amino derivatives by Halanaerobium praevalens and Orenia marismortui, obligatory anaerobic Bacteria of the family Halanaerobiales (Oren et al., 1991). Such halophilic anaerobes were also suggested to be useful for the production of acetic and other organic acids (Lowe et al., 1993). They may perform the first step in the degradation of pretreated lignite in a biogasification process to take place in underground salt caverns as cheaply available bioreactors (Oren, 1990). Anaerobic halophilic Bacteria have also been exploited in a pilot-scale field study to encourage microbially enhanced oil recovery in a hypersaline oil reservoir. Molasses and ammonium nitrate were added to the brines to stimulate microbial growth with the aim to selectively cause plugging of permeable regions in the reservoir and to prevent the development of sulfate reducing bacteria (Bhupathiraju et al., 1993).

11.3.3. Ectoines as stabilizers of macromolecules and as moisturizers The tetrahydropyrimidines ectoine and hydroxyectoine have gained considerable attention in recent years because they can protect and stabilize enzymes, DNA, membranes, and even whole cells against stress factors such as high salinity, thermal denaturation, desiccation and freezing (da Costa et al., 1998; Galinski, 1989, 1993, 1995; Galinski and Tindall, 1992; Louis et al., 1994; Margesin and Schinner, 2001; Ventosa and Nieto, 1995). The ectoines exert a strong in vitro stabilizing action on many otherwise labile enzymes, thereby increasing the shelf life and activity of enzyme preparations. For that reason they are sometimes termed "molecular chaperones". Their mode of action is still far from being completely understood (see also Section 8.5). The stabilization of proteins by such solutes is both based on a general protective action on the peptide backbone. This effect is relatively independent of the protein in question and depends both on the compatible solute applied and on a variable specific effect, which describes the interaction of individual amino acid side chains with the solvent and the co-solute. While all solutes tested provided some protective effect on enzymes against heating,

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freezing, and drying, the degree of protection depended on the type of solute chosen and the enzyme used as test system (Lippert and Galinski, 1992). Labile enzymes such as lactate dehydrogenase and phosphofructokinase have served as models to investigate their protective effect (Galinski and Lippert, 1991; Göller and Galinski, 2000; Knapp et al., 1999; Lippert and Galinski, 1992). In a study in which the protective effect of different compatible solutes (glycine betaine, trehalose, glycerol, praline, ectoines, and sugars) was compared, hydroxyectoine showed the highest efficacy for the protection of lactate dehydrogenase against freeze-thaw treatment and heat stress, whereas ectoine was the most effective freeze-stabilizing agent for phosphofructokinase (Lippert and Galinski, 1992). Hydroxyectoine was the most effective compound to improve survival of Escherichia coli during freeze-drying, air-drying and storage (Louis et al., 1994). The ectoines also support periplasmic expression of functional recombinant proteins in Escherichia coli under osmotic stress conditions (Barth et al., 2000). Ectoine and its derivatives have found interesting applications as moisturizers in cosmetics for the care of aged, dry, or irritated skin (Motitschke et al., 2000). The Merck Co., Darmstadt, Germany has recently introduced and reported multiple cosmetic benefits for the skin with respect to immune system of the Langerhans cells, formation of heat shock proteins, and protection of membrane integrity (Beyer et al., 2000). Ectoine also reduces the formation of "sunburn cells" in the skin following UV radiation (Bünger et al., 2000). Ectoine and hydroxyectoine are now commercially produced by Bitop (Witten, Germany) (see http://www.bitop.de). Ectoine is industrially produced from Halomonas elongata, and hydroxyectoine from Marinococcus M52. Chemical synthesis of ectoine can easily be achieved, but is not economically competitive with biotechnological production due to the price of the precursors. Chemical synthesis of hydroxyectoine is problematic because of the need to create two chiral centers. The basis for the commercial production is the so-called "bacterial milking" process (Galinski and Sauer, 1998; Galinski et al., 1993; Sauer and Galinski, 1998; Thomas and Galinski, 1996) (Figure 11.1). Bacteria are grown in a fed-batch fermentation process at a salinity of Once a sufficiently high cell density is reached, an osmotic downshock from 100 to is applied. As a result, about 80% of the intracellular ectoine or hydroxyectoine is secreted to the surrounding medium, probably through stretch-activated channels. Crossflow filtration techniques are then used to harvest the excreted solutes. Further purification is a two-step procedure based on cation exchange chromatography and crystallization. After the harvest of the solutes, salt is added to the culture to restore the original salinity, which leads to renewed synthesis of osmotic solutes, and these reach the original level within 10 hours. The "bacterial milking" procedure can then be repeated. A yield of 2 g ectoine per liter of medium per day can thus be obtained. For production of hydroxyectoine, Marinococcus M52 was grown in an exponential fed-batch culture with medium exchange. Cell densities up to 56 g dry weight per liter are employed, in which hydroxyectoine accounts for 13.5% of the dry weight. Hydroxyectoine can be harvested by Soxhlet extraction with methanol (Frings et al., 1995). To improve the growth yield, Krahe et al. (1996) grew Marinococcus M52 in dialysis reactors using cuprophane membranes, and cells were inoculated in an

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inner chamber in a complex medium based on fish peptone to which glucose was added as the main carbon source. After 62 hours a biomass yield of 132 g dry weight was achieved, which contained about 20 % hydroxyectoine.

Advances in fermentation technology and genetic engineering of moderate halophiles may allow large amounts of these compatible solutes to become available for different biotechnological applications. The genes involved in the synthesis of ectoine and its regulation have recently been cloned from different moderately halophilic bacteria (Cánovas et al., 1997; Galinski and Louis, 1999; Louis and Galinski, 1997; Min-Yu et al., 1993), opening the way toward the desired overproduction of the solute. Another intriguing potential application of ectoine as osmotic solute is the expression of the genes governing its production within agricultural crops such as wheat, rice and barley, thus enabling their growth in more saline soils. Transgenic tobacco cells in which the ectoine genes of Halomonas elongata had been cloned

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showed an increased salt tolerance in culture, even though the level of ectoine accumulated by the cells was low. The cells also showed an increased tolerance to hyperosmotic shock (Nakayama et al., 2000).

11.3.4. Production of biopolymers by halophilic Bacteria Bacterial exopolymers may be of great value in enhanced oil recovery processes because of their surfactant activity and bioemulsifying properties. Since the conditions existing in oil deposits are often saline to hypersaline, the use of salt-resistant surfactants may be advantageous. Pfiffner et al. (1986) isolated more than 200 bacterial strains from oil wells, soils in the vicinity of oil wells, effluents of sandstone cores, anaerobic sewage sludge and brine injection water, that produce extracellular polysaccharides anaerobically in a sucrose- mineral salts medium with up to NaCl at 50°C. The predominant cell type was an encapsulated Gram-positive, motile, sporeforming rod. One strain grew well up to NaCl, and produced a polysaccharide with pseudoplastic behavior (i.e., showing a decreased viscosity as the shear rate is increased), that was resistant to shear and thermal degradation. It showed higher viscosities at dilute concentrations and elevated temperatures than commercial polymers such as xanthan gum. The polymer is a charged heteropolysaccharide that contains glucose, arabinose, mannose, ribose, and low levels of allose and glucosamine. Production was optimal under anaerobic conditions in media containing NaCl. Halomonas eurihalina strain F2-7 produces large amounts of an extracellular polyanionic polysaccharide. This polymer is a potent emulsifying agent, which exhibits a pseudoplastic behavior. It is unusual at it reaches high viscosities and forms gels at acid pH. Therefore the polysaccharide (EPS V2-7) may have a range of potential applications in Pharmaceuticals, the food industry, and biodegradation (Béjar et al., 1998; Calvo et al., 1995; Quesada et al., 1993). The polymer displays a remarkable immunomodulating activity in vitro. It enhances the proliferativc effect of human lymphocytes as a response to the presence in blood of the anti-CD3 monoclonal antibody (Pérez-Fernandez et al., 2000). New Halomonas isolates from Morocco also produce interesting exopolysaccharides, and may find applications as emulsifiers with potential in the oil industry (Bouchotroch et al., 2000). An altogether different group of halophilic Bacteria that produces copious amounts of exopolysaccharides with properties of interest in biotechnology are the unicellular cyanobacteria designated as Aphanothece halophytica, Aphanocapsa halophytica, Cyanothece, etc. (De Philippis et al., 1998; Morris et al., 2001; Sudo et al., 1995). Extracellular carbohydrate synthesis is stimulated by nitrogen limitation and to a lesser extent by phosphorus limitation. An examination of the hydrodynamic characteristics of the Aphanothece exopolysaccharide, using viscosimetry and other physical techniques, showed the polymer to be xanthan-likc in its properties. Its relatively poor solubility, however, is a potential disadvantage (Morris et al., 2001).

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11.3.5. Enzymes from halophilic Bacteria A number of extra- and intracellular enzymes from halophilic Bacteria have been characterized, including amylases, nucleases, phosphatases, and proteases (Kamekura, 1986; Onishi, 1972a, 1972b, Onishi and Hidaka, 1978; Onishi and Sonoda, 1979; Onishi et al., 1983). Three intracellular esterases with a high affinity for short chain esters and a membrane bound esterase with affinity for butyric and caproic esters were isolated from a strain of Halomonas elongata growing in Danish bacon curing brines (Hinrichsen et al., 1994). An extracellular amylase-encoding gene amyH from Halomonas meridiana has been cloned. This gene is functional both in Halomonas elongata and in Escherichia coli (Coronado et al., 2000). The thermostable from Bacillus licheniformis could be expressed in Halomonas meridiana and in Halomonas elongata (Coronado et al., 2000). The from the hyperthermophilic Archaeon Pyrococcus woesei has been cloned and expressed in Chromohalobacter salexigens (Frillingos et al., 2000). Members of the genus Halomonas are therefore good candidates to be used as cell factories to produce heterologous extracellular enzymes. Halomonas elongata can express the ice nucleation gene inaZ of Pseudomonas syringae and produce active cellfree ice nuclei (Tegos et al., 2000). As Bacillus species are used for industrial production of many enzymes, the exploration of the potential of the halophilic endospore-formers may also yield new enzymes of interest.

11.4. APPLICATIONS OF HALOPHILIC EUCARYA The halophilic unicellular green alga Dunaliella is grown worldwide as a source of valuable chemicals. The most important product made is but other uses have been explored as well, including the production of glycerol and pyrolysis of Dunaliella biomass for the production of oil.

11.4.1. Commercial production of

from Dunaliella

As documented in Chapter 5.1, certain Dunaliella species (Dunaliella salina, Dunaliella bardawil) produce large amounts of when grown under suitable conditions (high light intensities, high salinity, nutrient limitation). Some strains may contain up to 10% and more of in their dry weight, including a large percentage of the 9-cis isomer. is a valuable chemical, used as a food coloring agent, as pro-vitamin A (retinol), as additive to cosmetics, and as a health food (Borowitzka, 1986). The antioxidant properties of have led to extensive research on its effect in animal model systems. The compound is taken up by the animal body as a source of retinol. Retinol, 9-cis retinol, and 9-cis were found in the liver of rats fed with Dunaliella (Ben-Amotz et al., 1988). There are reports that

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high average serum carotenoid levels are correlated with a lower incidence of cancer and cardiovascular disease. Low doses of the algal product are more potent than high doses of the synthetic all-trans in providing retinol to rats and chicks. The difference may be attributed to the high content of the 9-cis isomer of the natural product. The 9-cis is a more effective quencher of singlet oxygen and other free radicals than the all-trans form. Addition of Dunaliella to the diet of mice inhibited the formation or the progression of development of mammary tumors. The additive had no influence on the endocrine system parameters, showing that the effect was not due to changes in hormone levels. Increase of the homeostatic potential of the host animals as well as the antioxidant function of were suggested as the causes of the effect (Nagasawa et al., 1989, 1991). Dunaliella bardawil carotene promotes growth of normal mammary gland cells in mice but inhibits that of neoplastic cells (Fujii et al., 1993). However, there are also studies that have disputed the beneficial action of diet additives (Hennekens et al., 1996), and may even increase incidence of cancer and cardiovascular disease in smokers and workers exposed to asbestos (Ben-Amotz, 1999; The Alpha-Tocopherol, Beta-Carotene Study Group, 1994). The commercial cultivation of Dunaliella for the production of is one of the success stories of halophile biotechnology (Ben-Amotz, 1999; Ben-Amotz and Avron, 1981, 1983; Borowitzka et al., 1984). The first pilot plant for Dunaliella cultivation for production was established in the USSR in 1966 (Masyuk, 1968). Dunaliella is now grown throughout the world. Different technologies are used, from low-tech extensive cultivation in large lagoons to intensive cultivation at high cell densities under carefully controlled conditions (Ben-Amotz and Avron, 1989). Ben-Amotz (1999) listed the following companies that market Dunaliella carotene: NBT (Nature Beta Technologies, Eilat, a subsidiary of Nikken Sohonsha Co., Gifu, Japan) (intensive culture) (Figure 11.2). N.P.I. (Nutrilite Products Inc.), Calipatria, CA, USA, a subsidiary of Anmay Co., USA) (intensive culture) (http://www.nutrilite.com: last accessed 3 January, 2002). Cyanotech Co., Kailua-Kona, Hawaii, USA (intensive). Western Biotechnology Let., Bayswater, Western Australia, Australia, a subsidiary of Coogee Chemicals Pty., Australia (semi-intensive). Betatene Ltd., Cheltenham, Victoria, Australia, a division of Henkel Co., Germany (extensive) (now Cognis Australia Pty. Ltd.) (http://www.betatene.com.au; last accessed 3 January, 2002). Inner Mongolia Biological Eng. Co., Inner Mongolia, China (semi-intensive). Photo Bioreactors, PBL Ltd., Murcia, Spain (highly intensive, using closed photobioreactors). Tianjin Lantai Biotechnology, Inc., Nankai, Tianjin, in collaboration with the Salt Scientific Research Institute of Light Industry Ministry, P.R. China (intensive).

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In the extensive mode of operations no mixing is applied, and the level of control of the conditions is minimal. The salt concentration is increased as much as possible to minimize loss due to predation by ciliates, amebae or brine shrimp. Growth is slow and production is low, but so are operating costs. Ponds as large as 250 ha are employed in Australia (Borowitzka, 1999). In the semi-intensive mode used in Australia and China, ponds of up to 5 ha are used with partial control and no mixing. Intensive cultivation of Dunaliella is a high-technology operation, in which all parameters are carefully controlled. Raceways of up to surface are used which are slowly mixed by revolving paddle wheels. A pond of 20 cm deep and 3,000 area containing 15 g of will yield 9 kg of A production of about 200 mg can be obtained on yearly average. A highly intensive way of growing Dunaliella in closed bioreactors has also been developed. Even higher cell densities can be achieved while using immobilized cells. Laboratoryscale experiments with Dunaliella bardawil immobilized in calcium alginate beads or in microencapsules gave the highest cell concentrations in the microcapsule system (Joo et al., 2001). Factors to be controlled during the intensive growth of Dunaliella are salinity, nutrient levels, and pH. Uncontrolled autotrophic growth causes the pH to rise to

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values exceeding 10. Addition of is therefore used to control pH. A two-stage growth regime of Dunaliella bardawil for production has been designed. The first stage aims at the production of a high biomass in the presence of excess nutrients. In the second stage the culture is diluted to achieve nitrate limitation, and this leads to a massive induction of carotenogenesis. In the first stage, culture densities up to were obtained with a ratio of 5±1, after three-fold dilution the nitrate-deficient culture grew to a density of with and a ratio of 10±2 (Ben-Amotz, 1995). Predatory ciliates may cause problems in outdoor mass cultures of Dunaliella. Such ciliates grow at salt concentrations as high as and they easily tolerate sudden shifts in salinity. If necessary, chemical control with quinine sulfate at 10 mg can be applied to eliminate ciliates; this treatment did not cause significant damage to the algae (Moreno-Garrido and Cañavate, 2001). Different procedures have been devised for the harvesting of the cells and their further processing toward the purification of the In the extensive process, cells are harvested by flocculation and surface adsorption. The algae flocculate on addition of inorganic or organic flocculants such as alum (aluminum sulfate), ferric chloride, ferric sulfate, lime, or polysaccharides. Alum is extensively used for this purpose, and the is then extracted from the alum. In intensive operations centrifugation is commonly used to harvest the cells. Continuous flow stainless steel batch type centrifuges or self-discharging de-sludge type centrifuges are used that typically can be operated at flow rates of at 15,000 rpm or at 5,000 rpm. The water released in the centrifuge can be recycled after purification by oxidative treatment of the organic load and filtration. Biological treatment of the waste water may be optimized by supplementation with and and complete removal of residual glycerol can then be achieved after two days (Santos et al., 2001). The cell pastes obtained contain 20-40% cell material dry weight, dependent on the type of centrifuge used. Another method of harvesting, termed hydrophobic binding, is used by Betatene Ltd., Australia. It is based on the fact that above 4 M salts Dunaliella converts to cysts which have a hydrophobic surface coat. These coated cells readily adsorb to hydrophobic surfaces. The product is marketed as dry Dunaliella cells or as purified To extract the carotene, edible oil is used, sometimes accompanied by the use of organic solvents, liquid or crystallization. Purified is sold mostly in vegetable oil in concentrations between 1 and 20%. It is generally accompanied by other pigments present in the alga such as lutein, neoxanthin, zeaxanthin, violaxanthin, and cryptoxanthin, and these may amount to about 15% of the concentration. Powdered or microencapsulated is sold as health food or food supplement. Antioxidants may be added during spray-drying of Dunaliella to inhibit decay of by isomerization or oxidation, and especially to counteract photodegradative loss of the 9-cis form, which is more sensitive to heat and to oxygen than the all-trans form (Orset et al., 1999).

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11.4.2. Commercial production of glycerol from Dunaliella Another product that can be made from Dunaliella biomass is glycerol. Glycerol is accumulated in molar concentrations within the cells to serve as osmotic solute (see Section 8.4). A process for the commercial production of glycerol from Dunaliella was devised in 1981 (Chen and Chi, 1981). It was claimed to be economically feasible and to favorably compete with traditional procedures for glycerol production (as a byproduct from animal and vegetable oils or by hot chlorination of propylene). The harvesting was based on the use of a continuous screw press in which the cells are disrupted and intracellular fluid is squeezed out. The glycerol-rich liquid is then fed into an evaporator at 0.39 psi and 32 °C to yield a liquid of 90% glycerol, which is then further refined by distillation at 0.002 psi and 130 °C to a purity of 99% (Figure 11.3) (Chen and Chi, 1981). The NaCl is recycled by crystallization in an evaporation pond. In another process an algal paste is formed by settling and centrifugation. This paste is then suspended in water to induce osmotic shock to lyse the cells. The glycerol-rich liquid is subsequently separated by a filter press or centrifuge (Ben-Amotz, 1980; BenAmotz and Avron, 1981). In spite of the great potential of the process, glycerol production by Dunaliella is still not truly economically feasible.

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Other products from Dunaliella

Dunaliella protein has a similar composition to soy bean meal, but has a higher lysine content (Galinski and Tindall, 1992). It is therefore suitable for use as feedstock in mariculture (crab, shrimp, shellfish) and for livestock such as chickens. As a result of the absence of cell walls the cells are digestible. Catalytic pyrolysis of Dunaliella cell material al 200-240 °C produces an oil-like substance. The overall process is exothermic, and thus most of the thermal energy needed to initiate the reaction may be regained. A conversion of 22.3% of the algal protein was obtained at 350 °C to a product that contains 69.9% carbon. 7.7% hydrogen, and 7.3% nitrogen. Addition of KCl, and increased the yield to 27% with 75.5% carbon, 8.5% hydrogen, and 6.8% nitrogen (Goldman et al., 1981a, 1981b). At an estimated price of about $ 40 per barrel (Ginzburg, 1991) this process is not economically feasible at present. Dunaliella is also being used as an additive in cosmetic anti-wrinkle skin creams in combination with Dead Sea minerals (Ma'or et al., 2000). The algal cell preparation allegedly binds and ions. However, the authors state that "the low biosorption of calcium and magnesium obtained from the algal biomass, and the tendency to a low release of minerals at the normal pH of human skin (5.5) led to the conclusion that the advantage of these algae as a mineral vehicle for Ca and Mg is limited."

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SECTION 3

HYPERSALINE ENVIRONMENTS AND THEIR BIOTA

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INTRODUCTION

The following chapters discuss the ecology of the halophilic microorganisms whose properties have been presented in Section 2. The ecological approach, which was so prominently present in the early studies (see Chapter 1), has often been neglected in the past decades in favor of the in-depth physiological, biological and genetic studies, in which a few selected species are being used as models. Such studies therefore cover only a limited part of the tremendous diversity among the halophiles. It is also becoming increasingly clear that the list of halophiles that have been isolated and described (Chapter 2) encompasses only a small fraction of the true diversity to be found in hypersaline environments in nature. There has, however, been a revival in the interest in the ecology of hypersaline environments in the past decade (Norton, 1992; Oren, 1994, 1999). New techniques derived from molecular biology are now being applied to the study of salt lakes, salterns, and other high-salt ecosystems. These new approaches have broadened our understanding of the functioning of these ecosystems, and they have also shown that the kinds of halophiles dominant in nature are not necessarily those known from laboratory cultures. Hypersaline environments are no different from other ecosystems in this respect. There have also been a number of cases in which 16S rRNA gene sequences attributed to halophilic Archaea of the family Halobacteriaceae have been detected in environments where they were little expected (Chapter 17). Hypersaline environments can be divided into two groups: thalassohaline and athalassohaline. Thalassohaline environments originated by evaporation of seawater, and their ionic composition reflects that of seawater: is the dominant cation, the main anion, followed by and the pH is neutral to slightly alkaline. Hypersaline marine lagoons belong to this class of environments, as do solar saltern evaporation ponds. However, when seawater is concentrated by evaporation, different minerals precipitate long before NaCl saturation is reached. These include (as calcite or aragonite) and (gypsum). Changes in the ionic composition of the brine therefore do occur as evaporation proceeds. The salt composition of the waters of the Great Salt Lake, Utah, resembles that of seawater, in spite of the fact that the lake has been detached from the world ocean for thousands of years. Its properties can therefore still be considered thalassohaline. Many other natural brines have an ionic composition that differs greatly from that of sea watert, and these are termed athalassohaline environments. Alkaline soda lakes are highly depleted in the divalent cations and contain high concentrations of carbonate/bica