Advances in Microbial Physiology Volume 02

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

MICROBIAL PHYSIOLOGY Edited by A. H. ROSE Department of Microbiology University of Newcastle upon Tyne England

and

J. F. WILKINSON Department of General Microbiology University of Edinburgh Scotland

VOLUME 2

1968

ACADEMIC PRESS

LONDON and NEW YORK

2 0 JUN ?68

PRESS INC. (LONDON) LTD. BERKELEY SQUARE HOUSE BERKELEY SQUARE LONDON, W.1

U.S. Edition published by ACADEMIC PRESS INC. 111 FIFTH AVENUE NEW YORE, NEW YORE

10003

Copyright 0 1968 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the Dublishers Library of *Congress Catalog Card Number : 67-19850

PRINTED IN GREAT BRITAIN BY SPOTTISWOODE, BALLANTYNE AND CO. LTD LONDON AND COLCHESTER

Contributors to Volume 2 K. BERAN,Department of Technical Microbiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.

W. X. KELLEY, Department of Microbiology, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

JUNE LASCELLES, Bacteriology Department, University of California, Los Angeks, California 90024, U.S.A.

B. E.B.MOSELEY, Biology Division, Oak Ridge National Laboratory, Tennessee, U.S.A.

M. H. RICHMOND, Department of Moleculur Biology, University of Edinburgh, West Mains Road, Edinburgh 9, Xcotland.

M. SCRBECHTER,Department of Microbiology, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

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Contents Contributors t o Volume 2

.

V

The Bacterial Photosynthetic Apparatus. JUNE LASCELLES I. An Introduction to the Photosynthetic Bacteria and their Pigments A. The Photosynthetic Pigments . B. Environmental Effects on Pigment Synthesis . 11. The Structure and Location of Chromatophore Material . A. Discovery and Definition of Chromatophores B. Electron Microscopy of Cells . C. Chromatophore Material and Cell Membrane D. Chlorophyll and Membrane Content . E. Peripheral and Internal Membrane as Sites for Pigments F. The Vesicles of the Green Bacteria . G . Conclusions 111. Isolation and Composition of Purified Chromatophores . A. Isolation Procedures . B. Composition C. Fractionation of Chromatophores . D. Immunological Reactivity IV. Formation of the Photosynthetic Apparatus . A. Differences between Members of the Athiorhodaceae Grown Photosynthetically and Aerobically B. Adaptive Synthesis of the Photosynthetic Apparatus . C. Chlorophyll Synthesis D. Regulation of Chlorophyll Synthesis . References

.

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.

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1 3 6 8 8 8 12 14 16 18 19 19 19 21 24 26 27 27 29 31 34 39

The Plasmids of Staphylococcus aureus and their Relation to Other Extrachromosomal Elements in Bacteria, M. H. RICHMOND I . Introduction . A. Historical . B. Curing C. Stability . D. The Plasmid Location of Penicillinase Genes

.

.

. . . . .

43 43

44 45 48

...

CONTENTS

Vlll

.

11. Types of Staphylococcal Plasmid A. Plasmid-Borne Markers . B. Classification of Plasmids . C. Distribution and Abundance of Plasmid Types . D. Plasmid Nomenclature . 111. Transduction of Plasmids . A. Preparation of Transduciilg Phage B. Transductants Formed in Negative Recipients . C. Transductants Formed in Plasmid-Carrying Recipients . D. Compatibility E. The Role of Attachment in Recombination and Diploid Formation . F. Chromosomal Attachment of Plasmid Markers . G. ThePlasmidMap H. Size of the Penicillinase Plasmids . IV. A Comparison of Penicillinase Plasmids with Other Extrachromosomal Elements in Bacteria V. Acknowledgements . References

51 51 55 58 59 60 60 60 61 71

The “Life Cycle” of Bacterial Ribosomes. WILLIAM S. KELLEY

and

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.

.

.

75 78 80 83 84 86 87

MOSELIO SCHAECHTER

I. Introduction . . A. Problems Particular to the Study of a Ribosomal “Life . Cycle” . . B. Scope of the Review . C. Studies of Ribosome Structure and Composition . . * D. Cellular Localization of Bacterial Ribosomes . . 11. The Role of Ribosomes in the Control of RNA Synthesis . 111. The Production of Ribosomal RNA . . IV. The Production of Ribosomal Proteins . . V. The Assembly of Ribosomal Subunits . . A. Studies on Cells in Balanced Growth . B. Studies of Metabolically Inhibited Cells . C. I n Vitro Experiments on Ribosome Assembly . D. Conclusions . VI. The Formation of Functional Ribosomes . VII. The Participation of Ribosomes in Protein Synthesis . . . VIII. The Release of Ribosomes from Messenger RNA . IX. Conclusions . X. Acknowledgements . . References

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.

. .

89 90 91 91 97 99 101 107 112 112 115 124 126 126 131 133 136 137 137

ix

CONTENTS

Budding of Yeast Cells, Their Scars and Ageing. I . Introduction .

K. BERAN

. 11. Composition and Structure of the Cell Wall of Yeast A. Macromolecular Wall Components . B. Structure of the Wall . 111. Mechanism of the Primulin-Induced Fluorescence . IV. Budding of Yeast Cells and its Mechanism A. Budding of Yeast Cells as Observed in the Optical and Electron Microscopes . B. Examination of Buds and Scars Using the Fluorescence Microscope . C. Mechanism of Budding . D. Some Further Perspectives . . V. Ageing of Cells and Age Distribution in a Population A. Maximum Reproductive Capacity of an Individual Cell B. Age Distribution in a Population . References

.

.

143 145 145 147 148 148 148 149 154 161 162 162 164 169

The Repair of Damaged DNA in Irradiated Bacteria. B. E. B. MOSELEY

.

I . Introduction 11. Evidence that DNA is an Important Target in Radiation Inactivation . 111. Radiation-Induced Chemical and Physical Changes in DNA . A. Changes in DNA Caused by Ultraviolet Irradiation . B. Changes in DNA Caused by Ionizing Irradiation . IV. The Repair of Damaged DNA A. Photoreactivation . B. DarkRepair . V. Summary . References

.

174 176 176 181 183 183 187 191 192

195

Author Index

Subject Index

173

.

203

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=Photosynthetic Apparatus JUNE

LASCELLES

Bacteriology Department, University of Californ$a, Los Angeles, California 90024, U.S.A.

.

1 I. An Introduction to the Photosynthetic Bacteria and their Pigments A. The Photosynthetic Pigments . 3 B. Environmental Effects on Pigment Synthesis . . 6 11. The Structure and Location of Chromatophore Material . . 8 A. Discovery and Definition of Chromatophortttes . . 8 B. Electron Microscopy of Cells . . 8 C. Chromatophore Material and Cell Membrane . . I2 D. Chlorophyll and Membrane Content . . 14 . 16 E. Peripheral and Internal Membrane as Sites for Pigments . F. The Vesicles of the Green Bacteria. . . 18 G. Conclusions . . 19 111. Isolation and Composition of P u r s e d Chromatophores . . 19 A. Isolation Procedures . . 19 B. Composition . . 21 C. Fractionation of Chromatophores . . 24 D. Immunological Reactivity . . 26 IV. Formation of the Photosynthetic Apparatus . . 27 A. Differences between Members of the Athiorhodaceae Grown Photosynthetically and Aerobically . . 27 B. Adaptive Synthesis of the Photosynthetic Apparatus . . 29 C. Chlorophyll Synthesis . 31 D. Regulation of ChIorophyIl Synthesis . . 34 References . . 39

.

1. An Introduction to the Photosynthetic Bacteria and their Pigments

The unique physiological characteristic of the photosynthetic bacteria is their ability to grow anaerobically in the light, a property conferred upon them by their photosynthetic pigment system. Unlike green plant photosynthesis oxygen is not evolved in the bacterial process, and, connected with this, they require an exogenous reductant (Stanier, 1961 ;van Niel, 1962; Vernon, 1964; Gest, 1966). The different genera of photosynthetic bacteria characteristically use either reduced inorganic sulphur compounds, hydrogen, or organic substrates as reductant ;they 1

TABLE1. Some Characteristics of Photosynthetic Bacteria Photosynthetic growth supported by A

Group Green sulphurbacteria Thiorhodaceae Athiorhodaceae

Representative members

Chlorobium spp. Chloropseudomonas ethylicum Chromatium spp. Rhodopseudomonas spp. Rhodospirillum spp.

Sulphur compounds

+ c02

Organic acids alone

Ability to grow aerobically (dark)

Yes

NO

NO

>

Major form of chlorophyll Chlorobium chlorophyll 660 or 650

a 24

M

:: M

u)

Yes No

Yes Yes

NO

Yes (some species)

Bacteriochlorophylla Bacteriochlorophylla or b"

Bacteriochlorophyll 6 is found in a new species, Rps. viridis (Eimhjellenet al., 1963; Drews and Giesbrecht, 1965, 1966).

3

THE BACTERIAL PHOTOSYNTHETIC BPPaRATUS

also vary with respect to their ability to use carbon dioxide as sole carbon source (Table 1). The overall process of bacterial photosynthesis may be represented by Fig. 1. There is strong experimental evidence for this scheme as a general outline though the precise sequence of events is yet to be established (Vernonand Ke, 1966; Gest, 1966).The scheme in Fig. 1 fits observations made largely with Rhodospirillurn rubrum, and variations in detail are likely in other organisms.

Cytochromoid?.

I F

-

Ubiquinone

1

i

Ferredoxin

_____f

I

Flavoprotein

ADP+Pi

NAD

FIG.1. Possible pathway of light-induced electron flow in Rhodospirillurn rubrum.

It has been well established that particulate preparations from the photosynthetic bacteria catalyse anaerobically: (1) light-dependent synthesis of ATP in the absence of an external reducing agent (photophosphorylation), and (2) light-dependent formation of reduced nicotinamide nucleotides in the presence of weak reductants such as succinate or certain reduced dyes (photoreduction). By analogy with the photosynthetic apparatus of the plant chloroplast the bacterial system might be expected to be associated with some type of organized structure and these structures are the concern of this review.”

PIGMENTS A. THE PHOTOSYNTHETIC 1. Chemistry In common with other photosynthetic forms of life the bacteria have both carotenoids and chlorophylls. The variety of carotenoids among the

* The term chromatophorewill be used for the pigmented particles isolated from cell-free extracts. When referring to the putative photosynthetic structure in intact cells the less precise terms “chromatophore material” or “photosynthetic apparatus” will be used.

4 JUNE LASCELLES different species is great (Jensen, 1963; Schmidt et al., 1965). Their probable function is to harvest light at wavelengths which are not absorbed by chlorophyll and also to protect cells from photodynamic oxidation reactions (Stanier, 1960; Dworkin, 1958).

Fk

II

II

CH=CHz

H~C

H3C

N-

H3C +

I CHz I

7 .

H3cx$cH3 CH2

COOH

CH2- CH3

\

c:~cH3 CHz

p< CHz

CH2

CH2

I CH2 I

I

H 'Z

I

COOH Protoporphyrin

HC-C=O

I

COOCHs

COOH Mg-vinylpheoporphyrin

c(5

$533 I

-CHI

p'

I

COO.CH3

COO.CzoHsg Bacteriochlorophyll

FIG.2. Structures of protoporphyrin, Mg-vinylpheopoqhyrh a5 (protochlorophyllide a ) and bacteriochlorophyll.

So far, three types of chlorophyll have been recognized in photosynthetic bacteria (Fig. 2) of which bacteriochlorophyll a is the most widely distributed (Jensen et aZ., 1964; Allen, 1966).Uhlorobium chlorophylls 660 and 660, a nomenclature based on their red absorption maxima in ether, are unique to the green sulphur-bacteria (Holt, 1966).Significant

THE BAUTERLBL PHOTOSYNTRETIC APPARATUS

5

differences in their structure from that of bacteriochlorophyll a are : (a) the Chlorobium pigments have a dihydro- rather than a tetrahydroporphyrin ring structure ; consequently, the red maxima are shifted towards the blue; (b) they lack the -COOCH, grouping on the cyclopentanone ring ; ( c )they are esterifiedwith farnesol rather than with phytol ; (d) the 660 pigment has an alkyl substituent on the 6-methene carbon atom of the porphyrin ring. A recent development is the recognition of bacteriochlorophyll b in a new species, Rhodopseudomonas viridis (Eimhjellen et al., 1963; Drews and Giesbrecht, 1965, 1966). This pigment is characterized by a red maximum in acetone at 795 mp, but its structure is yet to be determined. Its discovery should alert workers to careful scrutiny of new isolates for yet more forms of chlorophyll. 2. Spectrum of Chlorophylls in vivo ; Reaction-Centre Chlorophyll

The early spectroscopic observations of Wassink and of French (see Rabinowitch, 1951)indicated that the bacterial chlorophyllswere bound

Wavelength (mp)

FIG.3. Spectrum of bacteriochlorophyll from R h o d o p s e u d m m qvheroides in &o (-) and in methanol (---).

6

JUNE LASCELLES

in vivo in the form of macromolecular complexes. The red absorption maxima of all forms of chlorophyll found so far in bacteria exhibit a marked shift of at least 100 mp towards the blue when extracted into organic solvents. Such a change can be reasonably attributed to a release of the pigment from a bound form. The in vivo spectra also show several maxima in the red whereas the extracted pigment shows only one red peak (Olson and Stanton, 1966; Fig. 3). The multiple peaks in the in vivo spectra suggest that chlorophyll molecules are in association with different complexes, and the physiological significance of these is now being clarified (Clayton, 1966). The work with preparations from Rhodopseudomoms spheroides suggests that the bulk of the chlorophyll, associated with a complex absorbing at 850 mp (P-850),functions merely to harvest light. The complex absorbing at 870 mp (P-870) accounts for only about 5% of the total pigment but appears to represent the photosynthetic reaction centre. This pigment, but not P-850, is bleached (i.e. oxidized) reversibly upon illumination ;this phenomenon is shown most clearly in preparations which have been treated by methods which preferentially destroy P-850 (Clayton, 1963,1966). The isolation of mutant strains, which have the normal complement of P-850 yet cannot grow photosynthetically since they lack P-870, provides the most convincing evidence for the role of P-870 as the photosynthetic centre (Sistrom and Clayton, 1964).A complex absorbing at 800 mp is closely associated with P-870 (Clayton and Sistrom, 1966). There is evidence for reaction-centre chlorophyll in other photosynthetic bacteria. I n the green sulphurbacteria it seems that bacteriochlorophyll a is the reactive form, though the bulk of the pigment of the cell is Chlorobium chlorophyll (Olson and Romano, 1962; Olson, 1966).

EFFECTS ON PIGMENT SYNTHESIS B. ENVIRONMENTAL Photosynthetic bacteria, in common with plants, have the capacity to regulate chlorophyll synthesis in response to the demands of the environment. I n the bacteria the key factors are light intensity and, in the case of the Athiorhodaceae, oxygen pressure (see Tables 3, p. 15, and 6, p. 28). The elegant studies of Cohen-Bazire et al. (1957) and of Sistrom (1962b) showed that cultures of Rps. spheroides and Rsp. rubrum growing anaerobically respond to an increase or to a decrease in light intensity by suppression or stimulation respectively of synthesis of the photosynthetic pigments. Consequently, cells grown a t high intensities contain less chlorophyll and carotenoid per unit of protein than do those grown with low illumination. Repression of pigment synthesis by oxygen was also observed when this gas was introduced into cultures growing under continuous illumination. Oxygen repression is critically dependent

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

FIG.4. Electron micrograph of section of photosynthetically grown Rhodospirillum rubrum (Vatter and Wolfe, 1958). CW = cell wall; CM= cytoplasmic membrane; Ch = chromatophore. Inset: isolated chromatophores; the line indicates 1 p. Reproduced by kind permission of the authors and the editors of the Journal of Bacteriology.

8

JUNZ LASCELLES

on the oxygen concentration and occurs only under high aeration. Many of the Athiorhodaceae form chlorophylland carotenoids when the oxygen concentration is low and, under such conditions, the specific pigment content may reach that of photosynthetic cultures (Lascelles, 1959 ; 1960a). Formation of the pigments in response to decreased oxygen pressure occurs in the dark; a light-dependent step is therefore not obligatory (or may not even occur) in chlorophyll synthesis by the bacteria.

11. The Structure and Location of Chromatophore Material A. DISCOVERY AND DEFINITION OF CHROMATOPHORES The term LLchromatophore” was coined by Schachman et al. (1952)for the relatively homogeneous, pigmented particles which were prepared by differential centrifugation of extracts of Rsp. rubrum. They contained the entire complement of photosynthetic pigments and were about 600 A in diameter. Particles of this size (about 1908)were not found in extracts of the organism grown aerobically and therefore devoid of photosynthetic pigments. It appeared that the chromatophores represented a specialized structure to house the photosynthetic pigments. Later studies by Frenkel (1956, 1958) showed that chromatophores isolated from Rsp. rubrum catalysed photophosphorylation and the photoreduction of NAD in the presence of succinate, as well as other photoreduction reactions, indicating that they represented the bacterial photosynthetic apparatus.

MICROSCOPY OF CELLS B. ELECTRON Electron microscopy of sectioned cells of photosynthetic bacteria showed them to contain structures similar in size and appearance to isolated chromatophores (Vatter and Wolfe, 1958). Rsp. rubrum and Rps. spheroides exhibited discrete membrane-bound vesicles (in diameter 500-1000 A and 400-800 A respectively) which appeared to be dispersed throughout the cytoplasm (Fig. 4). The association of the vesicles with the photosynthetic pigments was suggested by their absence from non-pigmented cells of Rsp. rubrum grown aerobically. It has since been firmly established with many organisms that the number and extent of the vesicular structures is directly related to the pigment content of the cells (Cohen-Bazire and Kunisawa, 1963; Drews and Giesbrecht, 1965; Gibbs et al., 1965; Holt and Marr, 1965c; Holt et al., 1966b). The vesicular structures found in Rsp. rubrum are not typical of all photosynthetic bacteria (Table 2 ) . Some exhibit stacks of paired

9

T H E BACTERIAL PHOTOSYNTHETIC APPARATUS

TABLE2. Appearance of Chromatophore Material in Electron Micrographs of Cell Sections Organism

Chlorobium thiosulphatophilum Chlorobium limicola Chloropseudomonas ethylicum

Appearance of structure

Oblong vesicles arranged around periphery, immediately under the cytoplasmic membrane ; 1000-1500 i% long, 300-400 i% wide Membrane-bound vesicles throughout cytoplasm

Chromatiuin strain D Chromatium okenii Thiospirillumjenseni Thiopedia sp. Vesicles and large lamellar structure Thiocapsa sp. Membrane-bound vesicles throughout Rhodospirillum rubrum the cytoplasm, 400-1000 d diameter Rhodopseudomoms spheroides Rhodospirillum molischianum Discrete lamellar structures at periRhodospirillum f u l v u m phery Rhodospirillum photometricurn Extensive lamellar structure disposed Rhodopseudomoms palustris around periphery Rhodopseudomonas viridis Rhodomicrobium vannielii

Reference 1 1 2

3 3 3 3 3 3,4, 5 4, 6 7,899 10 10 10 11 12

References: (1) Cohen-Bazire et al. (1964); (2) Holt et al. (1966a); (3) Cohen-Bazire (1963); (4) Vatter and Wolfe (1958); (5) Hickman and Frenkel (1959, 1965b); (6) Drews and Giesbrecht (1963); (7) Giesbrecht and Drews (1962); (8) Gibbs et al. (1965); (9) Hickman and Frenkel (1965a); (10) Cohen-Bazire and Sistrom (1966); (11) Drews and Giesbrecht, 1965; Giesbrecht and Drews, 1966; (12) Vatter et al. (1959).

lamellae, similar to the structures found in blue-green algae, and arranged variously according to the organism. I n Rhodomicrobium vannielii, for instance, the lamellae are arranged concentrically around the periphery of the cell (Vatter et al,, 1959) whereas in Rhodospirillurn rnolischianum (Fig.6#) the lamellae appear as discrete discs a t intervals around the periphery (Drews, 1960). Perhaps the most remarkable structures are he large oblong vesicles found in the green sulphurbacteria (Fig. ; Cohen Bazire et al., 1964; Holt et al., 1966a).These lie immediately under the peripheral membrane. Conclusive proof that these various structures are the site of the photosynthetic pigments is difficult to obtain in the absence of techniques for locating the pigments in cell sections. Indirect evidence has been provided in many cases by observing a correlation between the number and extent of the structures with the pigment content of the cells. Also, the appearance of pigmented fractions isolated from disrupted cells has been shown with some organisms to resemble the structures found in cell sections

a

FIG.5. Electron micrograph of a section of Chlorobium thiosulphatophilum showing the complex cell wall (w) with its rod-shaped extensions (ex), the cell membrane (m) and ellipsoidal vesicles (cv) adjacent to but distinct from the peripheral membrane (vm). Two large mesosomal elemcnts (M) and a granule of polymetaphosphate (p)are also visible. From Cohen-Bazireet at. (1964). Reproduced by kind permission of the authors, and the editors of the Journwl of Cell Biology.

FIG.6. Electron micrograph of a section of Rhodospirillurn rnolischianurnshowing lamellar invaginations o f the peripheral membrane. As the culture ages, the lamellae increase in number and become closely associated into groups ( a t D'). The space (G) within the folds then increases and separates adjacent sides of the folds. The distinct lamellae seen at D average about 160 A in width. From Hickman and Frenkel (1965a). Reproduced by kind permission of the authors, and the editors o f the Journal of Cell Biology.

12

JUNE LASCELLES

(Cohen-Bazire and Sistrom, 1966). Such a correspondence is not always easy to show, possibly because of the fragility of the structures. For instance, isolated chromatophores from Rsp. molischianum appear as welldefined particles which may be subunits of the intracellular lamellar structures (Hickman and Frenkel, 1965a). Recently, Giesbrecht and Drews ( 1966) have succeeded in isolating chromatophores from Rps. viridis in which the lamellar structure is preserved.

C. CHROMATOPHORE MATERIAL AND CELL MEMBRANE Assuming that the structures described so far represent chromatophore material, a problem which arises is whether they exist as discrete entities in the cytoplasm (analogousto chloroplasts and mitochondria) or whether they are an integral part of a continuous membrane system. If the latter is true, the preparations of isolated chromatophores arise presumably by comminution of the large membrane structure into quite homogeneous particles. The evidence is strong that chromatophores from many organisms exist in the cell as part of a membranous continuum. However, there is still controversy over whether or not discrete chromatophores can arise in vivo from such a continuum (Stanier, 1963; Hickman and Frenkel, 1965b; Gibson, 1966). The derivation of chromatophores from large intracellular structures was suggested some years ago by Newton and Newton (1957). Sonic treatment of Chromatiurn for varying periods of time showed that pigmented particles were not released immediately upon cell rupture but appeared at a relatively slow rate. Also, the chlorophyll appearing during the early stages of disruption was distributed equally between small and large particles, but with more prolonged treatment, it was found almost exclusively in the small particle fraction. These observations are consistent with comminution of large membrane structures into small pieces. The method of controlled sonic treatment for demonstrating the locus of an intracellular component (first used by Marr and Cota-Robles, 1957) has since been applied to other photosynthetic bacteria with similar results (Holt and Marr, 1965a; Holt et al., 1966a). The association of chromatophore material with membrane was shown more directly with lysed preparations of Rsp. rubrurnz(Tutt1e and Gest, 1959). The ghost fraction from osmotically lysed spheroplasts, prepared by lysozyme treatment, contained the bulk of the photosynthetic pigments and had photophosphorylation activity. The significanceof this early work was not adequately recognized until later developments in the field of electron microscopy, which suggested that chromatophore material is associated with internal membrane, arising by invagination of the peripheral membrane. Evidence for this

T H E BACTERIAL PHOTOSYNTHETIC APPARATUS

13

was first obtained with osmotically lysed Rsp. molischianum, in which the lamellar structures could be seen to arise by invagination and folding of the peripheral membrane (Giesbrecht and Drews, 1962). A similar origin for the vesicular structures of Rsp. rubrum and Rps. spheroides has been strongly indicated by many observations which may be summarized as follows. (1) The few vesicles to be seen in cells of low pigment content are located exclusively a t the periphery (Cohen-Bazire and Kunisawa, 1963). As the pigment concentration increases, the vesicles become more numerous and extend more deeply into the cytoplasm. I n these studies with Rsp. rubrum, the chlorophyll content was varied by growth a t different light intensities under anaerobic conditions. Progressive extension of the vesicles from the periphery into the cell interior can also be seen in cells from cultures sampled a t various stages of growth at fixed light intensity (Drews and Giesbrecht, 1965; Hickman and Frenkel, 1965b).As the cell population increases, the specific chlorophyll content increases and the solely peripheral distribution of the vesicles gives way to deeper penetration into the cytoplasm. (2) The dimensions and structure of the membrane surrounding the vesicles are similar to the peripheral membrane, both being defined as two dense parallel lines with a total thickness in Rsp. rubrum of 75-85 d (Cohen-Bazire and Kunisawa, 1963; Holt and Marr, 1965a). (3) Continuity of the peripheral membrane and vesicles has been observed (Fig. 7 ; Cohen-Bazire and Kunisawa, 1963; Drews and Giesbrecht, 1965; Hickman and Frenkel, 19658). Such continuity is most clearly seen in cells of low pigment content which show a peripheral distribution of vesicles ; as the pigment content increases, the peripheral membrane exhibits more numerous invaginations, similar in appearance

FIG.7. Electron micrograph of section of Rhodopseudomoms spheroides showing chromatophores arising by invagination of the peripheral membrane (Drews and Giesbrecht, 1965). Reproduced by kind permission of the authors and of the editors of Zentralblatt fur Bakteriologie.

14

JUNE LASCELLES

to the vesicles. Such progressive invagination of the peripheral membrane may be followed in non-pigmented cells (grown with high aeration) as they form pigment upon transfer to semi-anaerobic-dark or anaerobiclight conditions (Cohen-Bazire and Kunisawa, 1963; Giesbrecht and Drews, 1963). Interconnections between vesicles have also been observed (Giesbrecht and Drews, 1963; Holt and Marr, 1965a). Studies with osmotically lysed Rsp. rubrurn have also provided evidence of continuity between an internal membrane system with a vesicular appearance and the peripheral membrane (Boatman, 1964; Holt and Marr, 1965a). (4) Impressive evidence that the internal membrane seen by electron microscopy gives rise to isolated chromatophores has been provided by Holt and Marr (1965b, c). These experiments are discussed in Section I1E (Pa 17).

D. CHLOROPHYLLAND MEMBRANECONTENT Electron microscopy shows a considerable amount of internal membrane in photosynthetic bacteria of high pigment content. This raises the question of how much chromatophore material is represented by the membranes, and this question cannot be answered in the absence of a method for locating chlorophyll by electron microscopy. Various indirect approaches have, however, been used to determine quantitatively the amount of chromatophore material. Gibbs et al. (1965)calculated the volume of membrane by measurement of electron micrographs of sectioned cells of Rsp. rnolischianum, containing varying amounts of pigment. The chlorophyll content was correlated with the amount of total membrane (peripheral and internal) but a constant ratio between pigment and internal membrane was not observed. Direct analysis was also made of the amount of chromatophore protein isolated from cells of differing pigment content, with results which did not accord with those of electron microscopy. I n experiments where recovery of chlorophyll and protein was almost quantitative, the amount of protein in the purified chromatophore fraction did not match the amount of pigment. Thus, in cells with a four-fold difference in chlorophyll content, the amount of protein in the purified chromatophore fraction varied by only 1.2-fold (65 and 54% respectively of the total cell protein). This lack of correlation between chlorophyll concentration in cells and the quantity of isolated chromatophore protein has been a frequent observation (Cohen-Bazire and Kunisawa, 1960; Drews and Giesbrecht, 1965; Bull and Lascelles, 1963). Lipid phosphorus has been used as an index of membrane in photosynthetic bacteria; in these organisms, as in other bacteria, the

15 phospholipids are located predominantly in the membrane fraction (Lascelles and SzilBgyi, 1965). The lipid phosphorus content of Rps. spheroides is higher in pigmented cells than in those grown with high aeration and consequently virtually devoid of photosynthetic pigments (157 and 90 mpmoles lipid phosphorus per mg. protein, respectively). The association of chlorophyll synthesis with an enrichment in phospholipid was also suggested by kinetic experiments which showed a THE BACTERIAL PHOTOSYNTHETIC APPARATUS

TABLE3. Chlorophyll Concentration in Cells and Pursed Chromatophores ~

Organism

Rhodospirillum rubrum

Rhodopseudomonas spheroides Rhodospirillum molischianum Chloropseudomonas ethylicum

~

~

Chlorophyll concentraLight tion (mpmoles/mg. n intensity protein) i during >-’-r growth Crude Purified (foot cell chromatocandles) extract phores 200 2000 18.5 1020 2040 1 100 2400 6000 18.5 1020 2040 70 2000 10 100 1000 10000

29.8 9.9 24 13 9 39.5 11 6 4 27 20 5 111 26 475 178 111 92

55 22.5

}

~~

Reference Cohen-Bazire and Kunizawa (1960) Drews (1963)

17 82

Holt and Mam (1965~) 42

Drews (1963) 9 159 45

I

212 130

J

Gibbs et al. (1965) Holt et al. (1966b)

All organisms were grown photosynthetically at the light intensity shown

parallel increase in pigment and lipid phosphorus in cells transferred from high to low aeration. However, constant proportionality between the concentration of phospholipid and that of chlorophyll has not been found in analyses of photosynthetic bacteria of varying pigment content, with the exception of Rhodopseudomonas palustris (Cohen-Bazire and Sistrom, 1966). I n Rsp. rubrum, for instance, a six-fold difference in chlorophyll concentration was reflected by only a 1-9-fold increase in phospholipid. 3

16

J U N E LASCELLES

Analyses of the specific chlorophyll content of chromatophores prepared from cells containing large and small amounts of pigment has provided conflicting results. If there is proportionality between membrane and pigment, isolated chromatophores should have the same specific chlorophyll content, irrespective of the chlorophyll concentration in the cells. This expectation has not been generally realized; the chlorophyll-to-pigment ratios of most chromatophore preparations reflect the specific pigment content of the cells from which they were isolated (Table 3). The highly purified chromatophores of Holt and Marr (1965b), however, did show a constant ratio in all preparations except those from cells grown at very high light intensity, where destruction of pigment may have occurred (Table 3). On the whole, the analytical results suggest that a considerable increase in pigment can be accommodated without a substantial increase in the amount of the membrane matrix of the chromatophore material. This is not substantiated by electron microscopy which shows the membranes of photosynthetic bacteria to be of constant thickness (70-80 8) in cells of low and high pigment content. A change of chlorophyll concentration of the magnitude observed experimentally could not occur without an increase in the thickness of the membrane (Holt and Marr, 1965~).

E. PERIPHERAL AND INTERNAL MEMBRANE AS SITESFOR PIGMENTS The discrepanciesbetween the analytical data and electron microscopy might stem from an uneven distribution of chromatophore material between the peripheral and internal membranes. It is possible that the pigments are located predominantly in the internal membranes, which may arise by differentiation of the peripheral structure. Chromatophores isolated by the usual procedures may be composed of peripheral membrane, low in pigment, and of inner membrane representing the purest chromatophore material. There is evidence in support of a differentiation of the internal membrane from the peripheral membrane at least in respect to chlorophyll content. Many workers have found at least two pigmented bands (“heavy” and “light” chromatophores) upon density-gradient centrifugation of the particulate fraction from various photosynthetic bacteria (Hickman and Frenkel, 1959; Cohen-Bazire and Kuniaawa, 1960; Worden and Sistrom, 1964; Gibson et aZ., 1962).The specific chlorophyll content of the heavy material is less than that of the light fraction (Table 4), and consequently the latter has usually been designated as the purified chromatophore fraction. The derivation of the light material from internal membrane of Rsp.

17

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

rubram has been shown convincingly by Holt and Marr (1965b). The membrane fraction from cells disrupted by osmotic chock was resolved into three pigmented bands upon sucrose-gradient centrifugation (Table 4).The intermediate band was free of cell envelopes, but showed large membranous structures in the electron microscope ;upon sonic Greatment TABLE 4. Chlorophyll Content of Pigmented Fractiom after SucroseGradient Centrifugation

Method of preparation of extract French press Osmotic shock

Position of pigment on gradient (em. from top)

Chlorophyll content (mpmoles/mg. protein)

yoTotal

1.90 3.60 1.85 2.90 3.60

81 35 59 60 45

23.2 77-5 13.2 12.7 63.5

Data from Holt and Marr (196513). Extracts were prepared from Rhodospirillum rubrum grown at an illumination of 73 foot candles

these were fragmented into material similar in appearance, density and specific chlorophyll content to the typical chromatophores found in the light fraction. It was concluded that the intermediate band represented internal membrane, and that the light chromatophores arose from this by fragmentation. The origin of the pigment in the heavy fraction is more difficult to establish. Electron microscopy has shown a heterogeneous collection of fragments, including fractured cell envelopes filled with large masses of internal membrane (Holt and Marr, 1965b). This fraction contains a major proportion of the total chlorophyll, at least when isolated from French-press extracts of Rsp. rubrum and Rps. spheroides (Worden and Sistrom, 1964; Holt and Marr, 1965b; Table 4). Also, it can give rise to particles similar in density and chlorophyll content to light chromatophores by further passage through the French press or by sonic treatment. This could be interpreted as release and subsequent comminution of internal membrane which was not originally detached from the cell envelope. Another possibility is that the heavy chromatophore fraction is derived from the peripheral membrane. Worden and Sistrom (1964) observed that the amount of protein in this fraction from Rps. spheroides was constant irrespective of the pigment content of the cells, and amounted to about 24% of the total cell protein. The constancy suggested that the

18

JUNE LASCELLES

heavy material originated from a structure present in both pigmented and non-pigmented cells. The chlorophyll-to-carotenoidratio and the chlorophyll absorption spectrum of the heavy chromatophores differed from the light material, suggesting that the pigments in the two fractions were associated with different complexes. There is some evidence for differentiation of internal and peripheral membrane in non-photosynthetic bacteria, at least with respect to components of the respiratory apparatus. I n Azotobacter agilis, 60% of the NADHz oxidase was located in the internal membrane, and was released by osmotic shock followed by ballistic disintegration (Pangborn et al., 1962). The residual oxidase (located in the peripheral membrane?) remained firmly attached to the cell envelope. Succinate dehydrogenase in Bacillus subtilis, located by reduction of a tetraeolium dye combined with electron microscopy, was observed in both peripheral and internal membrane (mesosome) but the activity was more pronounced in the latter structure (Sedar and Burde, 1965).Kashket and Brodie (1963a,b) found a difference in distribution of “succinoxidase” and NADHz oxidase in the large and small particles prepared from Escherichia coli ; both fractions had NADHz oxidase activity, but “succinoxidase” was confined mainly to the large particle fraction.

F. THEVESICLES OF

THE

GREENBACTERIA

The ellipsoidal vesicles seen in electron micrographs of green sulphurbacteria have been shown fairly conclusively to contain chlorophyll by techniques similar to those used by Holt and Marr (1965b) with Rsp. rubrum (Holt et al., 1966a). The vesicles provide the most clear-cut example of a differentiated chromatophore structure distinct from the peripheral membrane. The surrounding membrane of the vesicles is thinner than that of the peripheral structure (50 and 80 A respectively) and electron microscopy has not shown evidence of attachment between the structures (Cohen-Bazireet al., 1964).Differences in the composition of the structures has been indicated by comparative studies of the location of succinate dehydrogenase, a typical component of peripheral membrane, and chlorophyll (Holt et al., 1966a). Sonic treatment of Chloropseudomonas ethylicum released chlorophyll from the cells more rapidly than the dehydrogenase, in accord with the association of the enzyme with the peripheral membrane and the pigment with the more readily released internal structures. Fractionation of the dehydrogenase and the chlorophyll has also been achieved by differential centrifugation of French-press extracts of Chlorobium thiosulphatophilum (Fuller, 1963). The enzyme was found in the fraction containing large fragments of membrane, whereas the pigment was predominantly located in the

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

19

small-particle fraction. Although the composition of the peripheral membrane and the vesicles appears to be quite radically different, the possibility still remains that the two structures may be connected by delicate attachments which are destroyed upon disintegration of the cells.

G. CONCLUSIONS A tentative conclusion which may be drawn from the present information is that chromatophore material is located at least partly in the internal membranes of some of the photosynthetic bacteria. Also, the constant ratio of chlorophyll-to-proteinfound in highly purified chromatophores points to a stoichiometric relation between this pigment and the internal membrane matrix-at least in Rsp. rubrum (Holt and Marr, 1965b).However, there is also evidence for the presence of chromatophore material in peripheral membrane, though it is not possible t o decide whether the composition of this material differs from that in the internal membrane. Systematic analysis with respect to enzymic and chemical content of the various chromatophore fractions may help in the solution of these problems. The concentration of succinate dehydrogenase and other membrane-bound enzymes may serve, for instance, as an indication of differentiation of the membrane structures. Clearly the primary requirements are for methods of separating and purifying the internal and peripheral membranes, followed by fractionation into their component parts. Rupture of cells by lytic methods may offer the most promising beginning to such studies.

III. Isolation and Composition of Purified Chromatophores A. ISOLATION PROCEDURES Problems of isolating a pure chromatophore fraction begin with the disruption of the cell envelope, and the harsh methods in common use give ample opportunity for the generation of artifacts by damage to delicatemembrane structures. Some possibilities to be considered in evaluating the purity and the relation of isolated chromatophores to the functioning photosynthetic apparatus in the,intact cell are as follows. (1)Components whichinvivo are loosely attached to the structure may be solubilized and lost during the purification ; for instance, ferredoxin, c-type cytochrome and the cytochromoid pigments (Orlando et al., 1961; Horio and Kamen, 1962). (2) Structures which are not associated with the photosynthetic apparatus may be fragmented and may fractionate with the chromatophores during purification. Soluble cytoplasmic

20

JUNE LASCELLES

constituents might also be non-specifically adsorbed to the isolated particles. (3) The chromatophore structure may itself become fragmented into subunits which may or may not represent the state of the structure in vivo. There are no firmly established criteria for assessing the purity of chromatophores. The pigment-to-protein ratio (specific pigment concentration), and homogeneity upon ultracentrifugation or by electron microscopy are useful guides. Another parameter, infrequently used, is photophosphorylation activity expressed in terms of protein and of chlorophyll (Frenkel and Hickman, 1959; Cohen-Bazire and Kunisawa, 1960). Disruption of cells by the French press is the most usual choice for the initial preparation of crude extracts. Perhaps the best method to be exploited so far is disruption by osmotic shock. With Rsp. rubrum this may be achieved by mixing the cells with glycerol followed by rapid dilution into buffer (Holt and Marr, 1965a).Glycerol-lysisis not effective with all photosynthetic bacteria; however, some, including Rps. spheroides, are lysed by treatment with lysozyme and EDTA (Tuttle and Gest, 1959; Lascelles and Szil&gyi,1965; Gibson, 1965a). Chromatophores can be separated from the crude particulate fraction by sucrose-gradient centrifugation which usually results in two pigmented bands, the “light” and “heavy” chromatophores (Frenkel and Hickman, 1959; Cohen-Bazire and Kunisawa, 1960; Holt and Marr, 1965b; Worden and Sistrom, 1964). It is the light fraction which is usually deemed to contain the pure material, on the basis of specific pigment content and homogeneity of particle size ; the position of this fraction from extracts of Rsp. rubrum and Rps. spheroides corresponds to a density of 1.14 g./ml. (Cohen-Bazireand Kunisawa, 1960;Worden and Sistrom, 1964). Centrifugation in a linear gradient of caesium chloride has also been used with Rps. spheroides (Gibson, 1965a). Ribosomes are a likely contaminant of chromatophores purified only by sucrose-gradient centrifugation. Such contamination was essentially abolished from Rps. spheroides preparations by centrifuging in 27% rubidium chloride prior to sucrose-gradient centrifugation (Worden and Sistrom, 1964). The purest preparations appear to be those from Rsp. rubrum which were subjected to sucrose-gradient electrophoresisafter centrifuging in a linear sucrose gradient (Holt and Marr, 1965b).The specific pigment content of these chromatophores was high (Table 3) but their photophosphorylating activity was not reported ; comparison of this activity with less extensively purified material would be interesting. Sedimentation coefficients reported for purified chromatophores include 1535 and 165s for Rps. spheroides (Worden and Sistrom, 1964;

21 Gibson, 1965a), 120s for Chromatiurn (Bergeron, 1959) and 116s for Chlorobium thiosulphatophilucm (Sykes et al., 1965). THE BACTERIAL PHOTOSYNTHETIC APPARATUS

B. COMPOSITION The detailed analyses of Chromatium chromatophores (Newton and Newton, 1957)set a precedent which, regrettably, has not been followed. Attention has been given mainly to the specific pigment content, which in most preparations reflects the pigment content of the cells from which the chromatophores were prepared (Table 3). Since the highly purified preparations from Rsp. rubrurn (Holt and Marr, 1965b) showed a fairly constant specific pigment content, the variations found in other preparations might be attributed to contamination of the pigmented fractions with non-chromatophore material. Except for the photosynthetic pigments the composition of isolated chromatophores (Table 5) is similar to that of membrane fractions prepared from non-photosynthetic organisms (Weibull and Bergstrom, 1958; Kodicek, 1963). The predominant components are protein and lipid; the small amounts of nucleic acid probably represent ribosomal contamination (Worden and Sistrom, 1964). The carbohydrate found in some preparations may arise largely from contamination by cell-wall components, and analyses of chromatophores should include hexosamine as an indication of such material. Galactolipids are associated specifically with the chloroplast in plants (Benson, 1964)but bacteria do not appear to have a unique type of lipid associated with their photosynthetic apparatus. Phosphatidylethanolamine was found by Newton and Newton (1957)to comprise the bulk of the lipid of Chromatiurn chromatophores. Phospholipids are apparently the predominant form of lipid in other photosynthetic bacteria, assuming that the lipid content of the whole cell (upon which most analyses have been made) reflects that of the isolated chromatophores. Most species of photosynthetic bacteria contain several types of phospholipid ; for instance, Rps. spheroides and Chlorobium thiosulphatophilum contain phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidic acid (Lascelles and Sziliigyi, 1965; Wood et al., 1965). Athiorhodaceae grown photosynthetically or aerobically in the dark exhibit no difference with respect to the types of phospholipid, their relative proportion or in their fatty acid components (Lascelles and Sziliigyi, 1965;Wood et al., 1965; Scheuerbrandt and Bloch, 1962; Erwin and Bloch, 1964).Photosynthetically grown cells however, do contain a considerably higher content of total phospholipid (Lascellesand Szilhgyi, 1965; Cohen-Bazire and Sistrom, 1966).

TABLE 5. Composition of Isolated Chromatophores Component Protein Lipid

1

Chlorobium thiosulphatophiluma*' 63 25

Chlorophyll yo dry weight Carotenoids Carbohydrate Nucleic acid Total phosphorus pmoles/mg. Lipid phosphorus protein Chlorophyll Carotenoids Haems mpmoles/ Flavin (acid extractable) Nicotinamide nucleotides Non-haem iron

I

7

9 4

98 19 1.5

Chromatium 61 22 (Phospholipid) 4 1.5 7 0 0.83 0.51 40 (155)' 17 (88)g 2.8 (3.7)' 0.5 0.9 12

Sykes et al. (1965). Hulcher and Conti (1960). Newton and Newton (1957). Bergeron (1959). Bull and Lascelles (1963). Gibson et al. (1962). Values in parentheses from Hulcher and Conti (1960); others from Newton and Newton (1957).

Rhodopaeudomonas ~pheroides'*~ 58 25

5 1 4 1 0.62 0.40 112 44 2.9

-

cl

a

z

F

u)

Bu

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

23

1. Catalytic Components

Analysis of purified chromatophores has shown the presence of cytochromes, flavines, and nictotinamide nucleotides (Table 5). Other electron-transfer catalysts can be presumed to be present, at least in preparations which photophosphorylate. Analytical data are not available for the ubiquinone content of chromatophores, though it is known to be present in high concentration in the cells of photosynthetic bacteria, particularly when grown anaerobically in the light (Fuller et al., 1961; Sugimura and Rudney, 1962; Carr and Exell, 1965). Its presence in chromatophores has been indicated by light-induced spectral changes (oxido-reductions) in the region 240-340 mp (Clayton, 1962a). Ferredoxin has now been found in a number of photosynthetic bacteria (Buchanan et aZ., 1965; Yamanaka and Kamen, 1965) and its reduction by illuminated chromatophores from Chlorobium thiosulphutophilum has been shown (Evans and Buchanan, 1965). Extracts of this organism and of Chromutium catalyse a ferredoxin-linkedreduction of NAD in the dark with hydrogen plus hydrogenase as source of reducing power (Weaver et al., 1965). The concentration of ferredoxin in chromatophore preparations has not, to the reviewer's knowledge, been determined but it is presumably present in preparations which show photoreduction of nicotinamide nucleotides. A non-haem iron protein has been found in Chromatium chromatophores, and may contribute to the large amounts of non-haem iron shown in the analyses (Table 5). This protein is quite distinct from ferredoxin and has a potential of +0.35 V ; its function is unknown (Bartsch, 1963). Similar iron proteins have been found in several speciesof Athiorhodaceae (De Klerk and Kamen, 1966). 2. Enzymic Activities

The photophosphorylation activity found by Frenkel and Hickman (1959) with purified chromatophores from Rsp. rubrum was from 110 to 175 pmoles Pi esterified per pmole of chlorophyll per hour. Photoreduction of NAD in the presence of succinate occurred at the rate of 23-26 pmoles NADHzper pmole of chlorophyllper hour. The photophosphorylation values were obtained in the absence of phenazine methosulphate and succinate, which stimulate this activity in purified chromatophores (Geller and Lipmann, 1960 ; Cohen-Bazire and Kunisawa, 1960). Most studies of photophosphorylation have been with chromatophores from Rsp. rubrum partly because this organism provides relatively stable preparations. Purified material from Rps. spheroides exhibits feeble activity (less than 1 pmole Pi esterified per hour per pmole of 3*

24

JUNE LASCELLES

chlorophyll)which is not enhanced by phenazine methosulphate (Gibson, 1965b).Crude extracts of the organism are also far less active in comparison with other organismsbut some improvement occurs when the extracts are made in the presence of ascorbate. Possibly in Rps. spheroides an essential factor(s)is more easily dissociated and inactivated by oxidation. Photophosphorylation activity is usually expressed in terms of chlorophyll, and has been found to vary inversely with the pigment content of chromatophore preparations (Cohen-Bazireand Kunisawa, 1960;Drews, 1964).When expressed on a protein basis, relatively constant values have been obtained with preparations of widely differing pigment content ;the rate is therefore limited by a dark reaction rather than by the primary photochemical event. Factors which may become limiting include some of the oxidation-reduction catalysts which may not be flrmly attached to the structure. Cytochrome c2, for instance, is readily leached from the chromatophores of Rsp. rubrum with consequentloss of photophosphorylation activity; this activity is restored by addition of the cytochrome to the depleted preparations (Horio and Kamen, 1962). Ferredoxin and other iron proteins are also likely to be lost in purification since they are found predominantly in the soluble fraction of cell extracts (Bartsch, 1963; Yamanaka and Kamen, 1965). Purified chromatophores from Rsp. rubrum have succinate dehydrogenase activity which shows essentially no variation in preparations from organisms of very different pigment content (Cohen-Bazire and Kunizawa, 1960). The enzyme might therefore be an integral part of the chromatophore structure, This is not, however, the case with preparations from the green sulphur-bacteria. The enzyme is located in particles which are separable from the bulk of the pigmented material by differential centrifugation (Fuller, 1963; Holt et al., 1966b).

C. FRACTIONATION OF CHROMATOPHORES 1. ChlorophylLProtein Complexes

The object of many fractionation studies with chromatophores has been to isolate a pure pigment-protein complex, but the physiological status of such complexes is uncertain in the absence of test systems to examine their photochemical properties. The green sulphur-bacteria have so far provided the purest preparations (Olson, 1966).Two soluble complexes with red absorption maxima at 770 and 809mp have been isolated from alkaline extracts of Chbrobium thiosuhphtophilum and Chloropseudomoms ethylicum. The material absorbing at 809 mp has been crystallized and consists entirely of protein and pigment. It has a molecular weight of 3.6 x 104, with three moles of

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

25

pigment per mole of complex. The pigment is apparently bacteriochlorophyll a whereas the bulk of the chlorophyllin the organisms is Chlorobium chlorophyll 660 or 650. In vivo experiments suggest that the 809 complex is probably the photosynthetic reaction-centre in the green bacteria (Olson and Romano, 1962; Olson and Sybesma, 1963). Detergent treatment of chromatophores has shown that there are differences between the various pigment-protein complexes in their manner of binding to the chromatophore structure. Light-harvesting forms of chlorophyll in chromatophores from Rps. spheroides, Rsp. rubrum and Chromatiurn are readily solubilized by treatment with deoxycholate or with Triton X- 100,whereasthe reaction-centre form remains attached to the particles (Clayton, 1962b; Bril, 1958,1960). The solubilized pigment is readily destroyed by light unlike the particle-bound material. 2. Multi-Enzyme Complexes

The pigment-protein complexes function in vivo in conjunction with oxidation-reduction catalysts and phosphorylation systems. An understanding of the arrangement of these components in the functional photosynthetic unit requires detailed studies of the individual catalytic and structural units; this means dissection of the chromatophore and, hopefully, re-assembly of the components in some semblance of their natural state. Fractionation studies have not yet reached this degree of sophistication, but have provided leads for future exploitation. Lipase treatment of chromatophores from Rsp. rubrum considerably increased their specific chlorophyll content without altering their behaviour on sucrose-gradientcentrifugation (Cohen-Bazireand Kunisawa, 1960). The treated material did not have photophosphorylation or succinate dehydrogenase activities, presumably due to loss or inactivation of essential components. Treatment of chromatophores of Rps. spheroides with cholate plus deoxycholate has given material which still exhibits photophosphorylation (Gibson, 1965~). The bile salts removed 70% of the lipid (mostly phospholipid) and the sedimentation coefficient dropped from 160s to 1205. The depleted material was unstable and was readily disrupted into two pigmented fractions in the presence of salts. These relatively mild procedures for subfractionation may be usefully extended in the future. Gibson suggests that core particles, containing the photosynthetic pigments and photophosphorylation system, are covered in vivo by a thin layer of lipid. This notion requires confirmation by refined techniques of electron microscopy. The non-ionic detergent, Triton X-100, appears to be the best weapon so far for disruption of chromatophores into subunits with biochemical

26

JUNE LASCELLES

activity. It was first applied by Bril(1958)who showed that it solubilized the 850 mp-form of chlorophyll from chromatophores of Rps. spheroides. The detergent has recently been used to fractionate chromatophores from Chromatium and Rsp. rubrum (Garcia et al., 1966a, b). Chromatium chromatophores were split into two pigmented fragments, the light (L) and heavy (H) fractions, separable by sucrose-gradient centrifugation. The H fraction contained reaction-centre chlorophyll (P-890) which exhibited the typical photo-oxidation response. It also catalysed the anaerobic photo-oxidation of reduced phenazine methosulphate in the presence of ubiquinone-6 and was rich in cytochromes. The L-fraction contained the bulk of the chlorophyll (P-800 and P-850) but was virtually devoid of P-890, cytochromes and of photo-oxidative activity. Electron microscopy showed membrane material in the L fraction whereas the H fraction consisted apparently of subunits. Treatment of chromatophores of Rsp. rubrum with Triton X-100 also gave heavy and light pigmented fractions (Garcia et al., 1966b). Both contained reaction-centre chlorophyll but the H fraction contained most of the total pigment. Only the L fraction catalysed the photoreduction of NAD (reduced dichlorophenolindophenol as electron donor) and had succinate dehydrogenase activity. A third fraction, banding above the L fraction, contained bacteriopheophytin rather than chlorophyll and had succinate dehydrogenase activity and cytochrome c. This material appeared to exist per se in the chromatophore, since such particles were not released by further treatment of H and L fractions with detergent. Electron microscopy showed subunits in the L, and membranous material in the H, fractions corresponding respectively to the H and L particles from Chromatium. The observations support the possibility that light-harvesting chlorophyll is located in the exterior membrane of the chromatophore, and the photochemically reactive form together with associated catalysts is situated in the interior-a logical arrangement.

REACTIVITY D. IMMUNOLOGICAL Not surprisingly, chromatophores are highly antigenic. Antisera have been prepared by injection of rabbits with Chromatium chromatophores ; these antisera contain antibodies which precipitate the pigmented particles (Newton and Levine, 1959). The antigens of the chromatophores apparently included components from the cell surface since the antisera also reacted with intact cells. The immunological reactivity of pigmented subfractions, derived by treatment with disulphide-cleaving reagents, has also been examined (Newton, 1962). Chromatophores from Rsp. rubrum also induce the synthesis of specific

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

27

antibodies which precipitated the photosynthetic pigments from crude cell extracts (Newton, 1960). Extracts prepared from pigmented cells, which had been aerated in the dark for a short period, lost their reactivity with the specific antiserum, suggesting that some dissociation of the reactive material had occurred. The value of immunology as a tool may become more apparent when highly purified chromatophores are used as antigen. Such techniques might, for instance, be applied to members of the Athiorhodaceae to determine whether the chromatophores contain specific proteins which are lacking in particulate fractions from non-pigmented cells.

IV. Formation of the Photosynthetic Apparatus Those Athiorhodaceae capable of either photosynthetic or aerobic growth provide an outstanding example among bacteria of intracellular differentiation in response to the environment. The adaptive phenomena exhibited by these organisms is analogous to the development of plant chloroplasts in response to light. I n each case, synthesis of the photosynthetic pigments is accompanied by major changes in the synthesis of enzymes and cofactors required for photosynthetic metabolism. The plant has the added sophistication of a specialized organelle, the chloroplast, to house the photosynthetic apparatus and this may represent an evolutionary progression from the bacterial chromatophore.

A. DIFFERENCES BETWEEN MEMBERSOF THE ATHIORHODACEAE GROWN PHOTOSYNTHETICALLY AND AEROBICALLY Analysis of members of the Athiorhodaceae grown in the dark with high aeration or anaerobically in the light shows that pigment synthesis is accompanied by many changes in enzyme content and in catalysts associated with photosynthetic electron-transport (Table 6). The cytological changes which accompany pigment synthesis have already been discussed. The high concentration of haemoproteins and of ubiquinone in cells grown anaerobically in the light is consistent with their function in photosynthetic metabolism. There is no evidence for chemical differences between the b- and c-type cytochromes found in photosynthetic and aerobic cells. However, a unique type of haemoprotein (cytochromoids, RHP) has been found in many photosynthetic bacteria grown anaerobically in the light (Kamen, 1963; Bartsch, 1963). Such pigments have two haem c prosthetic groups per molecule, and are characterized by their ability to combine with carbon monoxide. Their formation is apparently associated with the development of the photosynthetic apparatus since

w

OD

TABLE 6. Concentration of Components in Rhodopaeudomonas spheroides Grown Aerobically asd Photosynthetically Concentration in cells grown h

t

Aerobic-dark

Component

1

Bacteriochlorophyll Carotenoids Haemoproteins Total mpmoles/mg. Cytochrome b protein Cytochrome c CO-bindingpigment Ubiquinone (&lo) Lipid phosphorus Ribdose diphosphate carboxylase mpmoles product/ 6-Aminolaevulinic acid synthase 8-Aminolaevulinic acid dehydrase

>

Anaerobic-light*

0.1 0.5

48 22

0.20 0.08 0.06 0.06 3.02 90 60 20 45

0.59 0.17 0.19 0.23 6.64 157 1800 205 105

Reference 1 1 2

*The strainwedwas N.C.I.B. 8253. Cultures were grown in a malate-glutamatemedium (Lascelles,1959)with high aeration or anaerobically

in light of about 250 foot candles intensity.

References: (1) Bull and Lascelles (1963); (2) Porra and Lascelles (1965); (3) Carr and Exell (1965); (4) Lascelles and Szilhgyi (1965); (5) Lascelles (1960b); (6) Lascelles (19608).

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

29

they have not been detected in aerobically grown cells (Taniguchi and Kamen, 1965).However, a function for these peculiar haemoproteins in photosynthetic electron-transfer reactions is yet to be found. The terminal oxidase in aerobically-grown Rsp. rubrum may be a form of cytochrome 0,a carbon monoxide-binding pigment with a protohaem prosthetic group (Taniguchi and Kamen, 1965; Chance et al., 1966). It is not known whether this type of oxidase is also present in photosynthetically grown cells. Such organisms respire vigorously but the respiration could be mediated by a cytochromoidpigment (Horio and Kamen, 1962). An a type cytochrome has been found in a strain of Rps. spheroides when grown aerobically (Motokawa and Kikuchi, 1966).It exhibits the typical binding with carbon monoxide, and appears to function as a terminal oxidase. No evidence for this type of pigment was found in the strains of Rps. spheroides examined by Porra and Lascelles (1965),in which respiration was apparently mediated by an o-type cytochrome. Ribulose diphosphate carboxylase is an enzyme typically associated with photosynthetic metabolism. It is found in high concentration in members of the Athiorhodaceae grown anaerobically in the light, but is low or undetectable in aerobically grown cells (Lascelles, 1960b). Its presence in members of the Athiorhodaceae is anomalous since these organisms grow only on fixed carbon compounds. The carboxylase is present in members of the Thiorhodaceae grown on organic substrates though the concentration of the enzyme is lower than that in cells grown with carbon dioxide as sole carbon source (Hurlbert and Lascelles, 1963).

B. ADAPTIVE SYNTHESIS OF

THE

PHOTOSYNTHETIC APPARATUS

The versatility of some species of Athiorhodaceae makes them useful for study of the synthesis and regulation of the photosynthetic apparatus. The organisms can be grown devoid of the photosynthetic system, and its development can be followed upon subsequent incubation either anaerobically in the light or in the dark with low aeration. I n the anaerobic-light system, the photosynthetic apparatus is developed non-gratuitously since ATP production under anaerobic conditions depends on photosynthetic reactions (Schon and Drews, 1966). The low-aeration system has the advantage of allowing adaptation to proceed gratuitously, and, most important, it can be applied to mutants which cannot grow photosynthetically (Lascelles, 1959;Bull and Lascelles, 1963;Lascelles, 1966a, b)The photosynthetic apparatus formed by organisms grown in the dark with low aeration appears to be fully functional since chromatophores from Rsp. rubrum exhibit photophosphorylation just as actively

30

JUNE LASCELLES

as preparations from photosynthetically grown cells (Cohen-Bazire and Kunisawa, 1960).Also, electron microscopy has shown the same type of internal structure with increasing invagination of the peripheral membrane as the pigment content increases in response to decreased aeration (Cohen-Bazire and Kunisawa, 1963; Biedermann et aZ.,1967). The kinetics of development of individual components of the photosynthetic apparatus have been examined in Rps. spheroides as it adapts from conditions of high to low aeration. Haemoproteins and ubiquinone are formed in parallel with chlorophyll (Porra and Lascelles, 1965; Carr and Exell, 1965)but the appearance of ribulose diphosphate carboxylase is delayed (Lascelles, 1960b). The formation of enzymes of chlorophyll synthesis and of particulate protein is discussed later. 1. Photosynthetic Mutants

Numerous mutant strains of Rps. spheroides have been isolated which are blocked at single stages in the chlorophyll biosynthetic pathway and are therefore unable to grow anaerobically in the light (Lascelles, 1964). I n the low-aeration systems, such mutants accumulate chlorophyll precursors and the usual carotenoids (Griffiths and Stanier, 1956; Griffiths, 1962; Lascelles, 1966a). Carotenoid-less mutants, which make chlorophyll and are therefore capable of photosynthetic growth, have also been isolated (Griffiths and Stanier, 1956; Jensen, 1963). Another class of mutants (“albinos”) form neither carotenoids nor chlorophyll, and are consequently only able to grow aerobically (Griffiths and Stanier, 1956; Lascelles, 1966b; Drews and Schick, 1966). One such albino, strain L-57, has been examined in some detail (Bull and Lascelles, 1963; Lascelles, 1966b).Under low aeration, the organism does not form photosynthetic pigments or ribulose diphosphate carboxylase, whereas the wild type does so under these conditions. Both mutant and wild type, however, show the same increase in contents of b and c type cytochromes and a carbon monoxide-binding pigment; it is not known whether the haemoproteins formed by the mutant are identical with those of the wild type (Porra and Lascelles, 1965).The mutant synthesizes free haem but not magnesium tetrapyrroles from 6-aminolaevulinic acid, and presumably has the iron branch of the tetrapyrrole pathway (Lascelles, 1966b). I n accord with the absence of photosynthetic pigments, the mutant does not form internal membrane when grown under conditions which promote this in the parent strain (Germaine Cohen-Bazire and Rio Kunisawa, personal communication); nor does it show an increase in phospholipid content under these conditions (Lascelles and Szil&gyi, 1965).

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

31

C. CHLOROPHYLL SYNTHESIS There is some justification for airing a special interest in chlorophyll synthesis and its regulation, since the formation of this pigment seems to play a central role in the final elaboration of the chloroplast and of its bacterial counterpart. 1. Biosynthetic Pathway

Haem and chlorophyll synthesis share a common pathway to the stage of protoporphyrin, where divergence into the iron and magnesium branches occurs (Fig. 8). Knowledge of the intermediates of the magnesium branch is fragmentary and is derived largely by intuition from products accumulated by Chlorella and by Rps. spheroides blocked in chlorophyll synthesis either by mutation or by inhibitors of chlorophyll synthesis such as 8-hydroxyquinoline (Bogorad, 1966;Jones, 1963a, b, c, 1964; Lascelles, 1966a).The only step to be demonstrated so far in cellfree extracts is the methyl transferase which catalyses the reaction : Magnesium protoporphyrin +S-adenosylmethionine-t Magnesium protoporphyrin monomethylester + S-adenosylhomocysteine This activity was found in the chromatophore fraction of Rps. spheroides and other Athiorhodaceae (Gibson et al., 1963). 2 . Chlorophyll Synthesis and Chloroplast Development

The development of the chloroplast in plants is closely associated with chlorophyll synthesis; the evidence for this may be summarized as follows. (1) Formation of chlorophyll upon illumination of etiolated leaves or of Euglena is strictly dependent on protein synthesis since pigment formation is prevented by inhibitors of protein and of nucleic acid synthesis (Bogorad, 1966). Such inhibition of chlorophyll synthesis may be only partly attributable to the prevention of the development of enzymes required for the biosynthesis. Addition of actidione to Euglena after greening has begun drastically curtails chlorophyllformation, suggesting the requirement for a protein which functions in stoichiometric rather than in catalytic amounts (Kirk and Allen, 1965). (2) Protochlorophyllide a (magnesium vinylpheoporphyrin a s ) occurs in etiolated plants in combination with protein, the red absorption maximum being 650 mp ;this complex is located in the proplastids (Klein et al., 1964). Conversion of the pigment to chlorophyllide a involves the reduction of ring D of the tetrapyrrole nucleus and occurs upon illumination. Only the bound form of the pigment is reduced; free

8 Glycine pyridoxal PO4

+

___j

-8COa

8 6-aminolaevulinic acid

8 Succinyl-CoA

- 4C09

coproporphyrinogen I11 t-- uroporphyrinogen I11

-2COr

-4H

I coproporphyrin I11 - 6H

protoporphyrinogen ---+

Mg-protoporphyrin

+methyl

+2H

+Fe

protoporphyrin ---+

j+%

4 porphobilinogen

uroporphyrinogen I

uroporphyrin I11

I.

J.

- 4KHs

I

-6H

J. haems

uroporphyrin I

Mg-protoporphyrin

Mgprotoporphyrinmono- --+ --+ methyl ester

+ Ha0

Mg-2-vinylphaeoporphyrina+ ----+ chlorophyllide a ----+ (protochlorophyll a )

Mg-2,4-divinylphaeoporphyrin a5

+2H __f

+2H

2-devinyl-2-hydroxyethylbacteriochlorophyll iphytol chlorophyllide

FIG.8. Outline of the iron-and magnesium branches of the tetrapyrrole biosynthetic pathway.

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

33

protochlorophyllide(absorption maximum at 631 mp), accumulated from exogenous 6-aminolaevulinic acid, is not converted (Granick, 1963 ; Klein and Bogorad, 1964). The protochlorophyllide complex or holochrome has been purified from etiolated bean leaves as an apparently homogeneous protein of molecular weight about 600,000 and sedimentation coefficient of 1 8 s (Boardman, 1966). The complexed pigment is reduced to the chlorophyllide upon illumination, but the hydrogen donor is not known. The status of the protein moiety is far from clear. It is similar in properties to Fraction 1 protein which comprises about 25% of the dry weight of the chloroplast. This fraction may consist largely of ribulose diphosphate carboxylase ; purified preparations of this enzyme (molecular weight 515,000) closely resemble the holochrome protein in many physical properties (Trown, 1965). It is possible that the isolated holochrome may represent the protochlorophyllide combined with a smaller protein which has become associated with the carboxylase during purification. I n vivo association of pigment with the enzyme, a t least in its active form, does not seem to be required for chlorophyll synthesis, since mutants of Chlamydomonas which lack the carboxylase still form chlorophyll (Levine and Togasaki, 1965). (3) The light-induced conversion of the protochlorophyllide holochrome to chlorophyllidea is accompaniedby changesin the fine structure of the proplastids, the tubular elements becoming transformed into vesicles (Virgin et al., 1963). The phenomena appear to be related since the structural changes are induced specifically by light of wavelengths required to effect conversion of the pigment (Klein et al., 1964). There is also evidence that the final lamellar structure of the chloroplast is linked with the esterification of chlorophyllide a with phytol (Klein, 1962; Eilam and Klein, 1962). 3. Chlorophyll Synthesis and Membrane Formation in Photosynthetic

Bacteria I n bacteria there is evidence for an obligatory linkage of chlorophyll and protein syntheses. Pigment synthesis by Rps. spheroides in lowaeration systems is prevented by inhibitors of protein and nucleic acid synthesis or by deprivation of amino acids, purines or pyrimidines in the case of auxotrophic mutants (Lascelles, 1959; Bull and Lascelles, 1963; Higuchi et al., 1965; Biedermann et al., 1967). The dependence of chlorophyll formation on protein synthesis has also been shown with photosynthetic cultures of Rps. spheroides, which form pigment in response to a decrease in light intensity (Sistrom, 1962a, b) and with Rsp. rubrum upon transfer from high aeration to photosynthetic conditions (Drews, 1965).

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Some, at least, of the-enzymes required for chlorophyll synthesis are adaptive (see later) and their formation could account for the obligatory link with protein synthesis. However, pigment synthesis is still sensitive to inhibition of protein formation in cells containing the full complement of biosynthetic enzymes (Sistrom, 1962a, b ; Bull and Lascelles, 1963; Drews, 1965). There is other evidence which points to a specific association of chlorophyll synthesis with the formation of membrane protein. I n low-aeration systems, Rps. spheroides incorporates labelled amino acids into chromatophore protein at a faster rate than into the soluble protein fraction (Bull and Lascelles, 1963). The preferential incorporation into the particulate fraction occurs in parallel with chlorophyll formation, and appears to be specifically connected with it (Bull and Lascelles, 1963; Altshuler and Lascelles, 1967). The phenomenon is not shown when cells are incubated under high aeration, which represses pigment synthesis. Also, albino mutants do not show preferential incorporation when incubated under conditions which promote it in the wild type. It is possible that the preferential incorporation represents the formation of structural protein which accommodates the photosynthetic pigments in the membrane. Fractionation of the membrane proteins is clearly necessary to establish this.* The integration of chlorophyll and membrane formation is also suggested by the increase in phospholipid content in Rps. spheroides which occurs as pigment is formed under low aeration (Lascelles and Szilhgyi, 1965). The albino mutant, L-57, does not show this increase when incubated similarly. There is a distinct possibility that protein-bound intermediates participate in chlorophyll synthesis by the bacteria, and perhaps this protein may become an integral part of the pigmented membrane structure. Evidence for bound intermediates is provided by mutant strains of Rps. spheroides which accumulate chlorophyll precursors ; these are bound to the particulate fraction of cell extracts but are released by treatment with Tween 80 (Lascelles, 1966a).This procedure is too mild to release chlorophyllfrom its complexes;possibly, the phytol residue, which is lacking in the precursors, is needed for the firm integration of pigment molecules into the membrane structure. The less attractive possibility, t$at the bound precursors are artifacts of the fractionation procedures, cannot be dismissed. OF CHLOROPHYLL SYNTHESIS D. REGULATION Mechanisms for the regulation of chlorophyll synthesis are almost completely obscure, a state of affairs which can be partly attributed to ignorance of the enzymic steps. One firm fact is that 6-aminolaevulinic

* See note added in proof on p. 42.

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

35

acid synthase plays a central role in the regulation, being controlled by repression and by feedback inhibition, but the precise mechanisms of this control are unknown. 1. Repression of Biosynthetic Enzymes

The concentrations of some of the enzymes of chlorophyll synthesis rise and fall in response to environmental changes in a manner which is consistent with control of pigment synthesis by enzyme repression. I n Athiorhodaceae, the onset of chlorophyll synthesis in cells transferred from high to low aeration or to photosynthetic conditions is preceded by a 5-10-fold increase in 6-aminolaevulinic acid synthase and other early enzymes of the biosynthetic pathway (Lascelles, 1959, 1960a; Drews, 1965; Drews and Oelze, 1966; Higuchi et al., 1965).I n the converse type of experiment, synthesis of both enzyme and pigment is repressed when pigmented cells are subjected to high aeration (Lascelles, 1960a). There is reason to believe that the enzymes of the magnesium branch are totally repressed by high aeration. Gibson et al. (1963) could not detect the methyl transferase in non-pigmented Rps. spheroides. Also, whole cells of this organism do not form magnesium tetrapyrroles from 6-aminolaevulinic acid when grown with high aeration, though such compounds are formed by pigmented cells (Lascelles, 1966a, b). 2. Role of 6-Aminolaevulinic Acid Synthase

The parallel between the activity of 6-aminolaevulinic acid synthase and the chlorophyllcontent of Athiorhodaceae is consistent with a central function for this enzyme in regulating the amount of pigment formed under various conditions. Repression of this enzyme, however, cannot account for the immediate cessation of chlorophyll synthesis which occurs upon the introduction of oxygen into cultures growing anaerobically in the light and containing the full complement of biosynthetic enzymes (Cohen-Bazire et aZ., 1957; Lascelles, 1960a; Sistrom, 1965). A direct effect on the action of the enzyme is suggested by the fact that there is no accumulation of intermediates in such oxygen-repressed systems. However, there is no evidence from in vitro experiments that the synthase is inhibited by oxygen; rather, it tends to be activated by aeration of crude extracts (Burnham and Lascelles, 1963). One possibility is that oxygen acts indirectly enhancing oxidation of succinate via the tricarboxylic acid cycle, and thereby deprives the synthase of succinyl-CoA (Lascelles, 1964).Knowledge of intracellular pool levels of succinate and succinyl-CoA under various conditions is required to support this. Rapid destruction of 6-aminolaevulinic acid synthase may also be a factor in chlorophyllregulation. The enzyme is highly unstable in extracts

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and partly purified preparations from Rps. spheroides and other bacteria (Burnham and Lascelles, 1963 ;J. Lascelles, unpublished observations). The enzyme is also unstable in wivo, since there is rapid loss in activity when protein synthesis is prevented by inhibitors or by deprivation of amino acids (Bull and Lascelles, 1963). Destruction of the enzyme also occurs when cultures growing anaerobically in the light are transferred to highly aerobic conditions (Lascelles, 1960a). Metabolic instability of the synthase may be of more general significance, since the drug-induced enzyme in liver is rapidly destroyed upon termination of protein synthesis (Marver et aZ., 1966). A control mechanism which involves rapid degradation of a key enzyme is not intellectually satisfying, particularly as one cannot point to many comforting precedents. The present evidence, however, does not allow the dismissal of this possibility. Feedback inhibition of 3-arninolaevulinic acid synthase. I n plants, there is evidence for feedback control of 6-aminolaevulinic acid synthase, exerted by the protochlorophyllide holochrome complex (Bogorad, 1966). The amount of this precursor which is accumulated by etiolated leaves is only a small fraction of the amount of chlorophyll formed from endogenous sources upon illumination. When the light source is removed, the chlorophyllide accumulates again to the original concentration. Free protochlorophyllide does not affect the action of the synthase since its presence in high concentration relative to the complexed form does not prevent chlorophyll synthesis from endogenous substrates (Granick, 1963; Klein and Bogorad, 1964). There is indirect evidence for regulation of the action of the synthase by magnesium tetrapyrroles in bacteria. Rps. spheroides and Rps. mpsalata accumulate coproporphyrin I11 which arises by spontaneous oxidation of the biosynthetic intermediate coproporphyrinogen 111, when chlorophyll synthesis is limited by iron deficiency (Lascelles, 1956 ; Cooper, 1963). Coproporphyrin is also accumulated by Rps. spheroides in the presence of ethionine (Gibson et d.,1962) or under conditions of methionine limitation (Lessie and Sistrom, 1964). The formation of coproporphyrin is accompanied by inhibition of chlorophyll synthesis, and this effect is attributable to a block at the methyl transferase step which requires S-adenosylmethionine, derived from methionine (Gibson et a,?.,1963). &ccumulation of the porphyrin may result from the failure of a feedback control on the 3-aminolaevulinic acid synthase, exerted normally by a component of the biosynthetic pathway beyond the methylation step. The regulator substance cannot be chlorophyll itself since some chlorophyll-deficient mutants form coproporphyrin like the wild type when ethionine is present. A possible candidate for the hypothetical regulator substance is a protochlorophyllide-type of pigment associated with a specific protein, as may be the case in plants.

THE BACTERIU PHOTOSYNTHETIC APPARATUS

37

3. Regulation of the Branched Pathway

Tetrapyrrole biosynthesis by the photosynthetic bacteria provides a good example of a branched pathway stemming from common intermediates. I n addition to the iron and magnesium branches, there is the path leading to vitamin B12,which is present in relatively high concentration in many of these organisms (Cauthen et al., 1967).There must be control mechanisms which ensure the flow of precursors into the appropriate channels to maintain a molar ratio of chlorophyll, haems and vitamins B12of about 1:20: 2000. The magnesium branch is also under some independent control, at least in the Athiorhodaceae, since chlorophyll synthesis can be repressed without preventing the formation of haems and vitamins B12(Lascelles, 1966b; Cauthen et al., 1967). Feedback inhibition of 8-aminolaevulinic acid synthase by haem has been found with partly purified preparations from Rps. spheroides, with supporting evidence from experiments with intact cells for the operation of sucha controlinvivo (BurnhamandLascelles, 1963).Theeffect of haem on the 8-aminolaevulinic acid synthase does not clarify the regulation of the magnesium branch of the biosynthetic pathway. One possibility is that there are two forms of the synthase; one may be specifically concerned with chlorophyllsynthesis under the control of a component of the magnesium pathway, and the other may be associated with haem synthesis. Tenuous evidence for two enzymes (or possibly one enzyme located in different specialized environments) is provided by the albino types of mutant of Rps. spheroides (Lascelles, 1966b; unpublished observations). Such mutants form haems but do not synthesize magnesium tetrapyrroles. These observations alone point to a loss of the magnesium-branch enzymes, possibly under the control of a single gene. However, the albinos also fail to accumulate coproporphyrin under conditions which promote its formation by the wild type or by mutants which are blocked at single steps of the magnesium pathway. The ability to accumulate coproporphyrin may be a manifestation of the unregulated activity of the “magnesium-branch synthase”. Failure of the albino mutants to accumulate the porphyrin could therefore be attributed to the absence of this fork of the synthase. It might be added that there is as yet no direct evidence for two forms of the enzyme or for its compartmentation in the cell.

4. Genetic Control of the Photosynthetic Apparatus There is a complete lack of knowledge of the genetic regulation of the formation of the bacterial photosynthetic apparatus due to failure to find a suitable experimental system, such as recombination or transduction.

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We are therefore forced to resort to speculations based on information about regulation of analogous multi-enzyme systems. Albino mutants, such as Rps. spheroides strain L-57, exhibit many phenotypic changes resulting, apparently, from a single genetic lesion. Examples are known in other complex systems of interactions between individual components which affect their synthesis and organization in the final complex. I n the pyruvate oxidase complex of Escherichia coli, for instance, a mutation affecting the formation of a single enzyme may alter the rate of synthesis of other enzymes of the system and also their capacity to form complexes with each other (Henning, 1966). The closest parallel to the albino mutants is provided by bleached mutants of Euglena and respiratory-deficient strains of yeast and Neurospora. I n these organisms a single genetic lesion results in many changes in the synthesis of enzymes and cofactors of photosynthesis or respiration, coupled with the inability to form normal chloroplasts or mitochondria (Gibor and Granick, 1964; Schiff and Epstein, 1965; Sherman and Slonimski, 1964; Slonimski, 1967). I n Euglena and yeast, both cytoplasmic and nuclear DNA are involved in defining the synthesis of the complete organelle. I n the bacteria, an episomal factor could participate in the control of the photosynthetic apparatus. The discovery of a satellite DNA in some photosynthetic bacteria gives some hope in this direction but there was no evidence that this material was located in the chromatophore fraction (Suyama and Gibson, 1966). It is difficult to envisage how a single mutation can result in such a multitude of phenotypic changes as occurs in photosynthetic and respiratory mutants. Recent work with respiratory-deficient mutants of Neurospora has pointed to a central role for structural proteins in the formation and organization of other mitochondrial proteins (Woodward and Munkres, 1966; Munkres and Woodward, 1966). Structural protein isolated from the mitochondrial fraction of some cytoplasmic mutants differed from that of the wild type by single amino acid replacements. The resulting conformational change affected the capacity of the protein to combine with malate dehydrogenase and other mitochondrial components. The prixtiary effect of the mutation upon the Structural protein had other secondary consequences. The mutants were deficient in cytochromes a and b, but made cytochrome c in excessive amounts ;synthesis of riboflavin and unsaturated fatty acids was also affected, these compounds being present in higher concentration in the mutants than in the wild type. The implications of this work can be applied as a working hypothesis to the case of the development of the bacterial photosynthetic apparatus. One can imagine “foundation molecules”, possibly structural proteins, which have a co-ordinating role in the formation and organization of the

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

39

various components of the photosynthetic apparatus. It might be synthesis of these proteins which is primarily subject to repression or derepression. If chlorophyll formation proceeds via protein-bound intermediates, the protein moiety may be such a “foundation molecule”. For some clarification of the many mysteries we require to examine photosynthetic mutants and revertants in depth. Unfortunately, genetic transfer systems have yet to be discoveredin the photosynthetic bacteria but a diligent search might provide a reward for the brave. REFERENCES Allen, M. B. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 511. Academic Press, New York. Altshuler, T. and Lascelles, J. (1967). Arch. Mikrobiol. in press. Bartsch, R. G. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 315, Antioch Press, Yellow Springs. Benson, A. A. (1964). Annu. Rev. plant Physiol. 15, 1. Bergeron, J. A. (1959). I n “The Photochemical Apparatus, its Structure and Function”, p. 118. Brookhaven Natl. Lab., Upton, New York. Biedemann, M., Drews, G., Marx, R. and Schroeder, J. (1967). Arch. Mikrobiol. 56, 133. Boardman, N. K. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 437. Academic Press, New York. Boatman, E. S. (1964). J . cell Biol. 20, 297. Bogorad, L. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 481. Academic Press, New York. Bril,C. (1958). Biochim. Biophys. Acta 29, 458. Bril, C. (1960). Biochim. Biophys. Acta 39, 296. Buchanan, B. B., Evans, M. C. W. and Arnon, D. I. (1965). In “Non-Heme Iron Proteins”, (A. San Pietro, ed.), p. 175. Antioch Press, Yellow Springs. Bull, M. J. and Lascelles, J. (1963). Biochem. J . 87, 15. Burnham, B. F. and Lascelles, J. (1963). Biochem. J . 87, 462. Carr, N. G. and Exell, G. (1965). Biochem. J . 96,688. Cauthen, S. E., Pattison, J. and Lascelles, J. (1967). Biochem. J . 102, 774. Chance, B., Horio, T., Kamen, M. D. and Taniguchi, S. (1966). Biochim. Biophys. Acta 112, 1. Clayton, R. K. (1962a). Biochem. Biophys. res. C m m u n . 9 , 4 9 . Clayton, R. K. (1962b). Photochem. Photobiol. 1, 201. Clayton, R. K. (1963). Biochim. Biophys. Acta 75, 312. Clayton, R. K. (1966). I n “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 609. Academic Press, New York. Clayton, R. K. and Sistrom, W. R. (1966). Photochem. Photobiol. 5, 661. Cohen-Bazire, G. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 89. Antioch Press, Yellow Springs. Cohen-Bazire,G., Sistrom, W. R. and Stanier, R. Y. (1957). J . cell. comp. Physiol. 49, 25. Cohen-Bazire, G. and Kunisawa, R. (1960). Proc. nat. Acad. Sci. Wash. 46, 1543. Cohen-Bazire, G. and Kunisawa, R. (1963). J. cell Biol. 16, 401. Cohen-Bazire, G., Pfennig, N. and Kunisawa, R. (1964). J. cell Biol. 22, 207. Cohen-Bazire,G. and Sistrom, W. R. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 313. Academic Press, New York.

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Holt, S. C. and Marr, A. G. (1965a). J. Bmt. 89, 1402. Holt, S. C. andMarr, A. G. (1965b).J . Buct. 89, 1413. J. Buct. 89, 1421. Holt, S. C. and Marr, A. G. (1965~). Horio, T. and Kamen, M. D. (1962). Biochemistry 1, 1141. Hulcher, F. H. and Conti, S. F. (1960). Biochem. Biophys. res. Commun. 3,497. Hurlbert, R. E. and Lascelles, J. (1963).J. gen. Microbiol. 33, 445. Jensen, S. L. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 19. Antioch Press, Yellow Springs. Jensen, A., Aasmundrud, 0. and Eimhjellen, K. E. (1964). Biochim. Biophys. Actu 88, 466. Jones, 0. T. G. (1963a). Biochem. J . 86,429. Jones, 0. T. G. (1963b). Biochem. J. 88, 335. Jones, 0. T. G. (1963~).Biochem. J. 89, 182. Jones, 0. T. G. (1964). Biochem. J. 91,572. Kamen, M. D. (1963).I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 61. Antioch Press, Yellow Springs. Kashket, E. R. and Brodie, A. F. (1963a). Biochim. Biophys. Actu 78, 52. Kashket, E. R. and Brodie, A. F. (196313).J. biol. Chem. 238, 2564. Kirk,J. T. 0. and Allen, R. L. (1965). Biochem. Biophys. res. Commun. 21,523. Klein, S. (1962). Nature, Lond. 196, 992. Klein, S. and Bogorad, L. (1964).J. cell Biol. 22, 443. Klein, S., Bryan, G. and Bogorad, L. (1964).J. cell Biol. 22,433. Kodicek, E. (1963). I n “Recent Progress in Microbiology”, (N. E. Gibbons, ed.), p. 23. Toronto University Press, Toronto. Lascelles, J. (1956). Biochem. J . 62, 78. Lascelles, J. (1959). Biochem. J. 72, 508. Lascelles, J. (1960a).J. gem Microbiol. 23, 487. Lascelles, J. (1960b).J. gen. Microbiol. 23,499. Lascelles, J. (1964). Tetrupyrrole Biosynthesis and its Regulation, p. 106. Benjamin, New Y ork Lascelles, J. (1966a). Biochem. J. 100, 175. Lascelles, J. (196613). Biochem. J . 100, 184. Lascelles, 5. and Szildgyi, J. F. (1965).J. gen. Microbiol. 38, 85. Lessie, T. G. and Sistrom, W. R. (1964). Biochim. Biophys. Actu 86, 250. Levine, R. P. and Togasaki, R. K. (1965). Proc. nat. Acad. Sci., Wash. 53, 987. Marr, A. G. and Cota-Robles, E. H. (1957).J. Buct. 74, 79. Marver, H. S., Collins, A., Tschudy, D. P. and Rechicgl, M. (1966).J.biol. Chem. 241, 4323. Motokawa, Y . and Kikuchi, G. (1966). Biochim. Biophys. Actu 120, 274. Munkres, K. D. and Woodward, D. 0. (1966).Proc. nut. Acud.Sci. Wmh. 55,1217. Newton, J. W. (1960). Biochim. Biophys. Acta 42, 34. Newton, J. W. (1962). Biochim. Biophys. Actu 58,474. Newton, J. W. and Levine, L. (1959). Arch. Biochem. Biophys. 83, 456. Newton, J. W. and Newton, G. A. (1957). Arch. Biochem. Biophys. 71,250. Olson, J. M. (1966).I n “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, ads.), p. 413. Academic Press, New York. Olson, J. M. and Romano, C. A. (1962). Biochim. Biophys. Acta 59, 726. Olson, J. M. and Stanton, E. K. (1966).I n “The Chlorophylls”, (L.P. Vernon and G. R. Seeley, eds.), p. 381. Academic Press, New York. Olson, J. M. and Sybesma, C. (1963). I n “Bacterial Photosynthesis”, (H. Gest. A. San Pietro and L. P. Vernon, eds.), p. 413. Antioch Press, Yellow Springs.

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Orlando, J. A., Levine,L. and Kamen, M. D. (1961). Biochim. Biophy8. Acta 46, 126. Pangborn, J., Marr, A. G. and Robrish, S. A. (1962).J . Bact. 84, 669. Porra, R. J. and Lascelles,J. (1965). Biochem. J . 94,120. Rabinowitch, E. I. (1951). “Photosynthesis”, Vol. 11, part 1, p. 702. Interscience, New York. Schachman, H. K., Pardee, A. B. and Stanier, R. Y. (1952). Arch. Biochem. Biophys. 38, 245. Scheuerbrandt, G. and Bloch, K. (1962). J . biol. Chem. 237, 2064. Schiff, J. A. and Epstein, H. T. (1965). I n “Reproduction: Molecular, Subcellular and Cellular”, (M. Locke, ed.), p. 131. Academic Press, New York. Schmidt, K., Pfennig, N. and Jensen, S. L. (1965). Arch. Milcrobiol. 52, 132-146. Schon, G. and Drews, G. (1966). Arch. Milcrobiol. 54, 199. Sedar, A. W. and Burde, R. M. (1965). J . cell Biol. 27, 53. Sherman, F. and Slonimski, P. P. (1964). Biochim. Biophys. Acta 90, 1. Sistrom, W. R. (1962a). J . gen. Microbiol. 28, 599. Sistrom, W. R. (1962b).J . gen. Microbiol. 28, 607. Sistrom, W. R. (1965). J . B a t . 89,403. Sistrom, W. R. and Clayton, R. K. (1964). Biochim. Biophys. Acta 88, 61. Slonimski, P. P. (1967). Fed. Proc. in press. Stanier, R. Y. (1960). Harvey Lectures, 1958-1959, pp. 219. Stanier, R. Y. (1961). Bact. Rev. 25, 1. Stanier, R. Y. (1963).In “GeneralPhysiology of Cell Specialization”,(D. Mazia and A. Tyler, ede.), p. 242. McGraw-Hill,New York. Sugimura, T. and Rudney, H. (1962). Biochim. Biophys. Acta 62, 167. Suyama, Y. and Gibson, J. (1966). Biochem. Biophys. res. Commun. 24, 549. Sykes, J., Gibbon, J. A. and Hoare, D. S.? 1965). Biochim. Biophys. Acta 109, 409. Taniguchi, S. and Kamen, M. D. (1965). Biochim. Biophys. Acta 96, 395. Trown, P. W. (1965). Biochemistry 4, 908. Tuttle, A. L. and Gest, H. (1959). Proc. nat. Acad. S’ci. Wash. 45, 1261. Van Niel, C. B. (1962). Alznu. Rev. p b n t Physiol. 13, 1. Vatter, A. E. and Wolfe, R. 8. (1958).J . Bact. 75,480. Vatter, A. E., Douglas, H. C. and Wolfe, R. S. (1959).J . Bact. 77, 812. Vernon, L. P. (1964). Annu. Rev. plant Physiol. 15, 73. Vernon, L. P. and Ke, B. (1966). In. “The Chlorophylla”, (L. P. Vernon and G. R. Seeley, eds.), p. 569. Academic Press, New York. Virgin,H. I.,Kahn, H. A. andvonWettstein,D. (1963).Photochem. Photobiol. 2,83. Weaver, P., Tinker, K. andvalentine, R. C. (1965). Biochem. Biophys. res. Commun. 21, 195. Weibull, C. and Bergstrom, L. (1958). Biochim. Biophys. Acta 30, 340. Wood, B. J. B., Nichols, B. W. and James, A. T. (1965). Biochim. Biophys. Acta 106, 261. Woodward, D. 0. and Munkres, K. D. (1966). Proc. nut. Acad. Sci. Wash. 55,872. Worden, P. B. and Sistrom, W. R. (1964). J . cell Biol. 23, 135. Yamanaka, T. and Kamen, M. D. (1965). Biochem. Biophys. res. Commun. 18,611.

Note added in proof. Recently, preferential synthesis of membrane protein has been demonstrated in growing cultures of Rps. Spheroides as they adapt from aeroio-dark to anaerobic-light conditions (Gray, 1967). Pigment synthesis is also accompanied by an increase in the proportion of rapidly labelled RNA which is rapidly degrading in the presence of proflavin (Ferretti and Gray, 1967; Cost and Gray 1967.)

The Plasmids of Staphylococcus aureus and their Relation t o Other Extrachromosomal Elements in Bacteria M. H. RICHMOND Department of Nolecular Biology, University of Edinburgh, West Mains Road, Edinburgh 9, Scotland I. Introduction

. . .

A. Historical B. Curing C. Stability . D. The Plasmid Location of the Penicillinase Genes 11. Types of StaphylococcalPlasmid . . A. Plasmid-BorneMarkers . B. Classification of Plasmids . . C . Distribution and Abundance of Plasmid Types D. Plasmid Nomenclature 111. Transduction of Plasmids . A. Preparation of Transducing Phage . B. Transductants Formed in Negative Recipients C. Transductants Formed in Plasmid-Carrying Recipients . D. Compatibility . E. The Role of Attachment in Recombination and DiploidFormation F. Chromosomal Attachment of Plasmid Markers . G. ThePlasmidMap H. Size of the PenicillinasePlasmids IT.A Comparison of Penicillinase Plasmids with Other Extrachromosomal Elements in Bacteria V. Acknowledgements . References .

.

.

.

.

.

43 43 44 45 48 51 51 55 58 59 60 60 60 61 71 75 78 80 83 84 86 87

I. Introduction A. HISTORICAL It has been known for years that certain hereditary characters are unstable in some staphylococcal strains in the sense that they are lost from cultures at a rate greater than can reasonably be explained by normal spontaneous mutation rates ;this phenomenon implies either an abnormally high mutation rate-a genetic hot spot-in the genes 43

44

M. H. RICHMOND

concerned, or a high rate of loss of the entire gene responsible for the character in question, One of the first characters examined in this way was penicillinase production. Although the phenomenon had been mentioned by Voureka (1948),Barber (1949)was the first to examine this matter quantitatively. She showed that, of 32 hospital isolates examined for the stability of penicillinase production, 17 contained penicillinaseless variants when slopes of the strains had been restored for about 6 months a t 2’. Although these results were hardly obtained under “natural” physiological conditions, the instability of penicillinase production in some strains.of Staphylococcus aureus has been amply confirmed, notably by Fairbrother et al. (1954), Novick (1963), Borowski (19631, May et al. (1964) and Asheshov (1966a). However, although the loss of the ability to synthesize penicillinase occurs widely-and has even been shown to occur in vivo (Hamburger et al., 1958; 1960)-the reverse process had never been shown unequivocally to occur under natural conditions. I n recent years other “unstable” characters have been demonstrated in staphylococci. May et al. (1964)have shown that the genes responsible for tetracycline resistance are lost from staphylococci at high frequency, and this is particularly common among strains of the 52/52A/80/81 complex (Asheshov, 1966a; Asheshov and Winkler, 1966). Similarly, Mitsuhashi and his collaborators (Hashimoto et al., 1964; Mitsuhashi et al., 1965) have shown that erythromycin resistance may be an unstable character in some strains, as may resistance to mercury (Hg2+) (Richmond and John, 1964), cadmium (Cd2+),lead (Pb2+)and arsenate (H2As0r or HAsOf) ions (Novick, 1967). Servin-Massieu (1961) has reported that the type and amount of pigment produced by some staphylococcal strains may also be lost at a relatively high frequency, particularly under rigorous culture conditions. B. CURING Certain growth conditions may markedly accelerate the rate of loss of penicillinase genes from 8.aureus. Fairbrother et. at. (1954) showed that the incidence of penicillinase-less variants in certain strains was greatly increased by growth at 44”, and this result has been confirmed by May et al. (1964) and by Asheshov (1966a). These last two groups of workers also showed that the loss of the tetracycline-resistance genes was accelerated in some strains-particularly those in the 52/52A/80/81 complex (Asheshov and Winkler, 1966)-but that the purging effect of growth at elevated temperature was not universal, either for penicillinase or for the tetracycline-resistance genes.

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

45

The clear-cut experiments of Hirota (1960) on the curing of the F (the sex, or fertility) factor from Escherichia coli by acridine dyes has led a number of workers to try to cure staphylococci of their penicillinase genes in a similar manner. Hashimoto et al. (1968) reported that overnight growth of staphylococcal strains in the presence of 25 pg. acriflavine/ml. led to the appearance of penicillinase-less variants-from 0.1 to 3.5% of the total population-in 17 out of the 18 strains tested. Similarly, Harmon and Baldwin (1964) reported 6.2% of penicillinsensitive cells'in a staphylococcal culture grown overnight in the presence of 10 mg. [sic] acridine orange/ml. On the other hand, Novick (1963) has been unable to show any significant curing effe& of acridine dyes on staphylococcal penicillinase genes; nor have P. H. A. Sneath, E. Asheshov (personal communications), or the author, in independent experiments. I n view of these conflicting reports, it is important to try to assess which is correct:At the moment it seems probable that acridine dyes do not cure the penicillinase genes, and the most likely explanation for the discrepancy between the reports of different workers is that neither Baldwin nor Mitsuhashi studied a statistically sjgnificant number of clones from untreated cultures to establish an qccurate enough "control" rate against which to compare the rate of loss of the penicillinase genes after treatment. Another procedure which has been reported to cure genes linked to an F factor in E . co2i is to grow a thymine-requiring mutant of the strain in question in the presence of growth-limiting concentrations of thymine (Clowes et al., 1965). However, growth of a thymidine-requiring mutant of a penicillinase-producingstrain of S. aurew in the presence of limiting amounts of thymidine had no effect on the spontaneous rate of loss of the penicillinase genes (M. H. Richmond, unpublished observation). C. STABILITY Although it is clear from the work already described that the penicillinase- and tetracycline-resistance genes may be unstable in aureus, this instability cannot be demonstrated in all strains. The probability of loss of penicillinase genes from a staphylococcal culture can be calculated from the proportion of penicillinase-synthesizingorganisms in the culture at a given density by means of the equation :

s.

r = -(2 -P)" 2"

where r is the proportion of penicillinase-producing cells in the total culture, p is the probability of loss of a penicillinase plasmid, and n is the

46

M. R. RICHMOND

number of generations the culture has completed from the one-cell stage (Richmond, 1966a). Therefore, in a colony which has grown thirty generations and which is found on examination to contain 1 % of negatives, the penicillinase genes are lost at a probability of rather less than 1:1000 cell divisions. And if the proportion of negatives in the clone is lo%, then the probability of loss is about 1: 150.

000 limit

------1/25

I

l

l

1:24 000 1:6000 1:1500 1:375 1:12000 1:3000 1:750 1:187 Frequency of negatives

FIG.1. Stability distribution among 67 independently isolated hospital staphylococci of phage groups I and 111. Data from Richmond (1966a).

I n practice, it is relatively easy to determine the proportion of penicillinase-less variants present in 20,000-30,000 cells-yet an incidence of one negative/30,000 cells corresponds to probability of loss of about 1 :lo4 and this is still considerably greater than the normally accepted value of about 1 :los for the rate of spontaneous mutation. This gap of about lo4 between the spontaneous mutation rate and the rate of loss

THE PLASMIDS OF STAPHYLOCOCCUS AUR.EUS

47

that is readily measured experimentally has made it difficult to decide whether the penicillinase and the tetracyline genes are relatively unstable in all staphylococcal strains-but it seems unlikely. Figure 1 shows the stability distribution among 67 independently isolated hospital staphylococci from phage groups I and I11 (Richmond, 1966a). This experiment was carried out in such a way that 25,000 staphylococcal colonies were screened from each strain to determine the incidence of negatives. I n all, 33 of the 67 strains showed no penicillinase-lesscells in 25,000 colonies; so in these strains the penicillinase genes, if lost at all, disappeared with a probability of less than 1:lo4. Of the remaining 34 strains, three produced as many as 30 negative wlls/25,000 colonies. In this series of experiments there was no apparent correlation between a high degree of instability of the pencillinase genes and phage pattern. Asheshov (1966a) examined 50 strains of staphylococci for loss of penicillin and/or tetracycline resistance. Of the 50 strains, three showed a loss of one type of resistance when grown at 37’ which was not significantly increased by growth at a higher temperature. On the other hand 12 strains showedlossof resistance to penicillin when grown at 43.5” and, of the 12, three showed an independent loss of tetracycline resistance. All of the 12 strains which showed a high rate of loss of resistance markers at high temperature belonged to the complex of staphylococcal strains having ;t.phage pattern 52/528/80/81. I n these experiments, therefore, it is clear that both the penicillinase and tetracycline genes are unstable in some strains, but it is not possible to conclude that they are stable in any. Although penicillinase-less variants can only be demonstrated in a proportion of staphylococcal strains, there is one strain in which there is good evidence that the penicillinase genes are truly “stable”. This strain is PS80 (the propagating strain for typing phage 80) and Asheshov (1966b) has been unable to detect the appearance of penicillinase-less variants from this strain. Although about 100,000 colonies have been examined in a single experiment for the presence of negatives, this evidence would still be insufficient were it not for two other facts. First, despite the inability to show penicillinase-less variants, mercury-sensitive variants do appear at a reasonable frequency; and secondly, the sensitivity to ultraviolet radiation of phage capable of transducing the penicillinase genes from this strain suggests that the penicillinase genes in PS80 are chromosomal. This strain-and the state of the penicillinase genes in it-will be discussed in detail later (see pp. 49 & 78). Recently K. G. H. Dyke (unpublished experiments) has studied the stability of the penicillinase plasmids in the 97 “hospital” staphylococci 4

48

M. H. RICHMOND

studied early by Dyke and Richmond (1967). Here penicillinase-less variants have been obtained from 93 out of 97 strains; but in one of the remaining strains there is preliminary evidence for a chromosomal penicillinaseregion. It appears, therefore, that a site for the penicillinase genes on a plasmid is much more common than a chromosomal location among naturally-occurring staphylococci.

D. THE PLASMID LOCATION OF PENICILLINASE GENES The high rate of loss of the penicillinase genes from S. a w e m led Novick (1963) to suggest that they were not carried as part of the bacterial

chromosome but were present on an extrachromosomal piece of DNA, which he suggested should be called a plasmid. This term has been proposed by Lederberg (1952) for extrachromosomal particles of various types, and Novick felt it to be important to avoid the term episome which has a precise definition involving the ability of the genes to pass relatively freely backwards and forwards between the chromosomal and extrachromosomal states (see p. 84). Even now, when considerably more is known about the behaviour of penicillinase plasmids, free passage of the genes in question in and out of the chromosome has not been shown unequivocally and for this reason the term plasmid is retained-although the penicillinase plasmids of S. auretm have an obvious similarity in certain respects to the episomes of the enterobacteria (see p. 84). Novick (1963) based his claim that the penicillinase genes in S. aweus were usually extrachromosomal on the following points : 1. The high rate of loss from the cell. This property of extrachromo-

soma1 particles in bacteria had been noticed frequently (e.g. Watanabe and Fukusawa, 1961). 2. I n certain strains, other genetic determinants are linked closely to the penicillinase genes and these are nearly always lost at high frequency en bloc with the penicillinase genes. 3. Where it is possible to transduce the penicillinase genes, the linked genetic determinants are cotransduced with high efficiency. Usually there is a greater than 95% cotransduction of all characters at a frequency of about 1:lo5infecting phage. 4. When such transducing phage preparations are inactivated by ultraviolet radiation, the frequency of transduction falls exponentially with the irradiation dose at a slope similar to that of the ultraviolet-radiation inactivation slope for the plaque-forming activity of the phage itself. Of these criteria, only the last is really convincing although the

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS’

49

examination of strain PS80 (see p. 78) has produced circumstantial evidence to support the validity of the first three of the points cited. The conclusion as to whether a gene is chromosomal or extrachromosoma1 on the basis of its sensitivity to ultraviolet radiation when undergoing transduction is based on Arber’s (1960) experiments concerning the transduction of genetic markers between E. coZi strains using phage P1. I n these experiments, one type of donor strain carried a variety of chromosomal markers, while the other carried either the episomal F particle or prophage A. Transduction of the chromosomal markers to a suitable recipient by phage P1 occurred at a frequency of about 1:lo7 and this frequency could be increased about ten-€old by ultraviolet irradiation of the transducing phage. But the ability of phage P1 to transduce the extrachromosomal F or h characters occurred at a frequency of about 1:lo5 and decreased logarithmically with ultraviolet irradiation. The reason for this difference in the behaviour of transducing phage preparations depending on the original genetic location of the transduced genes is not well understood. One suggestion is that ultraviolet irradiation activates repair enzymes carried in the phage particles (or causes them to be formed in the recipient cell) and that this process has the effect of facilitating the integration of transduced chromosomal genes into the chromosome of the recipient, while having no effect on the transfer of extrachromosomal genes. Whatever the reason for the response of the transducing phage to irradiation, Arber’s observations-which were made on E. coZi-appear to hold for S. aureus as well. Korman and Berman (1962) showed that ultraviolet irradiation of transducing phage increased the frequency of transduction of streptomycin resistance-and this gene is believed to be chromosomal in 8.aureus. On the other hand, Novick (1963) showed that there was an exponential decrease in the transduction frequency of the penicillinase genes on irradiation under circumstances in which there was some additional evidence that the genes in question were located on a plasmid (points 1-3 above). Perhaps the most convincing evidence that experiments of the Arber type indicate an extrachromosomal location in X. aureus comes from a study of strain PS80 (Asheshov, 1966b).This strain is resistant to tetracycline and to mercury salts, and synthesizes penicillinase. Both tetracycline-sensitive and mercury-sensitive variants can be obtained a t a relatively high frequency, but the loss of these two characters always appears to be due to independent genetic events. On the other hand, penicillinase-less variants do not appear from this strain-at least at a rate greater than the experimental limit of 1:lo6. This evidence suggests, prima facie, that the mercury and tetracycline genes are carried on

50

M. H. RICHMOND

separate plasmids, while the penicillinase gene is chromosomal. Since a penicillinase-lessvariant cannot be obtained from PSSO, another strain must be used as recipient in transduction experiments, and strain 17855 which was sensitive to penicillin, tetracycline and mercury salts was chosen for this purpose. Propagation of typing phage 80 on PSSO leads to a phage preparation capable of transducing all these genetic markers, although the tetracycline marker is transduced at a frequency of about 1 :5 x l o 5 whereas the penicillinase genes are transferred only at a

0

25

50

7.5

Period of exposure to ultraviolet rodiation (inin)

FIU.2. Transduction frequency of resistance to penicillin (o),to HgCl2 (A) and to tetracycline (A)as a function of the period of exposure to ultraviolet irradiation of the transducing phase. Donor strain: StaphyZococcw aureus PS80. The phage survival curve is also shown (0). Data from Asheshov (1966a and unpublished experiments).

frequency of about 1 :lo8. This difference in rate, in itself, suggests the extrachromosomal location of tetR (Arber, 1960). Ultraviolet irradiation of the transducing phage preparation has the effect shown in Fig. 2. The transduction efficiency of the HgR and tetR markers falls exponentially, whereas transduction of the penicillinase genes, when unlinked to the Bg marker (seep. 52), is increased about ten-fold (Asheshov 1966b). The internal consistency of these results therefore supports the view that a high rate of spontaneous loss and a decrease of transducing efficiencyon irradiation are characteristic of an extrachromosomal gene in S. aureus.

THE PLASMIDS O F STAPHYLOCOCCUS AUREUS

51

11. Types of Staphylococcal Plasmid

A. PLASMID-BORNE MARKERS A number of different types of penicillinase plasmid have been identified on the basis of the genetic determinants they carry. Originally the different types of plasmid were distinguished according to the immunological type of penicillinase they synthesized, whether they carried resistance markers for mercury salts and erythromycin, and the extent to which the penicillinase was extracellular (Richmond, 1965a ; Novick and Richmond, 1965).Although not all the possible combinations of markers were found, in practice it was possible to detect the presence of ten different plasmid types (called 01 to K ) in varying abundance among hospital staphylococci (Dyke and Richmond, 1967). I n addition, tetracycline resistance is plasmid-borne but apparently is always on a plasmid separate from that carrying the penicillinase genes (May et al., 1964). The identification of more genetic determinants on staphylococcal plasmids (Novick, 1967) has further complicated the situation and, a t the moment, the following markers may be present in the plasmid state in S. aureus : 1. The Penicillinase Region

This is a group of genes responsible for the production of penicillinase.

It probably consists of a structural gene p (for penicillinase) and a gene involved in response to inducer, i (for inducibility) and one responsible for “setting the rate” of penicillinase synthesis, pr (for promoter) (Richmond, 1966b). Three immunological variants of the p gene have been identified (Richmond, 1965b).They lead to the synthesis of the A-, B- and C-types of penicillinase, and the genes responbible are called p A ,pBand p c respectively. 2 . The “Extracellukcrity” Region (exo)

This region determines the degree of extracellularity of the penicillinase under standard cultural conditions. I n the early work (Richmond, 1965a; Novick and Richmond, 1965) two characters were recognized-high ( > 25%) and low ( < 25%) extracellularity. However, measurement of the proportion of enzyme in the extracellular state in a number of hospital staphylococci (Fig. 3) shows a biphasic distribution in the quantity of extracellular enzyme (K. G. H. Dyke, unpublished experiments). It seems from these results that the boundary between “high” and “low” liberation should be set at 10 rather than 25%. Bearing in

52

M. H.RICHMOND

mind the difficulties of measuring the true quantity of an extracellular enzyme, and the doubts that must exist as to whether small amounts are truly “extracellular” (Pollock, 1962), it is even possible that the types of extracellularity shown by staphylococcal strains should be classified as all or none (i.e. + or -), rather than setting the boundary at some arbitrary level.

% penicillinase extracellular

FIG.3. The proportion of penicillinase present in the culture medium of 97 staphylococcal strains carrying various pencillinase plasmids.

3. Erythromycin Resistance (eroR)

This marker confers resistance to all macrolide antibiotics, the cells carrying the marker being about 100-1000 times more resistant to the antibiotic than those which do not. The marker is plasmid-borne only in a small minority of erythromycin-resistant staphylococcal strains. The biochemical basis of the resistance is unknown. 4. Resistance to Mercury Salts (HgR) This marker confers a six- to ten-fold increase in resistance to Hg2+ salts and their organic derivatives such as phenylmercuric nitrate. Resistance does not depend on the accumulation of -SH groups within resistant cells-a possible mechanism for resistance because of the avid reaction of -SH groups with Hg2+-but probably reflects a change in membrane properties which excludes the mercury ions from the cell interior (M. H. Richmond, unpublished observations). 5. Resistance to Cadmium Salts (CdR)

This marker is genetically distinct from the HgR marker described above and confers a twenty- to fifty-fold increase in resistance on cells which carry it. The biochemical basis of resistance is unknown. As with the HgR marker, cadmium resistance could involve accumulation of -SH compounds in resistant cells, but this does not seem to occur.

v

a

FIG.4. Staphylococcal colonies (a)before, and (b) after stainmg t o show the presence of penicillinme(darkcolonies). Note how the targot-likecolonies in Fig. 4a are those which do not synthesize penicillinme (Fig. 4b) and therefore do not ca.rrya penicillinme plasmid. Photographs are reproduced by kind permission of Dr. R. P. Novick.

01 0

54

M . H . RICHMOND

Once again, modification of membrane properties is the more likely cause of resistance; but the separate genetic identity of the HgR and CdR marker implies that the biochemical processes conferring resistance are distinct in these two cases. Staphylococci which are resistant t o Cd2+ are also resistant t o Zn2+-but with this ion the difference in the level of resistance is less than with Cd2+(Novick, 1967). 6. Resistance to Arsenate ( h a R )and Arsenite ( A s i R ) I o n s

These are the markers conferring resistance t o arsenate and arsenite ions respectively. Phosphate seems to exert a competitive protection against the inhibitory effect of arsenate but not arsenite, and consequently the phosphate composition of growth media is critical in the detection of arsenate-resistant staphylococci. 7. Other Plasmid-Carried Murkers

A number of other characters may be located on penicillinase plasmids but less is known about their distribution and character. Novick (1967)

FIG.5. The edge of a region of confluent staphylococcal growth on solid medium, stained to show penicillinase production (dark area). The arrows show small, faster growing penicillinase-less sectors at the edge of the growth. The surface of the growth is aIso studded with minute penicillinase-less papillae. Photograph reproduced by kind permission of Dr. R. P. Novick.

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

55

has detected a marker responsible for resistance to Pb2+which is genetically distinct from the Cd", HgR and Asa" regions, but little is yet known of the properties of this marker. Si&larly, certain plasmids seem to carry a determinant which expresses itself as a dominant sensitivity to Bi3+ions. Again, nothing is known about the biochemical basis of this bizarre observation. The carriage of certain plasmids seems to be associated with changes in colonial morphology. Figure 4a shows a mixture of unstained plasmidpositive and plasmid-negative clones to demonstrate this point. Those parts of the mixture carrying a penicillinase plasmid can be seen as the dark regions in the stained version of the same field (Fig. 4b). Although the colonial morphology appears to be plasmid-linked in these experiments, it is not expressed in every host to which the plasmid is transferred. The colonial morphology marker may, in fact, just be a rather special example of the differences in rate of colonial growth often found where growth of penicillinase-less variants is compared with their plasmid-carrying relatives (see Fig. 5).

B. CLASSIFICATIONOF PLASMIDS The characteristics of ten penicillinase plasmids which had been classified on the basis of the type of penicillinase synthesized, their resistance to erythromycin and mercury salts, and whether more or less than 25% of the penicillinase is excreted into the growth medium, are shown in Table 1. However, the recognition that the division between "low" and "high" extracellularity should be 10% and not 25%, and the discovery of the Cd" and Asa" markers, has meant that this classification has to be re-organized, and this is also shown in Table 1. 1. Plasmid a (A-type enzyme, e m s ; HgR; > 25% exo) is now divided into two different plasmid types: the more fully defined a-plasmid (A-type enzyme; eras; HgR; CdR;h a R ; > 10% exo) and a new h plasmid (A-type; eras; HgR; Cds; Asas; > 10% exo). 2. Plasmid /3 (C-type enzyme; e m s ; HgR; < 25% exo) is now (C-type enzyme ; e m S ; HgR; CdR; AsaR; < 10% exo). 3. Plasmid y (A-type enzyme; em"; Hg"; > 25% e m ) is now (A-type enzyme ; e m R ; HgR; CdR; Asa" ; > 10yoexo). 4. Plasmid 6 (B-type enzyme; e m s ; Hg"; > 25% exo) is now (B-type enzyme; e m s ; Hg"; Cd"; AsaR; > 10% exo). 5. Plasmid E (A-type enzyme; e m s ; Hgs; > 2 5 % e m ) becomes two different types of plasmid: the more fully defined eplasmid (A-type enzyme ;emS;Hgs ; Cds ; Asas ; > 10% exo) and a new p-plasmid (A-type enzyme ; eros ;Hgs ; Cd" ;Asa" ; > 10yoexo).

TABLE1. The “Original” (Richmond, 1965a;Novick and Richmond, 1965)and the “Revised” Plasmid Classifications Plasmid &

I

A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Eg2+ Reaction t o erythromycin Proportion of extracellular penicillinase

A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Hg2+ Reaction t o erythromycin Reaction to Cd2+ Reaction t o HASO& Proportion of extracellular penicillinase a

b

\

a

j

3

y

8

r

[

T

8

+

-

f

-

+

-

-

+

+

+ R S

R R

S S

S S

R S

-

R S

-

+ + - R S

R S

S S

~

K

I

-

+

Earlier version

S S

>25 <25 > 2 5 >25 >25 > 2 5 > 2 5 <25 <25 <25

+ - + - + - -

f

-

-

-

+ + + + - -

R S R R

R S R R

R R R R

S S R R

S S S S

S S S S

R S S S

R S R R

S S S S

> 1 0 <10 > 1 0 > 1 0 >10 > 1 0 > 1 0
+

+ 7

- S s s R

S S

R R

>10 >10

Present version

J

The 5 plasmid is often accompanied in the cell by CdRand AmRmarkers which appear to be chromosomal. In two out of four cases so far, plasmid 6 is accompanied by an HgR marker which appears to be chromosomal.

57

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

eras;

6. Plasmid 5 (C-type enzyme; Hgs; > 25% exo) is now (C-type HgS; Cds; Asas; > 10% exo). Plasmid 5 is often accomenzyme; panied in the cell by CdR and AsaR markers which appear to be chromosomal (K. G. H. Dyke, unpublished experiments). 7. Plasmid 7 (C-type enzyme; HgR; > 25% exo) now becomes (C-type enzyme ; eroS;EgR;Cds ; Asas ; > 10% exo). 8. Plasmid 0 (A-type enzyme; HgR; < 25% exo) now becomes HgR; CdR; AsaR; < 10% exo). (A-type enzyme; 9. Plasmid L (A-type enzyme; eroS; H f ; ~ 2 5 %exo) now becomes Hgs; Cds; Asas; < 10% exo). (A-type enzyme;

eras;

eras;

eras; eras;

eras;

eras;

The K-plasmid (C-type penicillinase; Hgs; < 25% exo) disappears from the classification as all five strains examined in the survey by Dyke and Richmond (1967) excreted between 15 and 25% of their enzyme into the medium. However, examples of this plasmid do exist within the staphylococcal population. Apart from these plasmids a number of other combinations of markers have recently been detected, although it has been decided not to add them to the classification until they have been investigated further. These plasmids and their characters are shown in Table 2. TABLE2. Genetic Markers Carried by Plasmids Which are Not Yet Included in the General Classification (see Table 1) Strain No. A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Hg2+ Reaction to erythromycin Reaction to Cdz+ Reaction to HAsO42Proportion of extracellular penicillinase

1060

3951

608

3892

-

-

-

+-

S S

R R

S S S R

S S

10

10

10

+

+

+ R

S

-

S S S S

s

According to this classification, therefore, a t least ten distinct penicillinase plasmids have been detected under natural conditions so far. I n addition to these plasmids, however, one other related plasmid is known. It is carried by strainPS80, and, althoughit lacks the penicillinase region, it is clearly related to the penicillinase series of plasmids by possessing CdR, h a R and HgR regions. I n this strain, the penicillinase ( p A )region is probably chromosomal. I n addition to the penicillinase plasmids, staphylococcal strains often carry a plasmid conferring tetracycline resistance. These are found most

38

M. H. RICHMOND

commonly in strains carrying the cc-penicillinase plasmid (the plasmid common in hospital staphylococci) but they also occur with E-, 5-, 8-, L- and A-plasmids.

C. DISTRIBUTION AND ABUNDANCE OF PLASMID TYPES Any attempt to assess the distribution of plasmid types in the natural population of staphylococci is precarious because of the difficulty of ensuring that repeated isolates of the same strain are not included in the survey. However, if strains of distinct phage pattern and wide geographical separation are chosen for the survey, the chance of duplicating strains is largely eliminated. After taking these precautions, K. G. H. Dyke (unpublished experiments) studied the distribution of plasmid types among 97 hospital isolates of phage group I and I11 strains, and his results are shown in Table 3. The CL-, E-, 5-, 9-,7-, L - , A- and p-plasmids were found in various proportions, although the a-plasmid was by far the most common. No correlation between plasmid type and phage pattern emerged. TABLE3. Distribution of Plasmid Types among 97 “Hospital” Staphylococci of Phage Groups I and I11 Plasmid type

No. of strains

cc

43 0 0 0 13 26

P

Y

6. E

5 e

1 1

rl

10 0 2 1

I

x K

P

TOTAL

97

a Apparently confined t o Group I1 strains and therefore absent from this series.

A previous survey has shown that the immunological B-type of penicillinase is always associated with staphylococci from phage group I1 (Richmond, 1965b). I n this case, 12 group I1 strains were tested, but in only one case was there clear-cut evidence that the penicillinase genes were extra-chromosomal ;that is that plasmid 6 was involved.

THE PLASMIDS OF STAPHYLOCOCCVS AW7REVS

59

Two plasmids ( p and y ) listed in Table 1 appear to be extremely uncommon. The p-plasmid has been isolated once only. This was in strain 147-a strain isolated in a food poisoning outbreak in the 1930s (Segalove, 1947).Its rarity nowadays may just reflect that environmental conditions, as far as the staphylococcus is concerned, may have changed somewhat since the 1930s. The y-plasmid is unusual in that it carries genes conferring resistance to erythromycin. This plasmid has been detected a number of times in Japan (Mitsuhashi et al., 1965), but these isolates may well be the same strain. It is interesting that this plasmid may be a recombinant between a penicillinase plasmid ( ? a )and a piece of bacterial chromosome carrying an erythromycin-resistance region. Furthermore, it may represent the first step in the accumulation of resistance markers in a single extrachromosomal element ; a process familiar enough to those concerned with the R-factors of the Enterobacteriaceae (see Datta, 1965, for a review of this topic).

NOMENCLATURE D. PLASMID The nomenclature now used for X.aurew penicillinase plasmids is an extension of that proposed by Novick and Richmond (1965). The first figure in the designation is the strain number, and this is followed by the Greek letter which describes the type of plasmid carried and which refers to the plasmid as a whole. The Greek letter is followed by letters and subscript figures indicating the markers which are carried on the plasmid. The symbols p A ,pBand p c refer to the structural gene for the immunological type of penicillinase while the symbol i, when used without letters and figure subscripts, refers to the penicillinase inducibility locus. This region may be considered t o consist of two parts : il, indicating that part of the i gene in which mutation gives rise to mutants of the magnoconstitutive type; and i t ,the region which givesrise to baso-constitutive mutants. (For details of the behaviour of these mutants, see Richmond. 1967b). Mutations in the p and i genes are numbered in series; e.g. pxiZ indicates the structural mutant No. 2 in the penicillinase A-type gene; and iijs indicates the constitutive mutant No. 6 in the i i region. Resistance to mercury and cadmium salts is designated as HgR and CdR respectively. Similarly resistance to erythromycin is designated eroR, t o arsenate, AsaR, and to arsenite, h i R . Strains which carry no penicillinase plasmid fall into two classes: those that are naturally-occurring plasmid-less strains and those which are known to have lost a plasmid. The former are designated simply by their strain number, e.g. 8325; and the latter have (N) added, e.g. 147 (N).

60

M. H. RICHMOND

I n laboratory strains, in which recombination has occurred betweer different plasmids, the designation indicates which of the markers comet from which plasmid if this is known. Thus 8 3 2 5 1 ~i+@. . HgR...y .eroR: designates strain 8325 carrying a recombinant plasmid, the penicillinasc and mercury resistance regions being contributed by plasmid E and erythromycin resistance by plasmid y. I n plasmid diploids, the respectivc components are separated by an oblique stroke, e.g. : 147(cc.i+p$.H g R . . y .eroR//3. iip;. H@ .eras) .

III. Transduction of Plasmids A. PREPARATION OF TRANSDUCING PHAGE So far the only method available for transferring staphylococcal plasmid-borne markers from cell to cell is transduction: all attempts t c discover a mating system in 8. aureus have failed to date. Broad13 speaking, two main methods of carrying out transduction experimentE have been used. One is to propagate a staphylococcal phage-usually one of the international series of typing phages-in the donor culture by lytic cycle; the other is to induce prophages carried in the donor by irradiating the cells with ultraviolet radiation. As far as the first method is concerned, typing phages 47, 53, 80 and 81 have been used most frequently, but phages 29, 52A and 79 alsa appear to be effective-at least for the transduction of the tetR marker (Pattee and Baldwin, 1961). The efficiency of transferring plasmid markers in this way is variabIe and may range from 1 :lo6 to 1 :lo8. If on the other hand, the transducing phage is obtained by ultraviolet irradiation, the transducing frequency obtained is much more variablebut can sometimes be as high as 1 :5 x lo3 (M. €€.Richmond, unpublished experiments). Although both of these methods of phage production have been used fruitfully, it is important to stress that transduction by a phage preparation propagated by lytic cycle is preferable. On the whole, phage obtained by this means is homogeneous whereas ultraviolet irradiation of a staphylococcal culture frequently induces a number 01 phages, only some of which will be able to transduce. However, for experiments in which it is desirable to transfer plasmids reciprocally between two strains, it may be necessary to rely on the phages which are in the prophage state in the strains concerned.

FORMED IN NEGATIVE RECTPIENTS B. TRANSDUCTANTS Transduction of the penicillinase genes was first described by Morse (1960) and Ritz and Baldwin (1958, 1961), and since that time it has

THE PLASMIDS OF S!l’APHYLOCOCCUS AUREUS

61

been shown that all the genetic markers detectable on penicillinase plasmids can be transduced (Novick, 1963; Richmond and John, 1964; Novick and Richmond, 1965). Similarly the tetracycline-resistance gene(s)can be transferred by appropriate transducing phage preparations (Pattee and Baldwin, 1961 ; Collins and Macdonald, 1962; Asheshov, 1966a), but the penR and tetRregions are never cotransduced. When the recipient in a transduction experiment is a negative or derived negative, the plasmid from the donor is transferred with a frequency of 1 :105-1 :los, and the incoming plasmid is normally held in the plasmid state in the recipient (Novick, 1963). However, cotransfer of all plasmid markers to every transductant does not always occur, and this implies that certain recipients receive an incomplete plasmid. This process, which has been called “plasmid dissociation” (Novick and Richmond, 1965), is normally uncommon but its incidence can be greatly increased by irradiation of transducing phage preparations. Table 10 (p. 74) compares the markers that were transferred to a recipient, with those present on the plasmid in the donor, in one experiment in which the transducing phage was irradiated with ultraviolet radiation (Novick, 1967). The nature of the genetic deletions obtained in this way throws some light on the order of the genetic markers on the plasmid, and this point is discussed later (p. 80).

C. TRANSDUCTANTS FORMED IN PLASMID-CARRYINGRECIPIENTS If the recipient cell already contains a penicillinase plasmid, one of a number of transductant patterns emerges depending on the types of plasmid involved, and on how soon the transductants are selected after mixing the recipient with the phage preparation. These patterns can be classified as follows : 1. Recombination or apparent displacement; 2. Formation of transient diploids; 3. Formgtion of normal stable diploids; 4. Formation of associated stable diploids. Broadly speaking, the first two of these patterns are found in transduction experiments in which the donor and recipient plasmids are of the same (or closely similar) type, whereas the latter two occur when the two plasmids involved are different. 1. Plasmid Recombination and Apparent Displacement

If the two plasmids involved in a transduction experiment are of the same type (e.g. =/a,/3//3, or y / y ) , and selection is delayed for 5-6 hr. after mixing the phage and the recipient cells, the transductants are normally haploid and contain a relatively large number of plasmid

M. R. RICHMOND

62

recombinants. A typical experiment of this type is as follows :

#(a.iipi) -+ 8325(~.i+p2,,) Donor Recipient

(1)

I n this experiment, the transductants were selected with penicillin (which allows survival of the i-p+, i-p- and i+p+genotypes but not i+p-), and the relative abundance of parental and recombinant plasmids is shown in Table 4. The donor parental phenotype and both possible recombinants were found, and the recombination between the i i andpZlz sites occurs with a probability of about 5%. At first sight, crosses of this type open up the possibility of mapping the plasmids by three-point crosses, but there are certain difficulties involved in the interpretation of such experiments and these are discussed in detail later (p. 80). i

TABLE4. Relative Abundance of Parental and Recombinant Transductants Obtained by Selecting the Cross $(a.i - p i ) -+ 8325 (a.i+pG2)with Penicillin

Genotype Parent (donor) (a .$ - p i ) Recombinants (a.i-pz2) (..i+pi)

No. of transductants

Frequency of transductants

% of donor rate

612 37 29

1:2~105 1:1,2~107 1 : 9 x 10s

6 4.8

-

Recombinants are also common when certain plasmids of different types are placed together in a cell by transduction. Pairs that behave in this way are a/y and yla. Even when a/. crosses of this general type are performed with plasmids marked in such a way that it is possible to enrich the transductants with diploids should they occur-for example in the cross

~(a.iipi.CdR.AsaS.eroR) ---f 8325(~.i+pZ,~. CdS.AsaR.eros) (2) selected with either cadmium + arsenate ions or arsenate + erythromycin, transient diploids occur to an extent of less than 2% of all the transductants, as long as selection is delayed about 6 hr. after the phage is mixed with the recipient. Table 4 shows that transductants in which the incoming plasmid ( a .i-pi) appears to have displaced the resident ( a .i+pxI2)plasmid are the most common transductants selected from cross (1). If, as seems likely, the recipient plasmid is held in the cell a t an attachment site (see later), then the apparent displacement of a resident by an incoming plasmid can occur in two ways. Either a recombination between the two plasmids occurs near the point of attachment but outside the range

63

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

of known genetic markers; or the two plasmids compete for the attachment site in the recipient cell. Which of these two processes is responsible for the apparent displacement found in transduction experiments of this kind is not yet known. 2. Transient Diploids

If transduction ( 2 ) (see above) is carried out and selected either with cadmium + arsenate ions, or with arsenate + erythromycin within 50 min. of mixing the phage and the recipient cells, doubly resistant transductant colonies are obtained at a frequency of about 1 :lo6. If these transductants are now picked into ice-cold liquid medium so that no growth is possible, and plated to give single colonies on a variety of selective media, the characteristics of the colonies that arise show clearly that the transduced cell that was originally plated on the double selective medium was diploid. Furthermore, the composition of the colonies grown from such cells under double selection shows that the diploid state has survived in about 10-15y0 of the cells to the time the original colony was picked and replated. Examination of the remainder of the colony shows that it is made up of doubly resistant recombinants and singly resistant segregants (Table 5). TABLE5. Typical Composition of Transductant Clones from the Cross +(a.i-ph.CdR.AsaS.eroR --t 8325(cc.i+p,l,.CdS.AsaR.eros) when Selected on Medium Containing (a) Cadmium+ Arsenate, and (b) Arsenate + Erythromycin

No. of clones examined

+

(a) Selected on Medium Containing Cadmium Arsenate Donor i-p: .CdR.Asa'.eroR Recipient i+p&. Cd' ,AsaR.ero' Recombinant , i - p z CdR.AsaR.ero' Diploid i-p: .CdR Asas. eroR/i+pz2.Cd' AsaR.e r 8

.

.

61 46

3

.

12

TOTAL

+

(b) Selected on Medium Containing Arsenate Erythromycin Donor i-p: .CdR.Asas.eroR Recipient i+pGa.Cd' AsaR.eroS Recombinant i - p t CdR,AsaR.eroS Diploid i - p i CdR.Asa'. eroR/i+p&.Cd'. AsaR.ero'

. .

122

38 35 1 12

.

TOTAL

96

Single transductant clones were emulsified in ice-cold medium and plated on nonselective medium t o determine the relative abundance of the various possible genotypes in each clone.

64

M. H. RICHMOND

The survival of the singly resistant segregants in these colonies was unsuspected. It appears to be due to the persistence of phenotypic protection against a selective agent for a time after the genetic element conferring resistance to that selective agent has been lost by segregation. The instability of this type of diploid is shown by the fact that a clone containing 10-15% of diploid cells, picked from a double selective plate, contains less than 10% of diploids after 4 hr. growth in a liquid nonselecting medium. By this stage, the clone is almost completely composed of segregants of either parental or recombinant genotype. But the fact that diploids survived even this long in the colony argues that transient diploids are not formed by abortive transduction but can survive a sufficient number of generations to contribute to the composition of a fully grown colony, provided double selection is maintained. On the basis of the results of this and the previous section, therefore, it appears that transduction of an a-plasmid (or an w y recombinant) to a cell already containing an a-plasmid leads t o the formation of transient plasmid diploids. While the diploid state survives, recombination may take place, but segregation to give a haploid cell, containing either a single parental or recombinant plasmid, occurs rapidly. Selection for both plasmid components of the diploid, if applied up to 50 min. after mixing the phage with the recipient cells, may lead to the survival of a predominantly diploid clone for a considerable time; but even this process fails ultimately to preserve a relatively pure clone of diploids for, as time goes on, the colony becomes composed increasingly of doubly resistant recombinants. If the selection is delayed until 6 hr. after transduction, most of the transient diploids have segregated and only cells carrying a single parental or recombinant plasmid survive. 3. Normal Diploids With certain pairs of dissimilar plasmids (e.g. alp, pla, Ply, yip) recombination is uncommon, and relatively stable plasmid diploids are formed more or less regardless of the delay in selecting the transductants. Formation of diploids of this type was fist s h o h in cross (3) selected with penicillin (Richmond, 1964, 1965c):

i-p;) 4 8325(a.i+pAp) (3) Subsequently, however, similar diploids have been constructed in the reverse direction by selecting either with erythromycin (if a y or a . . . y recombinant plasmid is carried in the donor: cross (3) ) or with cadmium or arsenate ions if Cds or Asas alleles are on the recipient r$@.

THE PLASMIDS O F STAPHYLOCOCCUS AURJCUS

65

(crosses (4) and ( 5 )) ;similar methods may be used to construct diploids involving the /3- and y-plasmids (Novick, 1967).

.

8325(u.i-p; . . y.eroR) ---f 147(/3.i+p&.eroS) 8325(a.i-p;. CdR) -+ 147(/3.i+p6.Cds) 8325(a.i-p;. AsaR) --f l47(/3.i+&.AsaS)

(4) (5) (6)

Broadly speaking, all normal a//3 diploids seem to show a number of characteristics in common whichever plasmid is in the recipient cell when the diploid is constructed, and these characters can best be described by referring to the behaviour of the typical diploid 147(a.i+pi . . . y.eroR//3.i-p&.eroS)(Table 6). It is true that the characters are not found in a//3diploids where certain types of regulatory gene mutation in the penicillinase region-notably those involving a micro-inducible or micro-constitutive mutation-are carried on one of the plasmids. But such diploids are described in detail by Richmond (1966b, 1967b) and are not discussed further here. (a) The rate of segregation of the normal diploid to the two possible haploid states-by loss either of the a- or the 13-plasmid-occurs with a probability which reflects the spontaneous rate of loss of a and /3 when present alone in a cell (Richmond, 1965~). With strains 147 and 8325, the rate of segregation of the plasmids is approximately 1 :1000 divisions, whether they are haploid or part of a diploid. Every diploid clone, therefore, tends to be contaminated with approximately equal numbers of haploid segregants. (b) If one plasmid in the diploid carries an intact i+(fully inducible) allele while the other carries i- (fully constitutive), the diploid is inducible. I n the diploid 147(a.i+pi . . . y .eroR//3.i-p;. eras) the uninduced level of enzyme expression in the diploid is about 8 enzyme units/mg. dry wt. organisms as opposed to about 3 units/mg. for the haploid strain 147(a,i+p+. . . y.eroR) (Table 6). Thus the i+ allele represses the expression of i- in the trans position in the diploid, but in this case is not quite as effective at repressing C-type enzyme expression as a similar region would be acting cis (Richmond, 1965~). I n a diploid carrying two if alleles [e.g. 147(a.ifp; . . . y .eroR//3.i+p& .eras) 1, the basal level is about 1 unitlmg. compared with 5 unitslmg. for 147(a.ifp1 . . . y.eroR) and 3 units/mg. for 147(/3.i+&.eroS).The presence of two i+ regions in a single cell therefore causes a somewhat lower level of expression of both structural genes than is found for a single structural gene in the haploid state (K. Smith, unpublished experiments). (c) I n i+/i- or i+/i+diploids, the A- and C-types of penicillinase 5

TABLE6. Phenotypic Characteristics of the Stable Diploid 147(u.i i p z . . .y .eroR/8.i&p;. eras) Penicillinase synthesis (unitslmg. dry wt. bacteria) I

uninduced Strain

Genotype

Immunological typeofenzyme

induced 7

A-type

C-type

'

A-type

C-type

263

-

p1

Inducible donor Constitutive recipient Diploid

147(u.;+p:. ..y.eroR)" 147(8.i&p$. era') 147(~.i+pi.. .y . eroR/p.i.&v$ .eras)

Segregants

147(a(.ifpf.. .y.eroR) 147(8.iG3p;.eras)

A C A+C

-

3

217

-

C

248 128

c-------v---J 86

A

-

147

2.4

-

-

264

293

-

275

291

a The expression of the (a.i + p t . . .y . eroR) plasmid is shown in strain 147 rather than in the normal host, 8325, to allow closer comparison with the expression of this plasmid as a part of a plasmid diploid in strain 147. b About half of this enzyme was immunological A-type and about half C-type.

I

B 3

THE PLASMIDS OF STAPHYLOCOCCUS A V R B V S

67

(characteristic of the a- and /?-plasmidsrespectively) are synthesized in approximately equal amounts in the absence of inducer (Richmond, 1965~).Furthermore, if the penicillinase genes on both the plasmids in the diploid can be induced (as is the case with i+/i- and i+/if diploids) then the A- and C-types of enzyme are also equally represented in induced cultures (Table 6). The maximum quantity of penicillinase that appears to be synthesized by an a//? diploid in either strain 8325 or 147 (with or without induction) is about the same as found in a fully induced or magno-constitutive haploid. This is best demonstrated by comparing the quantity of penicillinase synthesized by the diploid 147(a.iipJ/?.i-p6) with147(a.i-p;) and 147 (/?.i-p&) (Table 7). I n all these cases the maximum quantity of penicillinase synthesized amounts to about 1% of the dry weight of the cell. In a//?or /?/y diploids, both A- and C-type enzyme are liberated into the growth medium t o the extent found in haploid cultures (Table 8). Thus the proportion of A- and C-type enzyme in the growth medium of an a/@ (or @/a)diploid is unequal since about 45% of A-type enzyme is normally liberated from a haploid cell carrying an a-plasmid while only about 5% of the C-type enzyme is extracellular in a culture carrying the /?-plasmid alone. These five characteristics allow a tentative picture of the behaviour of penicillinase plasmid diploids of the normal type to be drawn, particularly with respect to penicillinase production. The fact that the two plasmid components of the diploid co-exist in the cell for relatively long periods without recombining, and the fact that the presence of two plasmids in a cell does not seem to affect the stability of either, suggests that, in this type of diploid at least, the plasmids are held in the cell by two separate attachment sites. According to this model, these sites would have the property of regulating the rate of plasmid multiplication to that of the host cell and also ensuring that each daughter cell received at least one copy of the plasmid at division. The loss of plasmids, to yield derived negatives, might then reflect the failure of either of these two processes. Although this model postulates a separation of the two plasmid components of a diploid so that they do not interact genetically, this need not apply to their products ; indeed the diploid characteristics described above indicate strongly that interaction of plasmid products does occur in a diploid of this type. First, the fact that an i+/i-diploid is fully inducible, despite the presence of the constitutive (i-) genotype, argues that the i-gene product is able to influence the expression of the structural gene in the other plasmid. Whether this trans effect of the 5*

TABLE7. Phenotypic Characteristicsof the Stable Diploid 147(cz.i;pi.. .y.ero*/P.i.&p; .eras) Penicillinase synthesis (unitslmg. dry wt. bacteria)

,

c

uninduced Strain Constitutive A-type strain Constitutive C-type strain Diploid

Genotype

} 147(cr.i;pz.. .y.eroR)” } 147(~.i~,.p;.eros) 147(~.i;pf...y.ero”/P.i&p; .eras)

Immunological , type of enzyme A-type

C-type

A-type

C-type -

A

307

-

320

-

C

-

261

-

242

288

A+C

281

&

161

.. .

induced

127

++

141

140

CI The expression of the (a. i ; p 2 . y ero’) plasmid is shown in strain 147 rather than in the normal, 8325, to allow closer comparison with the behaviour of the same plasmid when part of a plasmid diploid in strain 147.

F

w L-d

l

TABLE8 Liberatian of A- and C-type penicillinase to the Culture Medium by Various Haploid and Diploid Strains

iM

Amount of induced penicillinase (unitslmg. dry wt. bacteria) Strain

Genotype

Immunological typeofenzyme '

A-type

Inducible A-type strain Constitutive C-type strain Constitutive A-type strain i+/i-Diploid

147(a.i+p:. ..y.eroR) 147(j3.iG3p$.era') 147(a.i-p= ...y.eroR) 147(a.i + p i ...y .eroR/p.i&p$ .e r d )

A C A A+C

285

i-/i-Diploid

147(a.iipf.. .y.eroR/j3.i&,p$ .eros)

A+C

177

C-typ;

-

-

% Total enzyme in extracellular state ' A-type

C-type '

45

-

.

w

-

261

-

-

45 38

6 -

%

128

5

E

130

43

6

295

A C A

0

345 167

307

Segregants : Inducible A-type strain 147(a.i+pfI. .y.eroR) Constitutive C-type strain 147(8.iG3p$.ero') Constitutive A-type strain 147(a.i;pi .. y.eroR)

E

294 I

347

261

-

~

a

8 2 B

47

-

-

6

41

-

22

aa W

70

M. R. RICHMOND

i-gene product is mediated at the genetic level, as postulated by Jacob and Monod (1961), or at the ribosomal site of protein synthesis, is not yet clear. Secondly, the fact that haploid and diploid cultures produce the same amount of penicillinase protein suggests that there is a limit imposed by the cell on the level of expression of a plasmid-borne structural gene; and that, in the event of two structural genes being present together in the cell, there is competition between the two gene products for expression at the available sites. Since staphylococcal penicillinase is an extracellular enzyme (Richmond, 1963) and is probably synthesized at the bacterial cytoplasmic membrane, a limit to the number of sites on this structure that could be used for penicillinase synthesis could account for the ceiling in enzyme production that appears to exist both in haploid and diploid cells. But the fact that enzyme can be liberated to achieve either 50% or 5% extracellularity in the haploid state, or even a different degree of extracellularity depending on which plasmid is being expressed in a diploid, argues that the sites must be non-specific as far as their ability to process the products of different penicillinase plasmids is considered. 4. Associated Stable Diploids

When transduction experiments of the type shown in equations

(a),

(5) and (6) (p. 65) are carried out, about 95-98% of the transductants are normal stable diploids of the type just described. I n 245% of cases,

however, interaction of the two plasmids in the diploid occurs to form an “associated” diploid (Richmond, 1967a). This process has been shown to occur, as a rare event, when the a-plasmid is introduced into strain 147 which already carries a P-plasmid and may occur when the a- and #I-plasmids are present together in other strains. Such associated diploids differ from normal diploids in the following properties : (a) Both plasmids may be lost together from an associated diploidto form a derived negative-at about 10,000 times the rate found with a normal stable diploid, where the rate of loss of both plasmids is the product of the rate of independent loss of each component. (b) Propagation of phage on associated diploids leads to a preparation capable of cotransducing both plasmids from the diploid, Phage propagated in this way on normal stable diploids is capable of transducing either plasmid from the diploid but not both. (c) Associated stable diploids may revert to the normal stable type. When this occurs, segregation to the haploid may follow (rather

THE PLASMIDS OB STAPHYLOCOCCUS AUREUS

71

than to the derived negative-see point (a) and the haploid segregants obtained in this way are now found to containrecombinants, unlike segregants from normal diploids. The first two of these characters imply that, in this type of diploid, the a- and p-plasmids are behaving as a single unit capable either of being transduced by a single phage particle or lost together from the cell. The presence of a connexion between the two plasmids, at least at some stage of their existence, is implied by the third character listed above, namely the appearance of recombinants among the segregants after the diploid dissociates. The most likely model to explain associated diploids of this kind would be one in which the plasmids behaved as though they were circles (Fig. 6; Richmond, 1967a).An associated diploid would then be formed by any single crossover between the two component circles of the diploid, and this structure could then behave as a single unit both with respect to loss from the cell or cotransduction. Furthermore, recombinant plasmids would arise by any crossover in the double circle in the opposite sense from the first wherever it occurred in the structure. This model suggests that the a-plasmid should have two different attachment sites in the cell-one in which a is compatible with to form normal stable diploids, and the other which can only be used when a has recombined with p. This second site could well be the normal attachment site for /3 in strain 147.

D. COMPATABILITY The observation that certain pairs of penicillinase plasmids can co-exist in a single staphylococcal cell over considerable periods whereas, with other pairs, recombination and reversion to the haploid state is common, led Novick and Richmond (1965) to postulate the idea of compatibility. Two plasmid types which could co-exist in the same cell were said to be compatible, while those pairs that segregated rapidly, either with or without recombination, were called incompatible. And TABLE9. The Allocation of Various PIasmids to Compatibility Groups Compatibility group

I Plasmids Genetic region involved in compatibility maintenance

a,y,

I1 E,

mc1

5

B

mCn

Data taken from Novick and Richmond (1965) and Richmond (1965a). -**

72

M. R. RICHMOND

on the basis of this behaviour two compatibility groups were compiled (Table 9) and called variously Corn I and I1 (Novick and Richmond, 1965) andPZa I and I1 (Richmond, 1965a). According to this original definition, i-

i+

.-

I

HgR

;+

HgR

i+

FIG.6. A possible mechanism for the formation and separation of associated plasmid diploids in XtaphyZococcus aureus. (a) Normal diploid. (b) First stage of formation of associated diploid; apposition of the two circles. (c) Formation of the associated diploid by a cross-over. (d) Re-formation of two circles by a cross-over in the opposite sense from the first. (e)Final form: note the new position of the eroR marker in relation to p:. Data from Richmond (1967a).

THE PLASMIDS OF STW'HYLOCOCCUS AURBUS

73

therefore, compatibility merely described whether two plasmids could co-exist in a single cell long enough to be detected; the term implied nothing as to the mechanisms involved. Although the discovery of associated diploids (Richmond, 1967a) could be accommodated within the original definition of compatibility, the discovery of transient diploids (see above) has confused the situation since two a-plasmids, which were formerly thought not to be able to co-exist in the cell, are now known to be capable of forming a diploid, albeit transitorily ;the distinction between compatible and incompatible pairs of plasmids now appears as a matter of degree rather than a clearcut difference. It follows therefore that, if the compatibility idea is to retain its usefulness, it must be modified, preferably to reflect the possible mechanisms involved in maintaining plasmids in the staphylococcal cell. Broadly speaking, two types of model could explain compatibility : either two plasmids survive in a single cell because insufficient homology exists between their DNAs to allow rapid recombination; or the plasmids are held in the cell at different attachment sites while incompatible plasmids compete with one another for the same site. By and large, the first of these two possibilities seems unlikely. Apart from the fact that an attachment site may well be necessary to ensure the efficient distribution of plasmids to daughter cells on division, it seems probable that the a- and /3-plasmids must have considerable stretches of DNA base sequence in common since they both carry the genetic regions corresponding to the penicillinase r, and i genes, and the regions responsible for resistance to mercury, cadmium and arsenate ions. Although it is not possible to be dogmatic on this point, the insertion of the tac region into the E . coZi chromosome from the P'lac state occurs at places where the homology is almost certainly a good deal less than would be the case between the a- and /3-plasmids in S. aureus (Beckwith and Signer, 1966). Furthermore, a and /3 have the same number and type of genetic markers in common as do a and y , yet, on the original definition, the first two plasmids are compatible whereas the second two are not. As mentioned previously, an attachment site might be expected to have two main functions: to regulate the rate of multiplication of the plasmid to that of the cell, and to ensure that, after the plasmid had replicated, one copy was transferred to each daughter cell on division. If this model is correct, therefore, the site might not only influence the compatibility relationships of the plasmids, it could also affect stability. One possible site of attachment is the chromosome. And this could clearly explain the behaviour of a few staphylococcal strains (e.g. PSSO; Asheshov, 1966b: and perhaps S26; Poston, 1966) in which the

74

M. H. RICHMOND

penicillinase markers have all the properties of chromosomal genes. If the incoming markers are “attached” by integrating them into the chromosome, the problems of regulating their rate of synthesis and of transferring them correctly to daughter cells at division are largely solved, since both these processes would then be carried out as part of the general process of regulating chromosome replication and partition. But there is good evidence that, in most cases, transduced penicillinase genes-either in the haploid or diploid state-are still plasmid-borne and it seems that their properties exclude the chromosome as an attachment site, at least if attachment occurs by “recombination in” of the transduced markers. An alternative location for the attachment site is some point on the membrane. If this were so, then the plasmid site could be similar in properties to that used to regulate chromosome synthesis and partition in S. aurezcs; in many ways the plasmids could then be regarded as additional chromosomes. In fact the only difference between plasmid and the chromosome, apart from size, would then be that failure to replicate or partition the chromosome would be lethal to one daughter cell while the same lesion affecting plasmid multiplication would just result in a plasmid-less daughter cell. And this difference derives solely from the fact that chromosomal genes are essential for survival of the

.

TABLE 10. Dissociated Plasmids Obtained from ( a .i + p i .AsaR.PbR.CdR.EgR mC.eroR)by Treating Transducing Phage with Ultraviolet Light and Selecting the Transductants on (a) erythromycin agar, (b) cadmium acetate agar, and (c) penicillin agar. Data from Novick (1967).

Transductant genotype

(a) Erythromycin agar penR.AsaR.PbR.CdR.HgR.mC.eroR PbR.CdR.HgR.mC.eroR CdR.HgR.mC.ero’ HgR.mC.eroR mC.eroR

No. of transductants 1500 1

eroRa

1 13 1 17

(b) Cadmium acetate agar penB.AaaR.PbR.CdR.Hg’ .mC .eroR

2000

penR.AsaR.PbR.CdR.HgR.mC

(c) Penicillin agar penR.AaaR.PbR.CdR.HgR.mC .ero’ penR.AaaR.PbR.CdR.HgR.mC a

Selective marker

eroR

CdB

8

6000 2

This class of eroR transductants are integrated into the chromosome.

pefiR

THE PLASMIDS OF STAPHYLOCOCCUS AZ7R.EUS

75

cell whereas plasmid genes frequently are not except under special conditions. If penicillinase plasmids are maintained in staphylococcal cells by this type of mechanism, a genetic region correspondingto the attachment site should be present on the plasmid and this region should express itself both in terms of the compatibility and stability of the plasmid. I n practice, there is some evidence that both “compatibility” and “stability” are determined by genetic regions, and that they lie close together on the plasmid, or may even be different manifestations of the same gene. Novick (1967) has shown by studying the behaviour of deletions covering various regions of the y-plasmid that there is a region lying between H f l and eroR, which is necessary for the retention in the cell of the erythromycin-resistance gene(s)in the plasmidstate (seeTable 10). If this “compatibility maintenance” (mC) region is deleted, the cells can only retain the eroR marker if it becomes integrated into the chromosome. Fragments lacking the mC region are compatible with both a and 6, and Novick’s experiments at least point to a genetic region responsible for maintaining a plasmid in an extrachromosomal state in the cell. I n the a-plasmid there is also evidence for genetic regions involved in stability and compatibility which are closely linked genetically and seem to lie on the same region of the plasmid as Novick’s mC locus (M. H. Richmond, unpublished observations). Under certain circumstances, transduction of the a-plasmid to strain l47(N) gives transductants in which the a-plasmid is particularly unstable, being lost at a rate of about 1:50 divisions (Richmond, 1966a). If this “unstable” a is now transferred to another 147 strain-this time already carrying a 6-plasmid-recombination may occur as a very rare event to give “unstable” p-plasmids. The acquisition of the instability marker is always accompanied by a change in compatibility characteristics of 6. I n this case, the “unstable” ,l3 becomes compatible with a “stable” 6, but incompatible with a. These results suggest therefore that there are genetic regions governing “stability” and “compatibility”, and that these could well be the same or very close to one another. They also have the properties that would be expected of the plasmid-borne region involved in the attachment of the plasmid to the cell.

E. THEROLEOF ATTACHMENT IN RECOMBINATION AND DIPLOID FORMATION If the attachment hypothesis is correct, it becomes possible to suggest the mechanism underlying plasmid recombination and diploid formation, and therefore to give a more precise definition to the idea of compatibility. According to this hypothesis, incompatible plasmids are those using

76

M. € RICHMOND I .

the same attachment site, while a compatible pair use different sites. Thus a normal a/P diploid survives because the two plasmids are attached to different sites and do not normally interact genetically-at least to the extent of forming recombinants. However, as a rare event, one of the plasmids may leave its attachment site and recombine with the other plasmid to form an associated structure. I n the associated diploids that have been examined (Richmond, 1967a) it is the /3-plasmid which seems to leave its site to recombine with a, because the diploid has the stability characteristic of a rather than 8; that is-on the attachment site hypothesis-the associated diploid is attached via the a attachment site. The fact that transduction of the a-plasmid into a cell already carrying usually leads to normal rather than associated diploids presumably reflects a preference on the part of the incoming plasmid to take up an attachment site, if available, rather than to recombine into an existing plasmid. When an a-plasmid is transduced to a cell carrying cc (or a /3-plasmid the relevant attachment site is already filled and it to a cell carrying /I), is reasonable to suppose that the incoming plasmid has no alternative site to which to attach. However, the incoming plasmid does have a high degree of genetic homology with the resident plasmid, and therefore recombination between the two might be expected to occur. If the plasmids are circular, the &st recombination event between the two plasmids establishes an “open-eight” structure; that is an a/a diploid will be formed, and both a-plasmids will be held in the cell at the M. attachment site in the form of an associated diploid (Fig. 7). Once the incoming a is recombined with the resident plasmid in this way, the cell will remain diploid and the stability of the associated structure (i.e. its tendency to form derived negatives) will be determined by the resident a-plasmid. Associated a/a diploids of this kind will probably survive only for a short time since any recombination in the opposite sense to the first (Fig. 7) will establish two separate plasmids, one still attached to the site and the other “free” in the cytoplasm. However, after this process has occurred, the distribution of genetic markers on the attached and free plasmids will vary depending on the precise location of the two recombinational events involved. It is quite possible by this means to achieve an exchange of all of the recipient plasmid markers to the free state or vice versa. This mechanism can explain therefore the origin of the large number of transductants in which the incoming plasmid seems t o have displaced the recipient plasmid entirely and which were found among the progeny of cross (1)(see Table 4,and p. 61). If the mechanism of plasmid recombination does involve an “associated” diploid as an intermediate, the question arises as to whether the

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

77

penR

mC

S

S

(f)

penR

FIG. 7. A possible mechanism for recombination between similar plasmids in Staphylococcus uureu8. (a)Plasmid haploid with theplasmidattachedto amembrane site S. (b)Diploid immediately after the insertion of the second plasmid by transducing phage. (c) First stage of recombination; apposition of the two circles. (d)Second stage: formation of an attached associated diploid by a single cross-over. (e) Re-formation of two separate plasmids, one still attached and the other free, by a cross-over in the opposite sense to the first. (f) Final stage: separation of the two plasmids. Note the apparent transfer of all the genetic markers from the donor to the recipient and vice versa. Such a mechanism could account for the apparent displacement of a resident by an incoming plasmid.

it5

M. H. XICHMIIUNU

“transient” diploids found to occur in a/acrosses soon after transducing phage and recipient are mixed are these intermediates. Were they so, the two plasmid components of transient diploids should be “associated” at least in some cases. I n an attempt to test this point, the transient a/adiploid 8325(a.4-p:. CdR.Asas. eroR/a.i+pxiz.Cds .AsaR.eras) was constructed, and a clone rich in transient diploids emulsified and picked into 2 ml. saline. This saline suspension was then irradiated and the resulting phages used to infect 8325(N). Transductants were selected for the Cd and AsaR markers separately, to measure the frequency of transduction of haploids from the transient diploid, and also for CdR and AsaR together. The CdR.AsaR transductants occurred at about 1yoof the incidence of AsaR and 50% of CdR transductants. However, when the (CdR.AsaR) transductants were examined further, four out of five were found to be diploid in the sense that they segregated haploids containing various combinations of the Cd, Asa and ero markers carried by the parents, whereas the remaining clone was a haploid recombinant. It follows from these experiments therefore that associated diploids do exist, at least to some extent, among the “transient” diploid class, but they probably do not account for all of the transient diploids present. Another point that is, as yet, not properly explained in this experiment is why the resident plasmid (a.i+pxlz.Cds.AsaR.eras) is transducedmore efficiently to 8325(N) from the transient diploid than the recently introduced plasmid (a.i-pi. CdR.Asas. emR).

F. CHROMOSOMAL ATTACHMENT OF PLASMID MARKERS As mentioned previously, certain genes which are plasmid-borne in the great majority of naturally-occurring staphylococci may be chromosomal in others. Thus the i and p A genes appear to be carried on the chromosome in 8.aureus PSSO (Asheshov, 1966b) while the CdR, Asa” and HgR markers are still present in the extrachromosomal state. Similarly, Dr. K. G. H. Dyke has shown that one class of staphylococcal strains found in hospitals is resistant to cadmium and arsenate ions, produces C-type penicillinase, but is sensitive to mercury salts. Examination of these strains shows that the penicillinase p c and i genes are plasmid-borne but, in many cases, the AsaR and CdR markers are chromosomal. Poston (1966) has also shown that the penicillinase genes behave as though they were chromosomal in some strains of S. aureus. I n this case, transduction of the penicillinase-, streptomycinand tetracycline-resistance genes from strain S26. PR.SR. TR. to strain S26. Ps .Ss .Ts was stimulated by ultraviolet irradiation in a manner similar t o that described by Arber (1960). There was, however, no

THE PUSMIDS OF STAPHYLOCOCCUS AUREUS

79

evidence for cotransduction of the penicillinase, tetracycline or streptomycin genes in these experiments. It would appear from all of these results, therefore, that integration of the plasmid-borne markers into the chromosome of 8. aureus-at least in some strains-is perfectly possible. Although the penicillinase genes are chromosomal in PSSO, it is not clear whether this is so in all cells in a culture, or whether some transition between the chromosomal and the plasmid state is possible. I n an attempt to answer this question, E.H. Asheshov (unpublished experiments) has studied the transduction of the penicillinase and metalresistance genes from PSSO to a strain 17855(N). The parental strain 17855 was resistant to cadmium and arsenate ions, produced C-type penicillinase, but was sensitive to HgC12.I n this strain, the penicillinase genes are plasmid-borne but the Cd" and Asa" markers are chromosomal. Strain 17855(N)is the derived negative with respect to the penicillinase genes; that is, it is sensitive to penicillin and HgCI, but still resistant to cadmium and arsenate ions by virtue of its chromosomal markers. When strain 17855(N)is mixed with phage SO propagated on PSSO, and the transductants are selected with penicillin, A-type penicillinaseproducing transductants are found. But when these are scored to sea whether the HgR marker has been cotransduced, this is found to be so in 20% of the penicillin-resistant transductants, despite the fact that the p i gene is apparently chromosomal in PSSO whereas the HgR is extrachromosomal. When the transductants which had received the i and p genes only from PSSO were compared with those that had received i, p and HgR, it wa8 found that the penicillinase region seemed to be chromosomal in the first class of transductants but was in the plasmid state, and linked to HgR, in the second. This transduction experiment was then repeated but the transductants were selected for the Hg" marker and scored for penicillinase production. Once again about 20% of the HgR clones were penicillinase producers despite the apparently different locations of the penicillinase and Hg" genes in PSSO. Examples of i+pi.H f , i+pt.HgR and HgR transductants were then taken and examined to see whether a transducing lysate obtained from them was stimulated by ultraviolet irradiation in the manner described by Arber (see p. 49). Stimulation was found when the i+pi.Hgs transductant was the donor but not for ifpi. Hg" and Hg". This result confirms therefore the chromosomal location of the penicillinase region in the i+pi.HgS transductants and the plasmid location in the other two classes. The fact that a phage preparation was capable of cotransducing the i, p and Hg regions to the plasmid state (but not vice versa) from a strain in which the penicillinase genes are chromosomal and the mercury gene

80

M. R. RICHMOND

plasmid-borne suggests that, in some cells, the penicillinase region leaves the chromosome to enter the plasmid state. However, if this were so, it should be possible to obtain derived negatives from PSSO, but this has never been shown. Another possibility which must therefore be considered is that some cells in the PS80 culture become transiently diploid with respect to the penicillinase region, one copy of the gene being chromosomal and the other on a plasmid. Alternatively, the HgR region may become integrated in certain cells. Whatever the detailed mechanisms involved in these experiments may be, it seems clear that a 'given set of penicillinase and associated genes (e.g. CdR or AsaR) may be chromosomal in some strains and plasmid-borne in others. Up to now there is no absolutely unequivocal evidence that these genes may move from the chromosomal to the extrachromosomal state in a single strain, but the results are highly suggestive. The possibility of transition from the chromosomal to the plasmid state has a bearing on the relationship between the penicillinase plasmids of X. aureus and the episomes of E. COG, and this point is discussed further later (see p. 84). Further indirect evidence for the possible chromosomal location of the penicillinase genes in S. aureus has been produced by Harmon et al. (1966). They used a penicillin-sensitive, methionine-requiring mutant as recipient, and claimed that transduction of the penicillinase region from a suitable penR.met+ donor leads to the cotransduction of the penicillinase genes and the gene responsible for cystathionine synthesis, a gene involved in the methionine biosynthesis pathway. The authors claim that these experiments show that the penicillinase genes are truly episomal since they appear to be extrachromosomal in the strains in question yet are capable of mobilizing one of the genes of the methionine biosynthetic pathway. However, although the genes of the methionine path are thought to be chromosomal in 8. aureus, Baldwin and his collaborators give no experimental evidence on this point, and their conclusions must therefore remain suspect for this reason. Furthermore, attempts to repeat these experiments with Baldwin's strains have failed (R.P. Novick, personal communication).

G. THEPLASMID MAP The whole question of determining the order of the genetic markers on the penicillinase plasmids depends to a great extent on whether the plasmids are circular linkage groups or not, and to a less extent on their size. Standard methods of determining marker order by three point crosses tend to break down if the linkage groups are circles with the markers scattered around an appreciable portion of the structure

THE PLASMIDS O F STAPHYLOCOCCUS A U R 3 U S

81

(Stahl and Steinberg, 1964; Hopwood, 1965). Thus, although it is possible to order markers unambiguously on the circular E . coZi chromosome, this is only because the markers involved in most crosses lie on a small region of the chromosome compared with the whole structure. The evidence that the penicillinase plasmids of 8. aureus are circular is circumstantial. Circularity might be expected as with other extrachromosomal elements. The build-up and break-down of associated plasmid diploids (see p. 70) suggests a circular nature (Richmqnd, 1967a),and preliminary mapping experiments along the lines advocated by Stahl and Steinberg (1964) tend to confirm this view (M. H. Richmond, unpublished experiments). Campbell (1962)has suggested that the F particle behaves as a circle, and Broda et aZ. (1964)have obtained genetic evidence on the integration of F into the E. coli chromosome which is consistent with this view. Elsewhere, Mitsuhashi (1965) has published a recombinational analysis which shows that the R-factors may be circular. Furthermore, the DNA of some but not all phages-which themselves can be considered as extra-chromosomal elements of a special type-can be inferred to be circular from their behaviour in the ultracentrifuge (Fiers and Sinsheimer, 1962). I n view, therefore, of the possibility of circularity existing among extrachromosomal particles in general, and the penicillinase plasmids in particular, any mapping data obtained by conventional three-point crosses must be regarded with some suspicion. I n view of these difficulties, the best mapping data concerning the penicillinase plasmids comes from Novick’s (1967)experiments in which deletions of various regions of the plasmids are obtained by irradiating transducing phage preparations with ultraviolet radiation (see also p. 75). Starting with the plasmid y, which carried the penicillinase i and p genes, the markers for CdR,h a R ,PbR,HgR, eroR and the compatibility maintenance site (mC,),Novick obtained the deletions listed in Table 10 when the transductants were selected on a variety of different selective media. Although not absolutely unequivocal, these results imply a probable marker order : I

(i,p).AsaR.PbR.CdR.HgR.mC.eroR

A certain amount of additional evidence supports this view. Transduction

of the CL . . . y recombined plasmid to a negative recipient-e.g. 8325(N)-leads to the transfer of dissociated plasmids in a small proportion of cases. Since the parent plasmid carries the markers i , p A , AsaR, PbR, CdR, HgR, mC, eroR, and the most common dissociated plasmid transductant is i . p .AsaR.PbR.CdR.H g R .mC (Richmond, 1966b), these results are consistent with Novick’s (1967) marker order.

82

M, H. RICHMOND

Attempts have been made to determine the linkage relationships of some of the plasmid markers by recombinational analysis (Novick, 1967 ; M. H. Richmond, unpublished experiments). For this purpose transductional crosses were performed between donor and recipient strains carrying an a . . . y recombined plasmid on the one hand and an a-plasmid on the other. A typical experiment carried markers as follows :

+(a.iipA+.Hq'

. . . y.eroR)

-+ 8325(a.i+p,,,. HgR.eros)

Transductants were selected for eroR and scored for the unselected marker. Recombination between ero and p occurred to about 16% of

..y .era') +8325(cr.i+p&. H f .ero"). Dotted lines represent scorable cross-overs. (b), (c) and (d)Diagrammatic representation of the results obtained from the cross shown in (a). Certain recombinants are illustrated by heavy (more common) or dotted (less common) lines. The relative ratios of the two types of recombinant in the pairs shown in (a), (b) and (c) are shown in the right of the figure. Data from Novick (1967). FIU.8. (a)Diagrammatic representation of the cross $(G.i - p z . H#.

the eroR transductants, while recombination between i- and p - occurred at a frequency of about 0.2%. The relative incidence of certain ; / p cross-overs was used to establish the map order. The three pairs of recombinant classes and the routes used to form them are shown in Figure 8 where the more common cross is shown in bold face, and the less common as dashed lines. The relative incidence of recombinants in the relevant pairs is also shown in Figure 8. On the basis of a linear linkage group, the only order consistent with these findings is i .p.H g .ero, with the Hg marker closer to p than to ero. But other orders are possible if a circular linkage group exists. Be that as it may, the order obtained by

T H E PLASMIDS OF STAPEYLOCOCCES A E R E E S

83

recombination analysis is, at worst, compatible with the marker order obtained by Novick's (1967) deletion experiments. Subsequent recombination experiments, using suitably marked strains, have also shown that the Cd marker lies between p and Hglero, but once again the order is only unambiguous if a linear model is assumed. I n summary, therefore, the mapping data suggest that the likely order of markers on the y and a . . . y recombined plasmids is:

i . p .Asa. Pb .Cd .Hg .mC. ero although these positions may have to be revised in the light of further experiments. I n the case of plasmids, such as a and j3, which carry no eron marker, the order is almost certainly the same but for the absence of the ero region. Thus 01 and appear to carry a deletion of the whole ero region when compared with y and a . . . y.

H. SIZEOF

THE

PENICILLINASE PLASMIDS

Since penicillinase plasmids can be transferred from cell to cell by phage particles, an upper limit to their size will be set, to some extent, by the amount of DNA that can normally be accommodated in a phage head. Although no information is available on exactly how much DNA this amounts to in 8. aureus phages, the E. coli phages T, and T4contain DNA of molecular weight about 1.2 x lo8 (Davison et al., 1961 ; Cairns, 1961). This amount of DNA corresponds to about lo6 base pairs. Another indication of the size of the penicillinase plasmids comes from a comparison of recombination frequencies with the distance between two known point mutations in the penicillinase structural gene p A . The two-point mutants used in this work (No: 2 and No. 54)each cause a change in the nature of the penicillinase molecule synthesized, No. 2 causing insertion of isoleucine for threonine, and No. 54 causing an exchange of asparagine for aspartic acid (R. Ambler, unpublished experiments). The two amino acid positions involved in these mutants lie about 75 residues apart in the single penicillinase polypeptide chain (R. Ambler, unpublished experiments) and the mutational events are therefore about 220 base pairs apart in the gene, if colinearity of gene and protein is universal (Yanofsky et al., 1967). Since the recombination frequency between p y and p)54 is about 0.07 %, whereas recombination between p and ero is about 16% and between p z and i i is about 0.2% (M. H. Richmond, unpublished experiments), it can be calculated, assuming uniform recombination along the map, that the total distance between the ii' and ero is about lo6 base pairs. Thus, on this evidence,

84

M. H. RICHMOND

the amount of DNA between the extreme markers detected on the plasmids so far is about the same as found in the average phage particle, and amounts to about 1-2% of the DNA in the staphylococcal cell. It follows, therefore, that it is unlikely that there is much plasmid-borne genetic information outside the known genetic markers.

IV. A Comparison of Penieillinase Plasmids with other Extra-chromosoma1 Elements in Bacteria The plasmids of X.aureus share their extrachromosomal status with the resistance-transfer factors (R) (Watanabe, 1963; Datta, 1965), colicin (col) factors (Fredericq, 1957) and fertility- or sex-factors (F) (Hayes, 1953); and with the FP particles of the pseudomonads (Holloway, 1955, 1956; Holloway and Jennings, 1958; Holloway and Fargie, 1960). I n addition, a great range of other extrachromosomal elements are found in organisms other than bacteria; for example the kinetoplast and kinetosomes of protozoa (Jinks, 1964), the mitochondria of animal cells (Lehninger, 1964), and the chloroplasts and chromophores of plant cells (Kirk and Tilney-Bassett, 1967). However, these non-bacterial structures probably represent a completely different extrachromosomal situation from the bacterial elements, and they will not be considered further here. Ofthe bacterial extrachromosomal elements, the least is known about the pseudomonad PP particles. Broadly speaking, they appear to behave like the F-particle in E . COGin that they can infect FP- cells to the FP+ state, but little further information has appeared since the original publications at the end of the 1950s (Holloway, 1956; Holloway and Fargie, 1960). The main comparison must therefore lie between the staphylococcal plasmids on the one hand and the R-, F- and col-particles of the Enterobacteria on the other. The F-, cob- and R-factors share many properties. One consequence of their extrachromosomal location is that they can be lost spontaneously from a culture at a frequency greater than that expected from spontaneous mutation rates, and loss of the particles is usually associated with the loss of all of the genetic markers carried on them. Thus cultures carrying resistance-transfer factors always contain some drug-sensitive cells (Watanabe and Fukusawa, 1961) and the same is true for coZ+ (Fredericq, 1957) and F+cells (Hayes, 1963). Hirota (1960) showed that F+ cells could be efficiently purged of their F particles by growth in the present of sub-inhibitory concentrations of acridine dyes, and the same is true for cells carrying R-factors (Hirota and Iijima, 1957), although the drug is much less effective in this case. Stocker et al. (1963), however, showed that multiplication and transfer of the col factor is unaffected

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

85

by acriflavine. Sensitivity to acridine dyes, therefore, is not an obligate consequence of the extrachromosomal state. I n addition to their common extrachromosomal status, the -F, col- and R-factors share two other important properties. The first is the ability to promote their own transfer by conjugation, to cells which lack the equivalent particles, and the other is their ability to mobilize chromosomal markers and transfer them by conjugation. Bacterial conjugation is usually apparent as the unidirectional transfer of genetic information between two strains. This was first reported for the F-factor by Hayes (1952) and by Lederberg et al. (1952), and subsequently for the R-factors by Akiba et aZ. (1960). Similarly transfer of the coZ-factor by conjugation was found by Clowes (1961). I n all these cases, transfer was originally studied between strains of the same species, but it soon appeared that all the factors could be transferred widely among Gram-negative bacteria more or less regardless of species boundaries (Hayes, 1963; Datta, 1965). Normally cells lacking a given factor could accept it by transfer from a donor but the similarity of the COZ-, F- and R-factors is underlined by the fact that, in certain cases, the carriage of one type of factor modifies the ability of that cell to accept another (Clowes, 1963; Meynell and Datta, 1966). The ability of the extrachromosomal particles to mobilize parts (or in some cases all) of the bacterial chromosome implies that, a t least for some part of its existence, the P-, col- and R-factors integrate into the bacterial chromosome. In the case of the F-particle, this integration takes two forms: either a relatively stable interaction in which the factor becomes part of the chromosome and is able to promote the transfer of the whole chromosome‘to an appropriate recipient at high frequency by conjugation-the so-called Hfr state (Hayes, 1953); or a freely reversible integration which allows the F-particle to incorporate limited regions of the chromosome while remaining extrachromosomal for at least a great deal of the time-the so-called 3’’ state (Adelberg and Burns, 1959,1960).Both the col- and R-factors are capable of mobilizing the chromosome in a manner analogous to F-to form HfRT (Sugino and Hirota, 1962)and HfcolT (Stocker et aZ., 1963)strains; but, to date, a situation analogous to the P‘ state has not been found. However, the ability of the extrachromosomal particles to undergo a slowly reversible integration into the chromosome has many analogies to the integration of temperate bacteriophage, and Jacob and Wollman (1958) have coined the term “episome” to describe the genetic elements which behave in this way. The formal definition of this term is that episomes are genetic elements which are capable of undergoing a reversible integration into the bacterial chromosomewithout adding to the genetic complement of the cell (Jacob and Wollman, 1958). Since the col-, Ii- and R-factors

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fulfill these criteria, they, as well as temperate phage, may be regarded as episomes. I n summary, therefore, the COZ-, F- and R-episomes are predominantly extrachromosomal elements which can promote their own transfer by conjugation to suitable recipient cells and which can integrate reversibly into the bacterial chromosome. I n certain respects the penicillinase plasmids are similar to the colF- and R-episomes. They behave as extrachromosomal elements which are lost from the cell at relatively high frequency, and the unpublished experiments of E. H. Asheshov (see p. 78) suggest strongly that plasmidborne genetic markers can enter the chromosomal state in at least some strains of 8.azLreu8. This phenomenon could, therefore, be analogous to the integration of true episomes. The difference between the penicillinase plasmids and the E. COG episomes is most marked in their inability to promote conjugation. Conjugation has never been observed to occur in staphylococci, nor, for that matter, in any Gram-positive bacteria. Although it is possible that the absence of conjugation in staphylococci may reflect a fundamental difference in the structure of Gram-positive cells, rather than an innate difference in the nature of the extrachromosomal genes, this possibility does seem rather unlikely. By and large the penicillinase plasmids seem to form plasmid diploids more readily than do the episomes of E . coZi, but this difference may be more apparent than real. Although diploids of two F-particles do not seem to occur readily, this may be solely because possession by a cell of a single F-particle impedes the uptake of a second or, alternatively, recombination between the two F-particles may be very rapid. A closer analogy to the staphylococcal plasmid situation is provided by strains which carry more than one cob or R-factor. In these cases, the two episomes are probably held in the cell separately, by the use of distinct attachment sites, much as has been postulated for the a//3 plasmid diploids in S. aureus. All in all, therefore, the penicillinase plasmids seem to represent a very similar class of extrachromosomal elements to the COZ-, F- and R-episomes of the Gram-negative bacteria despite their inability to promote conjugation.

V. Acknowledgements I would like to express my thanks to Drs. Elizabeth Asheshov, Richard Ambler and Keith Dyke for telling me about some of their unpublished experiments and allowing me to report them.

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REFERENCES Adelberg, E. A. and Burns, S. N. (1959). Genetics 44, 497. Adelberg, E. A. and Burns, S. N. (1960). J. Bact. 79, 321. Akiba, T., Koyama, K., Ishiki, Y., Kimura, S. and Fukushima, T. (1960). Jap. J. Microbiol. 4, 219. Arber, W. (1960). Virology 11, 273. Asheshov, E. H. (1966a). J . gen. Microbiol. 42, 403. Asheshov, E. H. (1966b). Nature, Lond. 210, 804. Asheshov, E. H. and Winkler, E. (196G). Nature, Lond. 209,638. Barber, M. (1949). J . gen. Microbiol. 3, 274. Beckwith, J. R. and Signer, E. (1966). J. molec. Biol. 19, 254. Borowski, J. (1963). Ann. Inst. Pasteur 104, 535. Broda, P. M. A., Beckwith, J. R. and Scaife, J. (1964). Genet. Res., Camb. 5 , 489. Cairns, J. (1961). J. molec. Biol. 3, 756. Campbell, A. M. (1962). Adwanc. Genet. 11, 101. Clowes, R. C. (1961). Nature, Lond. 190, 986. Clowes, R. C. (1963). Genet. Res., Camb. 4, 162. Clowes, R. C., Moody, E. E. M. and Pritchard, R. H. (1965). Genet. Res., Camb. 6, 147. Collins, A. M. and Macdonald, S. (1962). J. Path. Bact. 83, 399. Datta, N. (1965). Brit. med. Bull. 21, 254. Davison, P. F., Freifelder, D., Hede, R. and Levinthal, C. (1961). Proc. mat. Acad. Sci. Wash. 47, 1123. Dyke, K. G. H. and Richmond, M. H. (1967). J. din. Path. 20, 75. Fairbrother, R. W., Parker, L. and Eaton, R. B. (1954). J. gen. Microbiol. 10, 309. Fiers, W. and Sinsheimer, R. L. (1962). J. molec. Biol. 5 , 424. Fredericq, P. (1957). Annu. Rev. Microbiol. 11, 7. Hamburger, M., Walker, W. F., Clark, K. L. and Carleton, J. (1958).Tram.Amer. Clin. Climat. Assoc. 70, 49. Hamburger, M., Carleton, J., Walker, W. F. and Clark, K. L. (1960). Arch. int. Med. 105,668. Harmon, S . A. and Baldwin, J. N. (1964). J. Bact. 87,593. Harmon, S. A., Baldwin, J. N., Wei-Chen Tien and Critz, D. B. (1966). Canad. J. Microbiol. 12, 973. Hashimoto, M., Kono, K. and Mitsuhashi, S. (1964). J . Bact. 88, 261. Hayes, W. (1952). Nature, Lond. 169, 118. Hayes, W. (1953). Cold Spr. Harb. Symp. p a n t . Biol. 18, 75. Hayes, W. (1963). “The Genetics of Bacteria and their Viruses”, Blackwell, Oxford. Hirota, Y. (1960). Proc. nat. Acad. Sci. Wash. 46, 57. Hirota, Y. and Iijima, F. (1957). Nature, Lond. 180, 665. Holloway, B. W. (1955). J. gen. Microbiol. 13, 572. Holloway, B. W. (1956). J. gen. Microbiol. 15, 221. Holloway, B. W. andFargie, B. (1960). J. Bact. 80, 362. Holloway, B. W. and Jennings, P. A. (1958). Nature, Lo&. 181, 855. Hopwood, D. A. (1965). J. molec. Biol. 12, 514. Jacob, F. and Wollman, E. (1958). C.R. Acad. Sci., Paris, 247,154. Jacob, F. andMonod, J. (1961). ColdSpr. Harb. Symp. qwcnt. Biol. 26, 193. Jinks, J. L. (1964). “Extrachromosomal Inheritance”, Prentice-Hall, New Jersey. Kirk, J. T. 0. and Tilney-Bassett,R. A. E. (1967). “The Plastids”, W. H. Freeman & Co., London & San Francisco. Korman, R. Z. and Berman, D. T. (1962). J. Bact. 84, 228.

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Lederberg, J. (1952). Physiol. Rev. 32, 403. Lederberg, J., Cavalli, L. L. and Lederberg, S. (1952). Genetics 37, 720. Lehninger, A. L. (1964). “The Mitochondrion”, W. A. Benjamin, New York & Amsterdam. May, J. M., Houghton, R. H. and Perret, C. J. (1964). J. gen. Microbiol. 37,157. Meynell, E. and Datta, N. (1966). Genet. Res., Camb. 7 , 141. Mitsuhashi, S. (1965). Gunma J. med. Sci. 14, 169. Mitsuhashi, S., Hashimoto,M., Kono, K. andMorimura, M. (1965). J.Bact. 89,988. Morse, M. L. (1960). Proc. nat. Acad. Sci. Wash. 45, 722. Novick, R. P. (1963). J. gen. Microbiol. 33, 121. Novick, R. P. (1967). Fez. Proc. 26, 29. Novick, R. P. and Richmond, M. H. (1965). J. Bact. 90,467. Pattee, P. A. and Baldwin, J. N. (1961). J. Bact. 82, 875. Pollock, M.R. (1962). I n “The Bacteria”, (I.C. Gunsalus and R. Y .Stanier, eds.), vol. 4, p. 121, Academic Press Inc., New York. Poston, S. M. (1966). Nature, Lond.210, 802. Richmond, M. H. (1963). Biochem. J. 88,452. Richmond, M. H. (1964). Proc. 6th Intern. Congr. Biochem., New York, p. 237. Richmond, M. H. (1965a). Brit. med. BUZZ.21, 260. Richmond, M. H. (1965b). Biochem. J. 94, 584. Richmond, M. H. (1965~).J. Bact. 90, 370. Richmond, M. H. (1966a). Postepy Mikrobiologie 5 , 371. Richmond, M. H. (1966b). J. gen. Microbiol. 45, 51. Richmond, M. H. (1967a). J. gen. Microbiol. 46, 85. Richmond, M. H. (1967b). J. rnolec. Biol. 26, 357. Richmond, M. H. and John, M. (1964). Nature, Lond. 202, 1360. Ritz, H. L. and Baldwin, J. N. (1958). Bact. Proc. 40. Ritz, H. L. and Baldwin, J. N. (1961). Proc. SOC.e q . BioZ. Med. 107,678. Segalove, M. (1947). J. infect. DW. 81, 228. Servin-Massieu, M. (1961). J. Bact. 82, 316. Stahl, F. W. and Steinberg, C. M. (1964). Genetics 50, 531. Stocker, B. A. D., Smith, S. M. and Ozeki, H. (1963). J . gen. Microbiol. 80, 201. Sugino, Y. and Hirota, Y. (1962). J. Bact. 84,902. Voureka, A. (1948). Lancet i, 62. Watanabe, T. (1963). Bmt. Rev. 27, 87. Watanabe, T. and Fukusawa, T. (1961). J. Bact. 81, 669. Yanofsky, C., Drapeau, G. R., Guest, J. R. and Carlton, B. C. (1967). Proc. nut. Acud. Sci. Wash. 57,296.

The “Life Cycle” of Bacterial Ribosomes WILLIAM S. KELLEY AND MOSELIOSCHAECHTER Is

.’

Department of Microbiology, Tufts University School of Medicine, Boston, iWassachusetts, U.S.A. I. Introduction . A. Problems Particular t o the Study of a Ribosomal “Life Cycle” B. Scope of the Review C. Studies of Ribosome Structure and Composition. . D. Cellular Localization of Bacterial Ribosomes. 11. The Role of Ribosomes in the Control of RNA Synthesis . 111. The Production of Ribosomal RNA . IV. The Production of Ribosomal Proteins V. The Assembly of Ribosomal Subunits . A. Studies on Cells in Balanced Growth . B. Studies of Metabolically Inhibited Cells . C. In Vitro Experiments on Ribosome Assembly . D. Conclusions . VI. The Formation of Functional Ribosomes . VII. The Participation of Ribosomes in Protein Synthesis . VIII. The Release of Ribosomes from Messenger RNA . IX. Conclusions . X. Acknowledgements References .

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89 90 91 91 97 99 101 107 112 112 115 124 126 226 131 133 136 137 137

I. Introduction During the last decade, bacterial ribosomes have been the object of intense study by many workers. At the time of this writing only a few generalizations about their function and origin seem possible. Bacterial ribosomes are ribonucleoprotein particles, essential for protein synthesis and perhaps involved in the control of RNA synthesis. I n any sense but the superficial little can be said about their function on a molecular scale. At present no generally acceptable mechanism has been described which accounts for the assembly of ribosomes in the bacterial cell. Obviously a field which is the subject of such intensive study and which contains so many unsolved problems is sure to evolve very rapidly. A 89

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review aimed solely at cataloguing data is likely to be quickly outdated and of limited interest. Therefore we have attempted to focus on a central theme, that ribosomes in growing bacteria undergo a “life cycle”. We will define this “life cycle” as the events of inception, maturation, and repeated functioning of a ribosome, and divide it into a series of biochemical reactions. Formally we shall consider each reaction as a separate event, although in the bacterial cell they are quite probably interrelated in a complex manner. These reactions are :

A. The synthesis of ribosomal (IRNA. -) B. The synthesis of ribosomal proteins. C. The formation of ribonucleoprotein particles that are precursors of complete ribosomes.

D. The assembly of these precursor particles into completed ribosomes.

E. The participation of ribosomes in protein synthesis. F. The release of ribosomes from messenger (m-) RNA into a pool of free particles.

Our review attempts a critical consideration of each step of the “life cycle”, and we shall discuss the steps in terms of the nature of the reactants and the reaction products. Where such information is available we shall also discuss aspects of the timing and regulatory mechanisms. By organizing the available information on this basis, we have created several redundancies. I n many cases some aspects of a set of experiments apply to more than one of our designated reactions. Hence, many experiments have been cited several times, each, we hope, in a different light. We hope that the net effect is not confusion or tedium but rather a story that can be read as a series of discrete but related chapters.

A.

PROBLEMS PARTICULAR TO THE STUDY OFA RIBOSOMAL “LIFECYCLE”

As in any field of scientific study, experiments designed to investigate ribosome assembly and function have problems of methodology peculiar to themselves. In general we would prefer to discuss data which describe the “life cycle” in cells in balanced growth, unimpaired by external inhibitors or by lack of an essential nutrient. Bacterial cells growing at a steady state are generally taken to represent a baseline of microbial ( 6 normalcy” and, ideally, we are aiming at understanding “normal” function. However, few experiments have actually been performed under such circumstances. Various strategies have been used to approach the problem. I n general, work is made difficult because a simple chemical characterization of a, ribosome is impossible. Instead, ribosomes are generally defined on the -’-

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basis of their sedimentation characteristics in preparative and analytical ultracentrifuges. Used alone, these methods specify little of the molecular nature of ribosomes and, in addition, are fairly insensitive for the detection of small numbers of precursor particles and intermediates in ribosome assembly. Therefore it has been necessary for experimenters to detect precursors by their kinetic behaviour in labelling experiments or by perturbing bacteria in such a way that they accumulate precursor or precursor-like particles. Both approaches have specific drawbacks. The mathematical interpretation of kinetic data is complicated by other dynamic cell processes. Perturbing growing cultures by various drugs and metabolic shift conditionshas indeed been a popular and fruitful approach to the study of ribosome assembly, allowing analytical study of the accumulated particles. Obviously, though, cells so inhibited or perturbed are not in balanced growth. Much work has been done in aitro and some of the most interesting recent data are the result of such studies. When these data apply at present to the in vivo process, they will be included in our discussion. B. SCOPEOF

THE

REVIEW

The bacterial ribosome’s “life cycle” as we have defined it involves more than a simple discussion of the individual reactions. Ribosome production and the formation of polysomes (the aggregates of ribosomes and m-RNA active in protein synthesis) are connectedand, sincethesyntheses of r- and m-RNAs are chemically similar processes, an adequate model must relate the regulation of synthesis of one to the other. The kinetics and mechanism of m-RNA synthesis and polysome formation will also be considered, therefore, but principally in light of how they affect the “life cycle” of a ribosome.

C. STUDIESOF RIBOSOME STRUCTURE AND COMPOSITION To introduce our review of the “life cycle” we shall begin with a brief summary of datadescribing ribosome structure on a physical and chemical basis. The first intensive study of the physico-chemical properties of bacterial ribosomes was conducted by Tissibres and Watson (1958) and Tissibres et al. (1959). These workers noted that 80-90% of the RNA of exponentially growing Escherichia coli was in the form of ribonucleoprotein particles. The RNAiprotein ratio of these particles was about 65 :35, and their structural integrity was apparently dependent on magnesium ions. I n alumina-ground extracts, particles with sedimentation coefficients of 30,50,70 and 100s were observed, the relative number of each depending

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WILLIAM S. EELLEY AND MOSELIO SCHAECHTER

on the magnesium ion concentration. These experiments indicated that reversible, magnesium-dependent aggregations took place in a manner summarized by the equation : Increasing Mg2+ -+ 2(305)+2(50S) + 2(70S) + (100s) Using purified 70s ribosomes Britten and McCarthy (1959) confirmed this interpretation by analysing the sedimentation behaviour of the particles at various Mg2+concentrations. These data are reproduced in

I

I

I

I

I l l l l

Magnesium acetate concentration

(M

x lo-’)

FIG.1. Variation in the quantity of the various forms of ribosomes as a function of the concentration of magnesium acetate. Data were obtained from Schlieren diagrams by cutting out and weighing the peaks from enlarged prints. Reprinted from the Carnegie Institution of Washington Yearbook 58,266 (1959), by permission of the authors.

Fig. 1.Note that, at magnesium concentrations of less than M , most of the 70s particles have dissociated to 305 and 50s subunits whereas, at concentrations of lop2 M and greater, the 70s and 100s species are predominant. Tissieres et al. (1960)have shown that in vitro protein synthesis proceeds optimally at M Mg2+.Further, McQuillen et aZ. (1959)and Schlessinger and Gros (1963)have shown that nascent proteins sediment in association with 705 ribosomes. For these reasons the 705 ribosomes are probably the ones which support protein synthesis. Electron micrographs (e.g. Hall and Slayter, 1959; Huxley and Zubay, 1960) of isolated ribosomes show the 305 and 505 particles clearly, and substantiate the physical data that each 70s particle contains one 305 and one 50s subunit; 100s particles appear as dimers of 70s particles joined through the 30s subunits (Hall and Slayter, 1959). An electron

THE

L

‘

CYCLE” ~ OF ~ BACTERIAL ~ ~ RIBOSOMES

93

micrograph of ribosomes arranged on m-RNA in a polysome is shown in Fig. 2 . Although most of the 30s and 50s particles aggregate to form 70s particles at magnesium ion concentrations of M and greater, not all do so. This small number of particles can be separated from the 70s particles by differential centrifugation. First noted by Green and Hall

FIG.2. Electron micrograph of a bacterial polyribosome with membrane particle attached. The preparation was positively stained with uranyl acetate. Magnification x 270,000. Reproduced by courtesy of Dr. Henry Slayter, Children’sHospital, Boston, Massachusetts.

(1961), these particles are called “native” 305 and 50s particles to distinguish them from the “derived” 30s and 50s subunits produced by reversible disaggregation of 70s ribosomes at lower magnesium concentrations. Recent data (Kelley and Schaechter, 1968) indicate that, in lysates of Bacillus megaterium prepared in buffer with a magnesium-ion concentration as great as 4 x lo-’ M, 5-10y0 of the total cellular ribosomes are found as 30s and 505 particles. 6

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

In lysates prepared by a gentler method of disruption than the alumina grinding used in the earliest studies, as much as 80% of the total ribosomal material of the cell is found in oligomeric aggregates of ribosomes and m-RNA, the polysomes. Analysis of such lysates shows that the relative number of polysomes,ribosomes and subunits varies with changes in the magnesium concentration of the lysing medium (Kelley and Schaechter, 1968). These results are shown in Pig. 3. Note that, at magnesium ion concentrations below approximately 6-8 x M , most of the 705 ribosomes dissociate into their 30s and 50s subunits, as shown by the earlier work of Britten and McCarthy (1959; see Fig. 1). However, polysomes seem to be considerably more stable at these magnesium-ion

Mg2+concentration ( M x lo-*)

FIG.3. Percentage of total cellular ribosomes of various types in lysates prepared at different magnesium acetate concentrations. Data were obtained by measuring areas of enlarged prints of Schlieren diagrams. Reproduced from Kelley and Schaechter (1968).

concentrations, and dissociate completely only when the magnesium-ion concentration is less than 2 x M . As we shall discuss below, these observations not only indicate a difference between 70s ribosomes and polysomes, but may also be important clues about the nature of the assembly process by which polysomes are formed. When discussing ribosomes and their subunits, it is expedient to regard them as homogeneous and identical to one another as one usually considers a population of enzyme molecules. I n fact, this need not be so since the S value of a particle is not a sensitive measure of all chemical differences.In subsequent sections of this review, the question of whether all ribosomes are identical will play an important role in our discussion. At present, chemical methods of analysis cannot resolve this point. We

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do know from several lines of evidence that the two classes of subunits, 3 0 s and 50s)differ from eachother bothin size and composition.Although their densities and hence their RNAIprotein ratios seem to be identical (Brenner et al., 1961)) the RNAs and proteins of the subunits differ. Kurland (1960) demonstrated that the 3 0 s subunits contain RNA molecules with an S value of 16 whereas 50s subunits contain 2 3 s RNA. This result and many later ones were confused by the fact that the usual methods of RNA extraction almost always produce some 1 6 s RNA from the 50s particles. Midgley (1965) has shown that this phenomenon is dependent upon details of the extraction technique, and that carefully extracted 50s subunits yield 2 3 s and not 1 6 s RNA. Analyses of the nucleotide composition of these RNAs (Bolton, 1959; Spahr and Tissi&res,1959; Midgley, 1962) show that both have virtually identical base composition. However, fingerprints of partial ribonuclease digests (Bolton, 1960; Aronson, 1962) and hybridization experiments (Yankofsky and Spiegelman, 1963) have demonstrated that the sequences of these two types of RNA are different. The 1 6 s RNA has a molecular weight of about 5 x lo5 and the 238, 1.1 x l o 6 (Stanley and Bock, 1965). This is consistent with each particle containing only one RNA molecule. I n addition to 2 3 s and 1 6 s RNA, another class of RNA first reported by Rosset and Monier (1963) has been found to be associated with all bacterial ribosomes. This class sediments at 55 and can be confused with t-RNA in sucrose density-gradients but can be separated from it by acrylamide gel electrophoresis (Loening, 1967). It differs from t-RNA by its base composition, greater chain length, lesser degree of methylation, near absence of rare bases, and lack of acceptor capacity for amino acids. It is associated with the 50s subunit. A t present, the function of this component is unknown. For references on this subject, see recent papers by Comb and Zehavi-Willner (1967) and Brownlee and Sanger (1967). The amino-acid content of the ribosomes has been analysed, and differences in the proteins of 3 0 s and 50s particles have been found. Many of the amino acids are basic and some polyamines are present (Spahr, 1962). End-group analysis of the peptides shows tlfat most of the Nterminal amino acids are methionine and alanine and, on the basis of bhe number of ends, the average molecular weight of a ribosomal protein was estimated as 2.5 x l o 4 (Waller and Harris, 1961). This value has recently been confirmed by studies of Moller and Chrambach (1967)) Moller and Widdowson (1967)) and Traut et al. (1967). Various chromatographic techniques have been used to examine ribosomal proteins (e.g. Spitnik-Elson, 1964) but the most fruitful one has been electrophoresis on polyacrylamide gels (e.g. Leboy et al., 1964; Traut, 1966). This techniques shows that there are thirty or more proteins in the 7 0 s particles, and that different proteins are found in the 30s and 50s subunits (Leboy

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

et al., 1964; Traut, 1966). Several such acrylamide gel columns are shown in Fig. 4.The work of Traut et al. (1967) demonstrates conclusively that there are at least nineteen distinctly different proteins of molecular weight approximately 2-5 x lo4 in a preparation of 705 ribosomes, thus eliminating the possibility that the different bands observed in electrophoresis patterns are the result of polymerization of a few simple peptide subunits. The pattern of bands appearing in the acrylamide gels varies among different E. coli strains (Leboy et al., 1964). The patterns change

FIG.4. Polyacrylamide gel electrophoresiscolumns of 50S, 70s and 30s ribosomal proteins. Reprinted from Traub et al. (1966a), by permission of the authors.

apparently as the result of bacterial mutations to ochre suppression (Reid et al., 1965) and amber suppression (Bollen et d., 1965). We conclude that the 30s and 50s particles differ substantially from each other in their protein as well as their RNA content. ' Apparently Mg2+and, to some extent, polyamines, function to hold the peptides and RNA complex in a discrete structure i n vitro. Goldberg (1966) and Choi and Carr (1967) have estimated that the in vitro molar ratio of bound Mg2+to phosphate is 0.5. This Mg2+is apparently not all tightly bound €or Rodgers (1964) showed that 35% of the Mg2+in 705

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97

ribosomes exchanges in 2 min. at 0” in vitro. He concluded that this fraction was responsible for holding the 30s and 50s particles together, the more slowly exchanging material being bound within the subunit. Lowering the Mg2+concentration by treatment with EDTA or dialysis against low-Mg2+buffer apparently allows a loosening of the structure, making the particles unfold and become less compact (Rodgers, 1966; Gesteland, 1966; Gavrilova et al., 1966; Weller and Horowitz, 1964). Polyamines also function to stabilize the RNA-protein structure and will substitute for Mg2+ to some extent in test-tube experiments (e.g. Cohen and Lichtenstein, 1960; Colbourn et al., 1961; Silman et al., 1965). Conversely, high ionic-strength conditions, such as those used in equilibrium density-gradient centrifugation with caesium chloride, can cause dissociation of RNA and protein even at 5 x 10W M Mg2+(Meselson et al., 1964).At lower Mg2+ concentrations, this dissociation can be blocked by the addition of cross-linking agents such as formaldehyde (Spirin et al., 1965). By regulating the Mg2+concentration carefully, it is possible to remove only some of the protein components and produce dense RNArich particles. Under ‘carefully defined conditions, the proteins and stripped ribosomes can be recombined to give particles that resemble the starting material (Lerman et al., 1966; Hosokawa et al., 1966; Staehelin and Meselson, 1966a; Spirin and Belitsina, 1966). Electrophoresis and binding experiments have shown that apparently certain proteins, perhaps surface ones, are removed in this way (Staehelin and Meselson, 1966b; Raskas and Staehelin, 1967; Traub et al., 1966a, b; Nomura and Traub, 1967). For further discussion of the structure of RNA and particularly of ribosomes, the interested reader should consult the monograph of Spirin (1964).

D. CELLULARLOCALIZATION OF BACTERIAL RIBOSOMES For an understanding of the “life cycle” of ribosomes, it would be helpful to know whether at any time ribosomes are localized in any particular portion of the bacterial cell. Such a relationship between ribosomes and other structural elements of the cell might shed light on the mechanisms which control ribosome assembly and function. At present, however, the infordation available is not clear-cut. To examine this question, only two methods are currently available, namely cell fractionation and electron microscopy of ultrathin sections. These methods have been applied to the cells of higher organisms with much success, but bacterial cells are singularly intractable to this sort of approach. Unlike the higher-order cells, bacteria lack well-defined intracellular compartments which aid in isolation of cell fractions. Also, unlike animal cells which are surrounded by a pliable but well-defined membrane, bacteria are encased in comparatively rigid girdles of cell-wall

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

FIG.5. Electron micrograph of an ultrathin section of Acinetobacter, a Gramnegative bacterium. Cells were pre-fixed with glutaraldehyde by the method of Glauert and Thornley (1966). Ribosomes are visible as dark bodies scatterednearly a t random throughout the cytoplasm. Reproduced by courtesy of Dr. Audrey Glauert, Strangeways Research Laboratory, Cambridge, England.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

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material which make them unsuitable for the more commonly used preparative methods. Thus, it is little wonder that the techniques applicable to animal cells have yielded little information when applied to bacteria, and that special methods have had to be developed. After differential centrifugation of bacterial lysates, it is common experience to find many ribosomes (upward of 50% of the total) in association with the cytoplasmic membrane (Schlessinger, 1963; McQuillen et al., 1959; Aronson, 1966).This finding has suggested that protein synthesis takes place on membrane-bound ribosomes. This recalls the situation in cells of higher organisms which possess a “rough” (i.e. ribosomerich) endoplasmic reticulum where much protein synthesis takes place. Some authors (Schlessingeret aZ., 1965; Van Iterson, 1966)have reported electron-microscopic evidence for intracytoplasmic filaments associated with ribosomal material. The nature of these filaments is obscure and they have not been isolated from the cells in purified state. I n sections of growing bacteria, there are often so many ribosomes that virtually the whole cytoplasm is packed with particles in no particular orientation. I n such preparations, it is not possible to determine a preferential localization within the cell. Sections of bacteria not replete with ribosomes usually show a fairly random localization of these particles. I n the electron micrograph shown in Fig. 5 , the ribosomes seem scattered about the cytoplasm and few are seen near the envelopes of the cell. Interestingly, ribosomes have never been actually observed within the bacterial nucleus in thin sections. Maaloe and Kjeldgaard (1966) have estimated that, within the bacterial nuclear region, the DNA strands are quite tightly packed so that ribosomes may be excluded on steric grounds.

11. The Role of Ribosomes in the Control of RNA The efficient and regular pattern of bacterial cell growth suggests that physiological processes are interrelated and, on some levels, may serve to regulate each other (e.g. Dean and Hinshelwood, 1966). There are several indications that ribosomes may play a regulatory role of this sort in the synthesis of ribosomal and non-ribosomal RNA. I n vitro ribosomes participate in RNA synthesis catalysed by DNAdependent RNA polymerase. Byrne et al. (1964) have shown that ribosomes attach to nascent RNA strands, that is, to incomplete molecules still attached to their DNA template. Shin and Moldave (1966) demonstrated that the addition of ribosomes to such a system increases the extent but not the initial rate of the reaction. Attachment of ribosomes to nascent RNA chains has also been demonstrated in several in vivo experiments. Using B. megaterium infected with

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phage at multiplicities that result in the inhibition‘of synthesis of host RNA, Schaechter and McQuillen (1966) showed that SO% of the newlymade RNA was attached to ribosomes. Since this RNA was made during a very brief “pulse”, these authors concluded that most of these RNA chains were still being synthesized. This finding implies that the ribosomes attach to the free end of the nascent RNA chains. Analogous results were reported by Das et al. (1967) in cultures of E. coli kept at 0”. If attachment of ribosomes is a necessary step in the completion of nascent RNA chains, there are several possible explanations for this requirement. Stent (1966) has proposed that ribosomes function by traversing the nascent RNA and by directly coupling RNA synthesis to protein synthesis. I n his theoretical model, such movement of the ribosomes would serve to pay out the newly-made portion of the RNA molecules from the DNA template. The absence of such co-ordinate polymerase action and ribosome motion might explain why a molecule of RNA polymerase appears to function only once in in vitro systems (Bremer and Konrad, 1964). Under these artificial conditions, there would be no mechanism for releasing RNA from the DNA template. There are some data to support this theory. Several groups of experimenters have observed that the synthesis of m-RNA is a polarized process beginning at the 5’ and ending at the 3’ end (Shigeura and Boxer, 1964; Bremer et al., 1965; Maitra et al., 1965). It has also been shown that m-RNA is translated in this same 5’ to 3‘ direction (Wahba et al., 1966; Terzaghi et al., 1966). The oldest portion of an RNA molecule is thus read first, meaning that the attachment of ribosomes to m-RNA and the initiation of protein synthesis can actually begin before synthesis of the messenger is completed. I n experiments with certain /3-galactosidase extreme polar mutants of E . coli, it was shown that when enzyme formation was impaired at the level of protein synthesis the corresponding messenger for /3-galactosidasewas not made (Attardi et al., 1963). These experiments suggest that protein synthesis and m-RNA synthesis are linked. This co-ordinate synthesis is not obligatory under all conditions. Bacteria treated with a variety of antibiotics which inhibit protein synthesis (e.g. chloramphenicol, tetracycline and streptomycin) continue to synthesize RNA at a rapid rate while protein synthesis is arrested. Experiments of Naono et al. (1966) using E. coli depleted of ribosomes by magnesium starvation showed that m-RNA production is proportional to the number of ribosomes within the cell. Ribosomes might control RNA production in other ways as well. Thus, the free ends of nascent m-RNA molecules may be sensitive to nuclease attack, and ribosome attachment might protect them. This is supported by the in witro findings of Takanami and Zubay ( 1964) and Takanami et al.

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(1965) that the portion of synthetic polyribonucleotides or phage RNA attached to ribosomes in vitro is resistant to ribonuclease. Another possibility is that the nascent end of the RNA molecule may inhibit RNA polymerase, as suggested by Maitra etal. (1964) and Krakow (1966)who found that in vitro RNA synthesis is stimulated by the addition of ribonuclease. Attachment of ribosomes may function mechanically to clear the RNA from the template. As mentioned above, in ultrathin sections ribosomes are never seen within the nuclear region of bacteria. Perhaps a ribosome attached to a nascent RNA chain serves as a “float”, keeping the RNA molecule from intertwining with the DNA and providing it with a cytological sense of direction. It is well to note that these mechanisms are not mutually exclusive and may actually function in combination. Some indirect evidence has also been presented for an inhibitory rather than a stimulatory role of ribosomes in the synthesis of ribosomal RNA. This suggestion is based on the observation that amino-acid starvation results in both an inhibition of r-RNA synthesis and an increase in the number of free ribosomes as polysomes break down (Morris and DeMoss, 1966; Ron etal., 1966b).

111. The Production of Ribosomal RNA Presumably, r-RNA is produced by the same mechanism as other cellular RNA, by the action of RNA polymerase on ribonucleotide triphosphates directed by a DNA template. The size and location on the genetic map of the genes concerned with r-RNA synthesis have been studied with hybridization techniques. I n principle, an RNA molecule is the complementary copy of theDNAstrand on which it was made. If mixed in vitro with denatured, single stranded DNA, the RNA will make a specific double stranded structure with its homologous region on the DNA. Such studies in E . coli (Yankofsky and Spiegelman, 1962a,b ;Attardi etal., 1965),B. megaterium (Yankofsky and Spiegelman, 1963) and B. subtilis (Oishi and Sueoka, 1965) have shown that the portions of the bacterial chromosome complementary to r-RNA are small. The two types of r-RNA do not compete with each other in hybridization tests in Bacillus species, indicating that they differ in nucleotide sequence (Yankofsky and Spiegelman, 1963; Oishi et al., 1966). I n E. coli some competition has been reported by Attardi et al. (1965). Various estimates of the fraction of the chromosome complementary to the r-RNA have been made on the basis of these studies. Yankofsky and Spiegelman (1962a, b) found that 0.2% of the E. coli chromosome was complementary to 235 r-RNA, corresponding to about ten cistrons; in B. megaterium they found values of O . l S ~ Oand 0.14% complementary ?

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to 235 and 1 6 s respectively, corresponding to about thirty-five and forty-five cistrons, respectively (Yankofsky and Spiegelman, 1963). Similar estimations have been made by Attardi et al. (1965). These estimates all confirm the fact that each chromosome contains few copies of the genetic loci complementary to r-RNA and that together they comprise less than 1% of the genome. The r-RNAs of various bacteria apparently have many nucleotide sequences in common. This is demonstrated by hybridization experiments between r-RNA and DNA from different bacterial species. The high degree of cross hybridization observed indicates that few gross evolutionary changes have taken place in this part of the bacterial chromosome in the organisms tested (Attardi et at,1965; Dubnau et al., 1965b; Doi and Igarashi, 1966; Takahashi et al., 1967). Experiments designed to determine the location of the r-RNA cistrons on the genetic map are difficult. This is due in part to the nature of most genetic mapping experiments. Mapping commonly involves estimations of biochemical changes in the ultimate gene product, a protein, or phenotypic properties resulting from such changes in the recombinant progeny. I n the case of r-RNA, we wish to map changes in the primary gene product, RNA itself. Since, as we shall discuss below, there is no conclusive evidence that, bacterial r-RNA codes for particular proteins, classical genetic tests are inadequate. As a consequence, mapping of the r-RNA cistrons is restricted to techniques which are relatively coarse and do not give map positions with the precision of the usual methods. Mapping experiments have been carried out by synchronizing bacterial cells with regard to the replication cycle of their chromosome. The time when ribosomal cistrons replicate may be assumed to be related to their location on the genetic map. If the experiment is carefully designed, at some point in the replication cycle each chromosomal locus is suddenly doubled and this, it could be assumed, is soon followed by a doubling in the amount of the corresponding RNA. If synchronized cultures are sampled at different times of replication, the r-RNA loci can be determined either directly by quantitative hybridization of the DNA with r-RNA or indirectly by measuring the increase in r-RNA production. Rudner et al. (1964, 1965) assayed the base ratios of RNAs produced at various times in the division cycle of synchronized cultures of E . cobi. They found that, at two times after synchronized growth had begun, the RNA synthesized had a composition like that of r-RNA. Two different strains of E . coli with different chromosomal localization of the sex factor were examined and the results of experiments with each strain were similar. By estimating the rate of chromosomal replication and assuming that it was uniform over the length of the genetic map of the chromosome, they inferred the positions of the two loci correspondingto

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r-RNA. Similar results were obtained by Manor and Haselkorn (1967~) in a study of RNA synthesis in E . coli fractionated according to cell size. An extensive study of the location of r-RNA cistrons was carried out by Cutler and Evans (1967).They synchronized the growth of E . coli and at different times added a “heavy” precursor of DNA (Bbromouracil). This permitted the isolation of specific segments of the genome by equilibrium density-gradient centrifugation. By hybridizing these segments with r-RNA they showed that there are two widely separated r-RNA-specific regions, one of which maps near the streptomycin locus. Synchronous culture experiments can also be performed with B. subtitis. With this brganism, synchronous replication may be achieved by

ade

16s, 2 3 s RNA thr I

I eu I

met I I

FIG.6 . Tentative positions of 16s and 23s RNA loci on the Bacillus subtilis chromosome. The positions of the markers for adenine (ade), threonine (thr),leucine (leu) and methionine (met) are shown. The designation for the RNA locus is not to be taken as an accurate representationof the relative size of this locus. Reprinted from Oishi and Sueoka (1965), by permission of the authors.

inducing germination of spores. Not only can the DNA of these cells be hybridized with RNA, but it can also be used to transform suitable B . subtilis mutants to locate other genetic markers. If the DNAs are labelled with a density marker such as 15Nor bromouracil before replication takes place in a “light” medium, the old “heavy” and new “light” DNA can be separated on the basis of their differences in density. Conversely, “light” spores can be germinated in “heavy” medium. Using this method, Oishi and Sueoka (1965) and Dubnau et at. ( 1965a) separated newly-replicated DNA from unreplicated DNA. The time of appearance of replicated markers was assayed by transformation and, on the assumption that it is related to the position on the chromosome, was used to construct a genetic map. By estimating the hybridization capacity of the newly-synthesized DNA, the authors were able to locate the r-RNA cistrons in the vicinity of the streptomycin locus. The possible significance of this finding is discussed in Section I V (p. 108). A genetic map obtained from these experiments is shown in Pig. 6. The significant result of this work is the demonstration that the regions of the chromosome responsible for r-RNA production have been localized even though their precise location has not been determined. The products of the reaction directed by these templates are 16s and 23s RNA. Early experiments (e.g. McCarthy and Aronson, 1961;

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McCarthy et al., 1962;-Takai et al., 1962) implied that 16s and 235 molecules might be assembled as oligomers of a small polynucleotide with a sedimentation coefficient of about 8S-14S. The subsequent discovery of the existence of the unstable m-RNA in growing bacteria (e.g. Gros et al., 1961; Bolton and McCarthy, 1962) suggested that small RNA molecules are mostly m-RNA or fragments thereof. More recent analytical work of Lindigkeit and Handschack (1965) is consistent with this interpretation. Some evidence has been presented indicating that newly made r-RNA does not have sedimentation properties exactly identical to those of mature r-RNA (e.g. Hayashi et al., 1966). I n the cases reported, sucrose-gradient centrifugation of RNA extracted from a mixture of mature and nascent ribosomes showed that the nascent 16s RNA sediments slightly more rapidly than the mature 1 6 s RNA molecules. Sypherd and Fansler (1967)have concluded that the secondary structures of the nascent and mature r-RNAs differ. Dubin and Gunalp (1967) have found differences in the degree of methylation of nascent and mature r-RNA. There is to date no report of bacterial r-RNA larger than 235. I n the cells of higher organisms the two kinds of r-RNA molecules are synthesized jointly as a 455 chain which is later split (e.g. Scherrer et al., 1963). This phenomenon may reflect the need in higher order cells for a transport system between the nucleus and the cytoplasm. Estimates of the amount of r-RNA produced at any given instant vary. I n B. subtilis, Levinthal et al. (1962) showed that about 20% of the RNA labelled during a short pulse of RNA precursors did not decay vhen the cells were treated with actinomycin D, and that it was largely r-RNA. Bolton and McCarthy (1962) showed that in Proteus wulgaris a larger fraction, about 65% of the newly-made RNA, was r-RNA on the basis of hybridization studies. These differences may reflect the variability of different bacteria since Salser (1966) showed that more unstable RNA is formed in B. subtilis than in E. coli. Whatever the exact proportion of r- to m-RNA synthesized in a short time, a great deal of r-RNA must be produced and this production is apparently directed by less than 1% of the chromosome. The production of 16s and 2 3 s RNA molecules seems to be quite well co-ordinated. Although they are distinctly different molecules they are produced in virtually the same numbers so that the molecular ratio is close to 1 :1 and their weight ratio 1 :2. It is not known how this balance is achieved. How long does a growing bacterium take to synthesize a molecule of r-RNA? While the answer to this question is not available, certain limits can be established from the fact that a bacterial cell in balanced growth must double its ribosome complement every generation. If, in E . coli

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growing with a doubling time of 30 min., there are about 10,000 ribosomes, five or six ribosomes must be made per second if the rate of ribosome synthesis does not vary throughout the cell-division cycle. Since the number of cistrons corresponding to each class of r-RNA has been estimated to be ten or less in E . coli (see above), it is possible to calculate that per cistron one r-RNA molecule is made every few seconds. Note that the process may be intermittent, and therefore intrinsically faster, and that the rate of output is not necessarily the same as the rate of synthesis of individual molecules. The rate of synthesis is given by the time required for one RNA polymerase molecule to transcribe the cistron. If several polymerases are engaged in the transcription of the same cistron, the time of synthesis will be longer than that of output. If, say, ten polymerases are working simultaneously, the time of synthesis would be ten times longer. These calculations have bGen presented in more detail by Kjeldgaard (1967). An experimental attempt to determine the rate of r-RNA synthesis was carried out by Zimmermann and Levinthal (1967). They postulate that the time required for making a 1 6 s RNA molecule is half that for making a 2 3 s molecule since the 16s is half as large. Very short-term labelling experiments indicate that this is the case. If a culture of B. subtilis is pulse labelled with radioactive uracil, the specific activity of the 30s particle containing the 16s RNA is initially higher than that of the 50s particle which contains 2 3 s RNA. This effect was demonstrated by allowing the maturation of ribosomal subunits to proceed while RNA synthesis was inhibited by actinomycin D. Invoking a number of logical assumptions, the authors interpret this to mean that in a short time, less than that required to synthesize a whole 2 3 s molecule, 16s molecules can be labelled and released from their template as completed molecules. After this short lag, uniformly labelled 2 3 s molecules are produced and the 50s and 305 particles achieve the same specific radioactivity. The experimental data indicate that the time required for the synthesis of a 16s molecule is about 18 sec. at 37”. By extension, an RNAmolecule with a molecular weight of 1 x lo6 would be made in about 30 sec. Another rate determination was performed by G. Mangiarotti, D. Apirion, D. Schlessinger and L. Silengo (personal communication) using E. coli grown in a medium containing a high concentration of sodium sulphate. Under these conditions, the organisms grow slowly and are readily lysed by gentle means. By analysing the RNA of newly-synthesized ribosomal precursors after various times of labelling, they determined the rate ofr-RNA synthesis. The time of synthesis for each class of r-RNA was 2 min. at 37”. The discrepancy between these values and those obtained by Zimmermann and Levinthal (vide supra) may be due to differences in the organisms studied and in the experimental plans.

-

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Within this range of times, comparable values have been estimated for the synthesis of m-RNA in a few specific cases (Imamoto et al., 1965; Leive, 1965b). Although all these measurements should be considered tentative they suggest that the rates per nucleotide for synthesis of mand r-RNA may be comparable. If this is so, the relatively small region of the DNA correspondingto the r-RNA cistrons must be transcribed many more times than the cistrons for typical m-RNA molecules. Ribosomal-RNA production seems to be controlled by a specific chromosomal locus called the RC locus (Stent and Brenner, 1961). This control was discovered by examining amino-acid auxotrophic mutants of E. coli. Most mutants are unable to grow or proguce RNA in the absence of their required amino acid. Synthesis of RNA by these mutants is under stringent amino-acid control. However, a certain class of E. coli amino-acid auxotrophs can synthesize appreciable amounts of RNA in the absence of the required amino acid. This RNA is complexed with protein into ribosome-like particles (see Section V, B, p. 115). These relaxed mutants are actually double mutants which have mutated in the RC locus as well as in the amino-acid locus. Many workers have demonstrated that RNA made by relaxed mutants is largely ribosomal (e.g. Dagley et al., 196213; Nakada, 1965a; Sypherd, 1965b). Chloramphenicol has been shown to relieve stringency in stringent strains (Kurland and Maal~e, 1962).I n the presence of high concentrations of chloramphenicol,protein synthesis is blocked and amino acids may become more available for regulation of RNA synthesis. Under these conditions, E. coli produces ribosome-like particles which contain RNA and little protein (e.g. Nomura and Watson, 1959). The ribosome concentration in a bacterial cell is proportional to the cell’s growth rate as shown in 8almonella typhimurium by Ecker and Schaechter (1963). Shifting a bacterial culture from a poor to a rich medium (shift-up) causes a rapid increase in RNA synthesis which takes place before other biosynthetic rates increase (Kjeldgaard et al., 1958; Koch, 1965).Much of the RNA synthesized immediately after the shiftup has the nucleotide composition of r-RNA rather than m-RNA (Mitsui et aZ., 1963).Aronson and Holowczyk (1965) showed that, although this RNA has the same base ratio as r-RNA, nucleotide sequence studies revealed that it is more heterogeneous. These experiments imply that the cell’s capacity for r-RNA production is not at its limit before the shift since its output increases drastically after the shift, before much new DNA carrying additional ribosomal cistrons or much protein such as RNA polymerase can be formed. Apparently the production of new ribosomes takes precedence over all other functions in adapting a bacterial cell to faster growth (Maal~e and K jeldgaard, 1966).

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Production of r-RNA can also be shut off by external perturbations. Infection of the bacterium with a virulent bacteriophage may interrupt host-RNA synthesis (Nomura et al., 1960, 1962; Schaechter and McQuillen, 1966). Certain drugs apparently depress r-RNA synthesis somewhat selectively. This phenomenon has been reported for levorphanol (Simon and Van Praag, 1964; Simon et al., 1966a) and for 2,4-dinitrophenol”(Simon et al., 1966b). In summary, r-RNA synthesis in growing bacterial cells probably proceeds by the transcription of small regions of the bacterial chromosome by an enzyme which we assume to be RNA polymerase. Transcription apparently takes place with high efficiency, for less than 1yoof the genome is transcribed. At any one time this small segment of DNA directs the synthesis of 20-60y0 of the RNA being synthesized. The process is apparently controlled by amino acids and can be interrupted by certain drugs. A plausible explanation of the kinetics of labelling of 16s and 235 RNA is that the r-lkNA cistrons are each being transcribed by several polymerase molecules simultaneously, and that this transcription is coordinated so that neither type of r-RNA molecule is produced in exces8.

IV. The Production of Ribosomal Proteins Ribosomal proteins can be identified only by their integration into ribosomal particles, hence studies of their synthesis have been a part of studies of ribosome assembly. It is generally assumed that they are synthesized by the same sort of mechanism its other cellular protein. There has been no demonstration of the existence of specific polysomes which synthesize ribosomal protein. The principal N-terminal amino acids of E . coli ribosomal proteins are methionine and alanirie, which are the most abundant N-terminal residues of other E . coli proteins (Waller, 1963,1964).This finding implies that N-formylmethionine, hypothesized to be the initiator amino acid in the synthesis of other proteins (Adams and Capecchi, 1966),serves the same function for ribosomal proteins. If these assumptions are true, ribosomes in effect have an autocatalytic role in the cell, synt.hesizing protein for the completion of other ribosomes. If ribosomal proteins are synthesized by the same mechanism as other proteins, some m-RNA must direct the synthesis. Here again we must argue by inference, for no demonstration of the chemical and physical nature of such a m-RNA has ever been made. One hypothesis is that r-RNA may itself serve as this messenger (Ecker, 1965; Nakada, 1965a; Roberts, 1965; Stent, 1966).Such an hypothesis is teleologically attractive for its economy. Several limiting assumptions must be made if it is to be applicable; we shall consider them below.

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If r-RNA acts as the messenger for ribosomal protein, the two molecules must have the same genetic locus on the bacterial chromosome. The experiments described in the preceding section allow tentative assignment for the location of the r-RNA cistrons on the genetic map. While a direct mapping of ribosomal proteins is not yet possible, mapping of properties apparently due to changes in their function can be carried out. Foremost among these changes is the sensitivity of bacteria to aminoglycoside antibiotics such as streptomycin. Indeed, as we noted in Section 111(p. 103), streptomycin sensitivity h a p s near the loci for r-RNA. A number of experiments have shown that the effect of streptomycin both in vitro and in vivo is to derange protein synthesis by interaction with the ribosome (Flaks et al., 1962a, b; Spotts and Stanier, 1961). This effect is apparently due to misreading of codons on the m-RNA (Davies et al., 1964),for the ability of the ribosomes to form polysomes is unimpaired (Cox et al., 1964; Davies, 1964). The site of action of streptomycin was originally designated as the 305 subunit of the 705 ribosome (Cox et al., 1964; Davies, 1964) and more recently has been found to be one of the core components of this subunit (Staehelin and Meselson, 1966b; Traub et al., 1966a).Other experiments (Leboy et al., 1964)have shown that K12 strains of E. coli differ from other strains in the electrophoretic properties of one 30s ribosomal protein. Although recent evidence (Furano, 1966) shows that this protein may not necessarily be associated with streptomycin sensitivity or resistance, transduction experiments have shown its locus to be near or identical to that of streptomycin sensitivity or resistam\ It is largely on the basis of arguments such as these that the r-RNA and protein loci appear to be related genetically. Further mapping data may resolve this question. Any theory which proposes that r-RNA codes for ribosomal protein must consider the number of r-RNA cistrons available for coding and the number of different proteins which are found in ribosomes. Electrophoresis of the proteins extracted from E. coli ribosomes show that there are about 30 protein components of a 705 ribosome (e.g. Leboy et al., 1964; Traut, 1966),at least 19 of which have distinct differences in their peptide sequences (Traut et al., 1967).A simple calculation shows that, if each ribosome contains a 16s and a 235 RNA molecule identical to those of all other ribosomes and one each of the 30 various peptides, then the amount of r-RNA is insufficient for coding for all the proteins. Assuming an average molecular weight of 350 for a nucleotide residue, a coding ratio of three nucleotide residues per amino-acid residue, and an average molecular weight of 120 for an amino-acid residue, the ratio of template RNA to protein product should be 3 x 350: 120 or about 9: 1. The 2 : 1 weight ratio of RNA/protein of ribosomes (Tissikres et al., 1959) is considerably smaller. Clearly then, if r-RNA serves as m-RNA for the

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production of ribosomal protein, not all ribosomes are identical. At the very least their RNAs must vary sufficiently to produce the variety of proteins obsekved. If each r-RNA cistron on the bacterial chromosome differs from the others there can, however, be enough variation in its information content to produce the diversity of peptides observed. This can be seen by further calculations. The estimated average molecular weight of a ribosomal protein is 2.5 x lo4 (Section 11, p. 95). This represents apolypeptide chain of 2.5 x 104/120 or 200 amino acidresidues. An RNA molecule of 600 nucleotide residues (molecular weight of approximately 2 x lo5)would be required to code for such a polypeptide. If 30 such ribosomal proteins exist, then approximately 6 x lo6 daltons of ,RNA would be needed to specify the required information. As we have seen above, the molecular weight of 2 3 s RNA is approximately 1 x lo6 and that of 16s RNA is approximately 5 x lo5, and several copies of each template are found in each genome. If each r-RNA molecule were translated several times and each cistron were different in sequence, then there would be sufficient information contained in the r-RNA cistjons to code for ribosomal proteins. Estimates of the number of times each RNA would need to be translated are of the order of six (Roberts, 1965) to ten (Stent, 1966). Another completely different hypothesis is that each ribosome contains the same RNA as all other ribosomes, but does not have a complete set of the 30 different proteins. Rather, it contains only those proteins coded for by its 1.5 x lo6daltons of RNA and has several copies of each of these. It also seems possible that ribosomes differ in both their RNA and proteins. This latter model has been discussed by Roberts (1965). At present there is no evidence to favour one of the above models to the exclusion of the others. Indeed, there is no compelling evidence for the messenger role of r-RNA. It seems entirely possible that non-ribosomal RNA may serve as messenger for the synthesis of all or some of the ribosomal proteins. Some evidence exists to show that ribosomes appear to be functionally identical. Among the best is an experiment by Nakada (1963)showing that ribosomes existing in the cell before /3-galactosidase induction function in the synthesis of the induced enzyme. This was demonstrated by a, socalled density transfer experiment. Bacteria were grown in amedium containing heavy stable isotopes before being transferred to a “light” medium containing inducer for &galactosidase and radioactive isotopes. 8-Galactosidase was formed under these conditions before any appreciable amount of new, light, radioactive ribosomes could be detected by equilibrium density-gradient centrifugation. Thus, the pre-existing ribosomes were active in synthesis of a protein entirely new to the cell. Similar conclusions can be drawn from the observation that phage v*

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proteins are synthesized by pre-existing ribosomes after bacteriophage infectionwhilehostRNAsynthesisis stopped (Brenner etal., 1961).Therefore, at least some of the ribosomes must be capable of participating in the synthesis of a variety of proteins. Conversely, there is no evidence for the existence of special classes of ribosomes responsible for the synthesis of specific proteins. The ability of r-RNA to serve as a template to direct in vitro amino-acid polymerization has been invegtigated. Purified RNA extracted from \ribosomes functions in amino-acid incorporation only in the presence of neomycin and after its secondary structure has been broken (Holland et al., 1966). There is contradictory information about the ability of rRNA extracted from the incomplete particles synthesized under relaxed conditions (see Section 111,p. 106)to stimulate amino-acid incorporation i n vitro. Otaka et al. (1964),Willson and Gros (1964)and Nakada (1965a) reported that this RNA was more active than mature r-RNA. Manor and Haselkorn (19674 and Sypherd (1967)confirmed these findings but attributed them to contamination by m-RNA. The physical state of ribosomal proteins before their incorporation into completed ribosomes is unknown. According to the schemes discussed above, if r-RNA serves as the sole messenger for ribosomal protein it must be translated several times before being assimilated into a ribosome. During the time between completion of the first ribosomal protein and the assembly of the ribosome, it is possible that the ribosomal proteins enter a pool of soluble proteins from which they are later withdrawn. If non-ribosomal RNA serves as messenger for ribosomal proteins, i t is also likely that such a pool exists. A number of experimental observations support such an hypothesis. Perhaps the most convincing of these is the fact that, under conditions of “relaxed” growth, RNA synthesis can proceed in the absence of protein synthesis and bacteria accumulate ribonucleoprotein particles. The simplest explanation of this phenomenon is that a pool of proteins exists to be complexed with newly synthesized r-RNA (Kurland and Maalrae, 1962). Recent findings of S. Osawa (personal communication) indicate that r-RNA may aggregate nonspecifically with various cellular proteins. This complicates the interpretation of these experiments. Under special conditions of inhibition, bacterial cells lose their ribosomes; studies of recovery from these traumata have suggested that ribosomal proteins are not entirely destroyed under these conditions but survive as a pool from which they can be re-utilized. When Mg2+is removed from the growth medium, bacteria lose virtually all their ribosomes without great loss of viability (Dagley and Sykes, 1957; McCarthy, 1961; Kennel1 and Magasanik, 1962). When Mg2+is restored to the medium, bacteria respond by synthesizing new r-RNA

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(e.g. Suzuki and Hayashi, 1964) and producing new ribosomes. If Mge+is made available but a required amino acid is withheld, the RNA synthesized is complexed with proteins yielding particles similar to those found under conditions of “relaxed” synthesis (Nakada and Marquisee, 1965). Sinde the cells could not have synthesized proteins under these conditions, the protein complexed with the RNA must have pre-existed in the cell. More recent data of Kennell and Kotoulas (1967a, b, c) and Marchesi and Kennell ( 1967) indicate that these interpretations may be oversimplified. These workers report that cell populations are not homogeneous under conditions of Mg2+starvation and that, while some cells lose their ribosomes, others retain 30s and 50s particles. The net content of particles per unit of culture remains constant throughout the starvation period. Although polysomes break down in these conditions, some protein synthesis does take place. These results point t o the complexities arising from starvation experiments. Results similar to those reported with Mg2+starvation have been found for phosphate starvation. Julien et aZ. (1967) found that phosphate starvation causes degradation of the ribosomes of bacteria; following the addition of phosphate, ribonucleoprotein particles can be formed in the presence of chloramphenicol. Also supporting the hypothesis that protein from degraded ribosomes can be complexed with newly made r-RNA are the data of Nomura and Watson (1959).I n their experiments, prolonged treatment of E . coli cultures with high concentrations of chloramphenicol caused eventual breakdown of the existing 705 ribosomes of the cell. The particles which accumulated under these Conditions were thought to result from complexing of newly-made RNA with the proteins from the degraded ribosomes. However, this interpretation may not be correct since S. Osawa (personal communication)recently showed that non-specific aggregation of r-RNA and cellular proteins can occur. Some evidence suggests that ribosomal proteins may be divided into several functional classes and that the pool size of these classes may differ. Nomura and Traub (1967),Traub et al. (1966a,b), Staehelin and Meselson (1966a, b) and Lerman et aZ. (1966) showed that a class of ribosomal proteins can be removed from ribosomes and, under suitable conditions, can be re-associated in vitro. These proteins seem to be surface components of the ribosomes and are necessary for various essential functions in protein synthesis. It is likely that intensive work now carried out on these proteins will shed much light on their function. Julien et d . (1967)have suggested that perhaps these proteins are added to ribosome precursors as the final step in the assembly process. The size of the pool of free ribosomal proteins has been estimated by Schleif (1967)to be small, consisting of less than 5% of the total proteins

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of the cell. This determination was carried out by comparing the rate of incorporation of labelled amino acids into ribosomal and non-ribosomal proteins in balanced-growth cultures. I n summary, ribosomal proteins seem to be produced by the same mechanism as other proteins. No conclusive evidence shows that r-RNA serves as messenger for the produc\ion of these proteins, although the total number of r-RNA cistrons in the bacterial genome is sufficient to code for the various ribosomal proteins. It is possible that ribosomal proteins exist in part as a pool available for later assembly into ribosomes; perhaps a special class of proteins exists which is added to the partially completed particles to give the finished product.

V. The Assembly of Ribosomal Subunits I n this section we will discuss the aggregation of r-RNA with ribosomal protein to produce the elemental ribosome structures, the 305 and 50s subunits. This subject has been investigated extensively but, as we shall see, details of the assembly mechanism are not yet understood. Most investigators assume that the 30s and 505 particles are assembled by similar mechanisms and that no specific enzymes or energy sources are required for the reaction. Experiments designed to study the assembly process have been confined almost entirely to the examination of precursor particles by sucrose-gradient centrifugation. Studies have been carried out both on bacterial cultures in balanced growth and on cultures in which growth is unbalanced by drug inhibition or by mutation. Before considering the assembly process, it is well to note that very little r-RNA exists free and uncomplexed in the bacterial cell. This suggests that, as soon as it is completed or while it is being made, it associates with proteins from a pre-existing pool. Alternatively, if r-RNA serves a messenger fudction, it may be complexed with ribosomes either during its synthesis or immediately after completion.

GROWTH A. STUDIESON CELLS IN BALANCED When cultures of various bacteria are “pulsed” with radioactive uridine or uracil, about two-thirds of the incorporated label is found as a broadly sedimenting component lighter than the 70s ribosomes. Figure 7 shows the appearance of such material in a lysate of B. megaterium centrifuged through a sucrose gradient (Schaechter et aZ., 1965). The remainder of the incorporated label is in the form of m-RNA bound to 70s ribosomes and sedimenting with the polysomes. The light material contains ribosomal precursors and some m-RNA as shown by hybridiza-

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tion experiments using homologous DNA. It is heterogeneous and appears as an unresolved band containing peaks of about 455 and 25s when fractionated further by sucrose density-gradient centrifugation (Fig. 8). These rapidly labelled precursors are sensitive to amounts of ribonuclease which do not affect mature ribosomes, as shown in Fig. 8. If labelling is extended for longer periods of time, it is seen that the label fist enters the 30s and 505 regions and only later the 705 ribosomes. This has been shown in E. coZi by McCarthy et aZ. (1962)and Nakada and Kaji

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FIG.7. Gradient centrifugation of a Iysate from pulse-labelledBacillus meguterium. A culture was labelled for two doubling times by addition of 1.5 pC/ml. 32PO:-. The culture was pulse-labelled for 30 sec. by addition of tritiated uridine. The lysate was layered on a 29 ml. 15-40% sucrose density-gradient and centrifuged for 130 min. at 25,000 rev./min. in an SW25 rotor (Beckman). The ordinate indicates counts/min. The top of the gradient is to the right. Radioactivity of 32P, -o-e-; 3H, ---0--0---. Reprinted from Sehaechter et al. (1965).

(1967) and in B. megaterium by ourselves. I n the case of B. megaterium, label does not enter the 70s material until after a delay of about 5 min. when the culture is growing at 37" with a 30 min. doubling time (W. S. Kelley and M. Schaechter, unpublished observations). The only extensive study of the kinetics of synthesis of ribosome precursors and their assembly into ribosome subunits is the pioneering work of several investigators at the Department of Terrestrial Magnetism of the Carnegie Institution of Washington. These investigators studied the assembly process in E. coZi and used DEAE column chromatography and

114

WILLIAM S. EELLEY AND MOSELIO SCHAECHTER

sucrose-gradient centrifugation at M Mg2+to resolve the precursor components. They interpreted their data on the basis of a mathematical model for the incorporation of radioactive uracil into the various cell

4000 2000

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FIG.8. Gradient centrifugationof a lysate from pulse-labelledBacillus megaterium. A culture was labelled for two doubling times by addition of 0.4 pC/ml. s2PO,8-. The culture was pulse-labelledfor 30 sec. by addition of 200 pC 3H-uridine.A lysate was prepared by the method of Schaechter et al. (1965) and divided into two portions. One was layered directly on a 10-35% convex exponential sucrose densitygradient; the other was treated with 1 pg. pancreatic ribonuclease /ml. in the cold and then layered on another identical gradient. Gradients were centrifuged 34 hr. at 5' b a n SW39L rotor (Beckman).The top of the gradient is to the right. The ordinate indicates counts/min. a2P0,8-, ---o---o-; 3H, ---0-0-. The top gradient is the sample treated wi6h ribonuclease.

fractions. I n addition to the original publications (Britten and McCarthy, 1962; Britten et al., 1962; McCarthy et al., 1962), two reviews of their study are available, to which the interested reader is referred (Britten, 1963; Roberts et aZ.,1963). Their analysis makes it possible to compare

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

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data obtained from different experiments by normalizing both the expressions for the growth rate and specific radioactivity. Assuming a model in which the uracil label flows through two sequential precursors called “eosome” and “neosome”, a plot of experimental values for specific activities of each precursor at various times is well fitted by the theoretical curves. Since these studies were carried out before the existence of labile m-RNA was demonstrated, the mathematical model was constructed on the assumption that there is no significantturnover of RNA. Recent work by Nierlich (1967), Salser (1966) and Koch (1967), to which the reader is referred for details, has shown that the turnover of m-RNA is a major contributor to the kinetics of labelling of RNA. I n brief, the reason is the following. When a labelled precursor enters a cell it is mixed with the unlabelled constituents of the low-molecular-weight pool and is withdrawn from this fraction to be incorporated into RNA. This pool is replenished with unlabelled precursors arising from the decay of the pre-existing m-RNA. The net effect is to mask the kinetics of labelling of RNA which would take place if the pool components were solely of external origin. These considerations make it apparent in the light of newer knowledge that a detailed kinetic analysis constitutes a formidable task. However, the overall conclusion of the Carnegie group that ribosomes are assembled in a step-wise fashion, has been confirmed by other means. Recent re-examination of the precursor particles in E . coli has revealed there are at least two precursors to the 5 0 s subunit and at least one to the 30s subunit (S. Osawa, personal communication ; G. Mangiarotti, D. Apirion, D. Schlessinger and L. Silengo, personal communication).

B. STUDIES OF METABOLICALLY INHIBITED CELLS As indicated above, alarge number of studies have followed an alternative approach to examine the assembly of ribosomes. These studies have focused on the accumulation of RNA-protein particles in bacteria which have been metabolically inhibited. I n what follows we propose to present a sampling of the results that seem most pertinent. The earlier literature of this field has been thoroughlyreviewed by Osawa (1965). Our summary will deal with each type of particle separately. 1. RCR“ Particles A large number of studies have used the so-called “relaxed” mutants, which do not have the usual “stringent” requirements for amino acids in RNA synthesis (see Section 111, p. 106). The mutation in the RC locus does not influence the assembly of ribosomes in the bacterium under normal conditions. However, when the mutation is allowed to express itself, that is, when a required amino acid is withheld, ribonucleoprotein

116

WILLIAM S . KELLEY AND MOSELIO SCRaECHTER

particles accumulate (Dagleyetal., 1962a,b). Neidhardt and Eidlic (1963) showed that the accumulated particles contain 16s and 23s RNA. If the “relaxed” mutant requires methionine, the RNA of the particles produced by “relaxed” synthesis in the absence of methionine is undermethylated (Mandel and Borek, 1963; Dubin and Giinalp, 1967). However, if the bacterium requires a different amino acid such as histidine, methylation is apparently complete (Manor and Haselkorn, 19674. The

I

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FIG.9. Sucrosedensity-gradientanalysis of “relaxed” particles in a cell extract prepared from Escherichia coli RCRe’.cells after 60 min. methionine starvation in a glycerol-containingmedium. 1%-Uracilwas present during 10-50min. of methionine starvation. The sedimentationcoefficients of the major fractions are indicated in thegraph. -0-o-indicates extinctionat 260mp; -~-o-,radioactivityof material precipitated with trichloroacetic acid. Reproduced from Nakada et al. (1964), with permission of the authors..

particles seem to be fragile in conditions which do not affect normal ribosomes. They are broken by sonication (Dagley et al., 1963),shear forces generated by the French pressure cell (Sypherd, 1965a) and low concentrations of pancreatic ribonuclease (Dagley et al., 1963;Sypherd, 1965a; Manor and Haselkorn, 1967b). Various sedimentation coefficients have been reported, including 18s and 25s (Nakada et al., 1964; Manor and Haselkorn, 1967a); ZOS, 30s and 455 (Sypherd, 1965a); 205 and 235 (Dagley et al., 1963).Chemical analyses indicate that these particles are

THE ‘‘IJXE CYCLE” OB BACTERIAL RIBOSOMES

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poorer in proteins than are the normal ribosomes ;their RNA/proteinratio is about 3 :1. Figure 9 shows a sucrose density-gradient profile of “relaxed” and naturally occurring particles. Whether these particles have any in vivo function is unknown. Nakada (1965a)reports that, in vitro, these particles can incorporate amino acids in the absence of 705 ribosomes and that the product is a heavier particle that resembles a completed ribosome. Muto et aZ. (1966)have been unable to confirm this report. Sypherd (1967) has observed that addition of “relaxed” particles to an amino acid-incorporating system in the presence of 705 ribosomes stimulates the production of acid-insoluble peptides. However, this stimulation may be due to the presence of extraneous m-RNA in the preparation of “relaxed” particles. The RNA of the “relaxed” particles seems to be slightly different in structure from that of mature ribosomes. The undermethylated particles can be methylated in vitro (Gordon and Boman, 1964) or the RNA extracted from them can be methylated (Manor and Haselkorn, 1967a). Methylation of this RNA in vitro does not affect its capacity to serve as a hemplate for in vitro amino-acid incorporation ; also, undermethylated “relaxed” RNA seems to have about the same in vitro template activity as “relaxed” RNA which is normally methylated, but neither seems to function better than RNA from mature ribosomes (Manor and Haselkorn, 1967a). Sypherd and Fansler (1967) have observed that “relaxed” RNA differs slightly from the RNA of mature ribosomes in sedimentation and when separated chromatographically on a methylated albuminkieselguhr column. If further RNA synthesis is inhibited by a chemical such as 2,4-dinitrophenol, the RNA of these “relaxed” particles apparently is digested by the cell but more slowly than m-RNA (Nakada et al., 1964). This also seems to occur if E . coli are infected with virulent phage T4 (Turnock, 1966). This latter result implies that the “relaxed” particles need the synthesis of non-ribosomal RNA for stabilization. When protein synthesis is re-initiated after restoration of the required amino acid, the amount of material in “relaxed” particles decreases and the number of ribosomes increases (e.g. Dagley et al., 1962a; Turnock and Wild, 1964; Nakada, 1965a; Sypherd, 1965b). After restoration of methionine to a starved methionine RCR” auxotroph, the RNA of the particles becomes fully methylated (Nofal and Srinivasan, 1966), and is incorporated into mature ribosomes (Nakada et aZ., 1964). During this process, the RNA “?natures” in its configuration and the unique properties of the “relaxed” RNA in sucrose-gradient centrifugation and methylated albumin-kieselguhr chromatography are lost (Sypherd and Fansler, 1967). This process takes place without degradation of the “relaxed” particles, and apparently is due to preferential

WILLIAM 8. KELLEY AND MOSEUO SUHAEUHTER 118 synthesis of ribosomal proteins which are added to the particles allowing them to mature into complete ribosomes (Ennis and Lubin, 1965b). Among the most interesting experiments performed with “relaxed” particles are those done by Nakada and Unowsky (1966). They demonstrated tha%,when these particles are mixed with ribosomal protein, ribosomal subunits are formed. This reaction requires no biosynthetic activity and no energy source. Particles produced in this way function as ribosomes in in vitro protein-synthesizing systems, incorporating phenylalanine under the direction of polyuridylic acid. This finding strongly suggests that “relaxed” particles are identical or very similar to normal ribosomal precursors.

2. Mutants Defective in Ribosome Assembly

Two mutants which accumulate ribonucleoprotein particles smaller than 705 ribosomes during normal growth conditions have been studied. The first of these is a derivative of E. coli 15reported by MacDonald et al. (1967). It grows slowly in ordinary media and has unusually large RNA/ protein or RNA/DNA ratios. Much of this excess RNA is found as a particle which sediments at approximately 435. The second mutant is derived from E. coli K12. During exponential growth, this organism also accumulated 435 particles (Lewandowskiand Brownstein, 1966). The mutant of MacDonald et al. (1967)is streptomycin-resistant.Aswe have noted i n the section on ribosomal protein (p. 108), streptomycin sensitivity seems to have a suggestivebut uncertain nexus with ribosomal structure. Since the streptomycin-resistance locus maps near the regions of the ribosomal RNA cistrons, the two may be related or even i d e n t i 4 It is possible that some structural differencesin the ribosome resulting in streptomycin resistance might result in formation of incomplete rib@ somes of this sort. 3. Particles Resulting from the Addition of Drugs that

Inhibit Protein Synthesis

Inhibition of protein synthesis by various drugs, such as chloramphenicol, puromycin and others, can result in the formation of protein-poor subribosomal particles which are similar to the “relaxed” particles in several ways. The most extensively studied are the particles formed in the presence of chloramphenicol,usually referred to as “chloramphenicol particles”. (a) Chloramphenicol Particles. Chloramphenicol is a potent inhibitor of protein synthesis in bacterial cells. When added to cells growing in

THE “LIFE CYULE” OF BACTERUL RIBOSOMES

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minimal media, it initially causes an acceleration of RNA synthesis (Gale and Folkes, 1953; Wisseman et aZ.,1954). This has been interpreted as being the result of increased availability of amino acids when protein synthesis is blocked (Kurland and Maalere, 1962). Hence, the effect of chloramphenicol is analogous to the RCR“ mutation allowing RNA accumulation to take place in the absence of protein synthesis. The accumulated RNA is in the form of ribonucleoprotein particles of sedimentation coefficient about 245 and 315 (Nomura and Watson, 1959), containing 75% RNA and 25% protein, and appearing as two species of 14s and 18s in some experiments (Dagleyand Sykes, 1959).As mentioned before, some of this protein may be spuriously attached (S. Osawa, personal communication). Seemingly, these particles are more fragile than ribosomes for they break down easily during sonication (Dagley and Sykes, 1960 ; Aronson and Spiegelman, 1961), ribonuclease treatment, and at low Mg2+concentrations (Nomura and Watson, 1959). The ultracentrifuge studies of Kurland et al. (1962) indicated that the particles were loosely coiled polyelectrolytes, sensitive to high salt concentrations, and sedimenting at 18s and 255 in low ionic-strength buffer. The smaller particle contained 16s RNA and the larger, 23s RNA. More recent studies have shown thqt this RNA is undermethylated (Gordon et al., 1964) like the RNA of RCR“particles, and apparently differs in minor nucleotide composition from that of mature ribosomes (Dubin and Gunalp, 1967).

During the extended period of chloramphenicol treatment necessary to produce large amounts of this material, some of the 705 ribosomes break down (Nomura and Watson, 1959). Approximately one-fourth of the RNA of chloramphenicol particles produced by this treatment is derived from degraded 70s ribosomes. The source of the protein in these particles is unclear, but it too may be derived from pre-existing particles, from pre-existing pools of nascent ribosomal protein, or from the adventitious attachment of non-ribosomal proteins. With these complicated factors in operation, it is difficult to determine if the aggregation of “chloramphenicol RNA” with pre-existing protein represents a normal mode of ribosome assembly. Figure 10 shows a tracing of Schlieren patterns obtained in ultracentrifuge studies of these particles. The short-term effect of chloramphenicol is bacteriostatic, and E . coli can survive in the presence of high concentrations of this drug. When the inhibitor is washed away, the cells recover permitting study of the fate of this so-called “chloramphenicol RNA”. It has been shown that at least some of the RNA synthesized in the presence of chloramphenicol is converted to mature ribosomes without degradation when the inhibitor is removed (Dagley et al. 1962b; Nomura and Hosokowa, $965). Lerman et aZ. (1967) showed that

W J L L U S. E E L L E Y AND MOSELIO SCHAECHTER 120 chloramphenicol particles can combine with ribosomal proteins to produce particles that are physically indistinguishable from 705 ribosomes. Thus, it appears that the particles produced during chloramphenicol inhibition might be some sort of precursor to mature ribosomes. However, this need not mean that they represent a natural precursor particle since they may be shunted off the normal biosynthetic pathway. (b) Pwomycin Particles. Puromycin is another inhibitor of protein synthesis. Its chemical structure resembles that of an aminoacyl t-RNA and

FIG.10. Ultracentrifuge pattern of an extract of chloramphenicol-inhibited

Escherichia coli prepared using buffer containing M Mg2+. 1 indicatesa soluble component ( 5 s ) ; 2,505 particles; 3,70S particles; 4 and 5, “chlorampheniool particles” (24Sand31s).ReprintedfromNomuraand Watson( 1959), bypermission of the authors.

it blocks completion of polypeptides by replacing -aminoacyl t-RNA, resulting in the formation of a peptide bond u h h puromycin as the carboxy terminal group (Traut and Munro, 1964). When bacteria are inhibited by puromycin, they accumulate ribonucleoprotein particles of smaller sedimentation coefficient than 305 ribosomes. Reported sedimentation coefficients for these particles vary from 15-185 and 18-258 (Nakada, 1965b)to 205 and 25s (Hosokawa and Nomura, 1965).At least a two-fold net increase in bacterial RNA content can be observed in the absence o f m y increasein protein synthesis (Dagleyetal., 1962a,b). When puromycin-inhibited cells are allowed to recover in the absence of the

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drug, the RNA of these particles is incorporated into mature ribosomes. During this recovery phase there seems to be a preferential synthesis of ribosomal protein (Sells, 1964; Hosokawa and Nomura, 1965; Nakada, 1965b). (c) Particlesformed in the. Presence of Streptomycin and Chlortetracycline. Similar accumulation of ribonucleoprotein particles resulting from inhibiting protein synthesis has been observed with streptomycin (Dubin, 1964; Dubin and Giinalp, 1967)and with chlortetracycline (Holmes and Wild, 1965a, b). These effects have not been studied as extensively as those mentioned above. 4. Particles Resulting from the Synthesis of R N A Containing Unnatural Bases Other metabolic inhibitors can cause accumulation of unnatural ribonucleoprotein particles by altering cellular RNA. Two of these inhibitors are 5-flumouracil and 8-azaguanine which are assimilated into the RNA as analoguesfor naturally occurring bases. I n the experiments of Aronson (1961), 5-fluorouracil was incorporated into particles which sedimented with the 305 and 505 ribosomal subunits and were incorporated into ribosomes when the analogue was removed. Although a report by Kono and Osawa (1964)implied that the RNA of such particles was incorporated into ribosomes unchanged, more recent reports have shown that it is unstable after the fluorouracilis removed (Andoh and Chargaff, 1965; Hills and Horowitz, 1966). Similarly the “relaxed” particles of an RCRez mutant produced in the presence of 5-fluorouracil are not incorporated into ribosomes after the inhibitor is removed although other “relaxed” particles formed in the absence of fluorouracil are (Nakada, 19658). The normal nucleotides of 5-fluorouracil-RNA can be re-utilized for synthesis of ribosomes afte5 an excess of uracil is added to the bacterial culture, whereas 5-fluorouracil is excluded (Andoh and Chargaff, 1965;Iwabuchi et al., 1965). A series of reports on the incorporation of 8-azaguanine are found in the literature, e.g. Dagley et al. (1962a, b), Otaka (1960),Otaka et ab. (1962), and Chantrenne.and Devreux (1960). Very little is known about the nature of the accumulated material except that it is unstable when the analogue is removed. 5 . Effects of Deprivation of Certain Ions on Ribosomal Assembly

I n some instances, upsetting the ionic balance of the growth medium of a bacterial culture can cause the accumulation of particles which resemble those described in the inhibition studies mentioned above. For

122 W I L W 9. KELLEY AND MOSELIO SCHAECHTER example, an E . coli mutant that requires high concentrations of K+, when starved of K+, accumulates unstable particles resembling the chloramphenicol particles (Ennis and Lubin, 1965s). If the K+ level is restored to that of a normal growth medium, these particles are rapidly .converted to ribosomes in much the same fashion as “relaxed” or chloramphenicol particles (Ennisand Lubin, 1965b). If bacteria are deprived of Mg2+they lose practically all their ribosomes and resynthesize them when the cation is restored (see Section IV, p. 110). Suzuki and Rayashi (1964) reported that, during recovery from Mg2+ starvation, bacteria accumulate new ribonucleoprotein particles similar to those formed in the presence of chloramphenicol. This effect is more manifest if an amino acid is withheld after the Mg2+is restored, thus preventing protein synthesis (Nakada and Marquisee, 1965). Phosphate starvation produces similar effects. After extensive deprivation, cellular RNA breaks down to soluble oligonucleotides (Maruyama and Mizuno, 1965, 1966). When phosphate is restored to the medium, synthesis of r-RNA takes place and large amounts of precursor particles accumulate which are gradually converted to mature ribosomes (Julien et al., 1967). This conversion seems to occur even more rapidly in the presence of chloramphenicol, showing that under these conditions ribosome synthesis apparently can take place from pre-existing proteins and nascent RNA.

./& &bP

6. Metabolic Shifts Designed to Promote Selective Synthesis of Ribosome Precursors As we described in Section-I11 (p. 106), bacterial cultures may be “shifted up” fro‘m a poor to a rich growth medium by nutritional supplementation. When this shift takes place, the rate of RNA synthesis exceeds that of synthesis of other macromolecules. Several reports indicate that, under these conditions, the principal RNA species formed is r-RNA, as shown by its nucleotide content (Mitsui et al., 1963; Aronson and Holowczyk, 1965). Kono et al. (1964) and Iwabuchi et al. (1965) used sucrose-gradient centrifugation to examine extracts of cells harvested soon after shift-up and found evidence for the formation of a series of ribonucleoprotein particles which they concludedwere precursors of mature ribosomes. The RNA made at various times after the shift-up was mostly 16s and 235 RNA and was active in stimulating amino-acid incorporation (Otaka et al., 1964).Growth in the presence of chloramphenicolor 5-fluorouracil added after the shift-up showed that particles aimilar to the chloramphenicol and fluorouracilparticles described above could be found (Kono

THE ‘‘LIFE UYULE” OF BACTERIAL RIBOSOMES

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FIU.11. Extinction at 260 mp (0-0) andradioactivity (0---0; counts/min.) of ribosome precursors synthesized in Escherichia ooli during a “shift-up”. Sedimentation analyses of crude extracts in pulse-labelling experiments are shown for 15 sec. (a)and 1 min. (b). Starved cells were transferred to 40 ml. of “shift-up” medium. [SH] Adenosine (100 pC) was added 6 min. after the transfer, and incubation continued for 15 sec. and 1 min. Extracts were treated with DNase ( 5 pg.) and centrifuged at 25,000 rev./min. for 9 hr. Reprinted from Kono and Osawa (1964), by permission of the authors.

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

and Osawa, 1964; Iwabuchi et al., 1965; Hayashi et al., 1966). These authors concluded that there were at least two stages in the assembly of each ribosome subunit. I n one case (Kono and Osawa, 1964) newlysynthesized ribonucleoprotein particles sedimented as a rather broad and heterogeneous band in sucrose gradients at S values between 1 5 s and 305 (Fig. 11). Osawa (1965) interpreted these data as suggestingthe existence of several ribonucleoprotein intermediates in the synthesis of 305 and 505 subunits. It seems difficult to determine the actual number of intermediates since there could be a broad continuum of incomplete particles differing from one another by a few proteins. Until analytical techniques which would separate such intermediates from each other more efficiently are developed, it will not be clear how many intermediate steps do exist. For further discussion of this work, the reader is referred to Osawa (1965). 7. A General View of Ribosome Assembly Based on "Relaxed" Syn-

thesis, Drug Inhibition, Recoveryfrom Ion Deprivation and Shift . Experiments The one underlying theme in all of the studies cited above (pp. 115-124) is (hat under certain conditions bacterial protein synthesis can be severely limited or-stopped entirely while RNA synthesis can continue. I n the apparent abeence of protein synthesis, ribonucleoprotein particles appear. I n our discussion of ribosomal protein production we considered the possibility that a pool of protein could exist within the cell and be combined with r-RNA synthesized at a later time, and we described experiments which support that idea. I n the section to follow, we will discuss some in witro experiments with ribosomes that also support the idea that ribosome assembly can take place when a pool of ribosomal protein and a partially completed ribosomal particle are allowed to combine.

C. I n vitro EXPERIMENTS ON RIBOSOME ASSEMBLY

A number of experiments reported recently have indicated that it is possible to remove certain proteins from ribosomes, and that these proteins can be replaced to generate active ribosomes. Several groups of workers have performed similar experiments based on the observation of Meselson et al. (1964) that partial degradation of ribosomes can be accomplished by centrifugation in 5 M caesium chloride solution (Fig. 12). By this treatment, it is possible to produce particles of 4 2 s and 235 from the 605 and 305 particles respectively (Staehelin and Meselson, 1966a; hosokawa et al., 1966). These particles have lost some of their proteins which can be recovered at the meniscus of the caesium chloride gradients. These particles are inactive in in vitro amino acid-incorporating systems.

THE “LIFE CYCLE” OF BACTERIBL RIBOSOMES I

‘ 5 x 10-3MI

125

I

Fraction number

FIU.12. Effect of Mg2+concentration on the distribution of ribosomesfrom Escherichia coli in caesium chloride density-gradients. The Mg2+ concentration and peak densitiesare indicated for each distribution. Reprinted from Meselson et al.(1964), by permission of the authors.

When the 425 and 23s particles are recombined with their respective “split protein” fractions, they reconstitute ribosomes which are active in vitro. The proteins separated by the caesium chloride treatment differ electrophoretically from those remaining in the ribosomal “core” (Traub et al., 1966b; Gesteland and Staehelin, 1967). When particles are reconstituted from 235 “cores” of ribosomes from bacteria resistant to streptomycin, and the “split proteins” of ribosomes from a sensitive strain, in,

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W I L L I A M 8. EELLEY AND MOSELIO SUHAEUHTER

vitro amino-acid incorporation using these particles is unaffected by streptomycin. If the cmverse reconstitution experiment is performed, the particles produced are sensitive to streptomycin, thus indicating that streptomycin sensitivity is a function of the “core” of the 305 subunit (Staehelin and Meselson, 1966b; Traub et aZ., 1966a).By similar studies, it has been shown that the “cores” themselves do not bind m-RNA (Raskas and Staehelin, 1967) and that distinct components from the “split proteins” from 305 and 505 particles are responsible for t-RNA binding, m-RNA binding and in vitro amino-acid incorporation (Nomura and Traub, 1967).Similar dissociationexperiments have been carried out by Lerman et al. (1966)which demonstrate step-wise dissociation and reaggregation under slightly different conditions. These experiments strongly suggest that ribosomes can be assembled by step-wise addition of specific proteins to an RNA core.

D. CONCLUSIONS I n conclusion, we might say that the assembly of ribosomal subunits is the most studied and yet the least understood of those processes we have discussed so far. The reaction apparently proceeds via a number of precursor particle stages, and can be arrested at intermediate degrees of completion. The precursors which accumulate seem to be the result of the complexing of an r-RNA chain with various ribosomal proteins. It seems likely that the time for completing a ribosome is sufficientlylong that, at any time, a substantial portion of the cellular RNA exists as a pool of precursor particles.

VI. The Formation of Functional Ribosomes I n the preceding section we discussed the reactions by which 305 and 505 ribosomal subunits are formed from r-RNA and ribosomal proteins. The purpose of this section is to consider the mechanisms by which these subunits combine to give functional ribosomes. Of all sections of our review this is the most speculative since, until recently, little work has been done on this important step. As we described in Section V (p. 113), in labelling experiments radioactivity accumulated in 305 and 50s particles before appearing in mature ribosomes (McCarthy et aZ., 1962; Nakada and Kaji, 1967). In these experiments, polysomes were broken by shear forces and the functionally mature ribosomes were found as 705 particles. These data indicate that free 305 and 50s particles are intermediates on the pathway of ribosome assembly.

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Particles identified as sedimenting in the 305 and 505 regions of sucrose gradients are actually a mixture of two kinds, “old” and “new”. “New” particles arise by ribosomal-neosynthesis while “old” ones result from the cyclic functioning of ribosomes in protein synthesia. The direct participation of the 30s and 505 subunits in the ribosomal cycle was demonstrated by Kaempfer et al. (1967). Their work was based on the previous observation of Meselson et ab. (1964) that ribosomal particles are preserved for at least three generations of cell growth. Kaempfer et al. (1967) showed that the 50s and 305 subunits rather than the 705 particles are conserved, and that the 705 particles dissociate into 505 and 305 components which re-aggregate at random during cell growth. This was demonstrated by growing bacteria in a medium containing heavy isotopes and transferring them to light medium. Zonal centrifugation in sucrose gradients and equilibrium centrifugation in caesium chloride revealed that the heavy ribosomal subunita formed before transfer, and the light ribosomal subunits formed after transfer, were distinctly s e p e rate entities whereas the 705 ribosomes found after the transfer were actually combinations of heavy and light subunits. All four possible combinations were found; heavy 50 and heavy 30, heavy 50 and light 30, light 50 and heavy 30, light 50 and light 30. So-called “chase” experiments add supporting evidence to this scheme. I n the chase experiment, uracil- or uridine-labelled bacteria are washed free of excess labelled base or nucleoside or are presented with a large excess of non-labelled molecules to dilute the soluble labelled precursor. During subsequent growth, RNA synthesized from this precursor is unlabelled or of low specific activity. If a particle containing RNA represents a rapidly synthesized and utilized intermediate in the biosynthesis of ribosomes, its specific radioactivity will decrease drastically during a chase. On the other hand, if a particle is a component of a cyclic metabolic pathway, during a chase its specific radioactivity will tend towards the same value as that of the other components of the cycle. This value will decrease for all components at the same rate as new material is synthesized. The specific radioactivity of the 305,505 and 705 particles decreased at approximately the same rate during a chase experiment. This has been shown for E . coli by Aronson et ab. (1960) and by G. Turnock (personal communication) and for B . megatehm by ourselves. There is no defkitive information about the properties of the two kinds of particles sedimenting in the 305 and 505 regions in sucrose gradients, the “old” and the “new”. They seem to be physically inseparable although the data of G. Turnock and R. E. MacDonald (personal communication) indicate that, i