MICROBIOLOGICAL REVIEWS, June 1978, p. 357-384 Vol. 42, No. 2 0146-0749/78/0042-0357$02.00/0 Copyright i 1978 American Society for Microbiology Printed in U.S.A. Genetics of VENETIA A. SAUNDERSt Department ofBiochemistry, University ofLiverpool, Liverpool L69 3BX, United Kingdom

TR ODU TION 357 PHYSIOLOGICAL ASPECTS 358

EVOLUTIONARY CONSIDERATIONS ...... 358 STUDIES WITH MUTANTS ...... 359 Survey of Mutants of the Rhodospillae 359 Resistant mutants 359 Auxotrophs 359 Pigment mutants 359 Electron transfer mutants 361 Temperature-sensitive mutants 362 Morphological mutants ...... 362 Miscellaneous mutants 362 Biosynthesis and Related Phenomena ...... 362 Towards Elucidation of Electron Transport Systems ...... 364 GENETIC ORGANIZATION 366 EXTRACHROMOSOMAL DEOXYRIBONUCLEIC ACID .367 Occurrence .367 Functions Evaluated 368 BACTERIOPHAGE AND BACTERIOCINS .369 Isolation and Characterization of Bacteriophage and Cyanophage .. 369 Bacteriocinogeny .370 "GENE TRANSFER AGENT"' OF RHODOPSEUDOMONAS CAPSULATA 371 Discovery and Properties 371 Mapping Genes for Bacteriochlorophyll and Biosynthesis ... 372 TRANSFORMATION 372 CONJUGATION 373 CONCLUDING REMARKS .374 LITERATURE CTED .375

INTRODUCTION laid on physiological processes of the photosyn- Photosynthetic have been effectively thetic bacteria which are amenable to investi- gation via the construction and subsequent anal- used for many years as model systems for inves- tigating and related metabolic ysis of specific mutants. Indeed, this kind of has contributed to the phenomena. Although this has led to a consid- approach substantially erable accumulation of biochemical and bio- current volume of literature regarding these physical data concerning the mechanics of bac- photosynthetic organisms. terial photosynthesis (for example, 26, 27, 58, 73, The photosynthetic procaryotes presently the algae), 101, 173, 174), there has, until recently, been a comprise cyanobacteria (blue-green conspicuous lack of complementary genetic in- the prochlorophyta, and the green and purple formation. A profound analysis of the genetics bacteria (179). (For the purposes of this review, of the photosynthetic'bacteria would certainly the photosynthetic bacteria refer to the green Four families are recog- enhance their present status as research tools. and .) This would, in turn, afford unique opportunities nized within the green and purple groups: the for exploring the development and function of Chlorobiaceae (green and brown sulfur bacte- energy-conserving systems. ria), the Chloroflexaceae (filamentous gliding green the Chromatiaceae sul- One of the primary aims in studying the mo- bacteria), (purple lecular biology of photosynthetic bacteria is to fur bacteria), and the Rhodospirillaceae (purple determine the nature, arrangement, and activity nonsulfur bacteria) (179, 180). This review of focuses on the latter family, reflecting of those genes specifying the photosynthetic ap- necessity limits of An attempt is paratus. Accordingly, this paper considers pro- the existing knowledge. made to of the gess made towards this goal and outlines poten- also integrate pertinent aspects cyanobacteria in line with reports of a close tial avenues of inquiry. Some emphasis will be affinity between these and other procaryotes t Present address: Department of Biology, Liverpool Po- (for example, 36, 43, 53, 59, 227, 229, 253). lytechnic, Liverpool L3 3AF, United Kingdom. For more extensive coverage of the genetics of 357 358 SAUNDERS MICR0O3IOL. RE:v. the cyanobacteria refer to recent reviews of were supposedly responsible for early biological Wolk (273) and Delaney et al. (43). oxygen production and hence for the dramatic evolutionary consequences stemming from this PHYSIOLOGICAL ASPECTS transition in the gaseous environment (18, 30, Photosynthetic bacteria are unique amongst 65, 168). Cyanobacteria have for some time been phototrophs in respect of the anaerobic nature cited as likely candidates for endosymbiotic of bacterial photosynthesis. No oxygen is precedents to photosynthetic plastids of certain evolved in the process, and oxidizable substrates eucaryotic cells (136, 146, 226, 237, 244). More other than water serve as electron donors (70, recently, it has been suggested that ancestors of 177, 224, 259). On the other hand, the cyanobac- some contemporary respiring bacteria may have teria typically exhibit oxygenic photosynthesis evolved from some purple nonsulfur photosyn- in a process comparable to that operative in thetic bacteria by atrophy of their photosyn- photosynthetic eucaryotes (65, 112). Most pho- thetic capacity. Likewise, certain of the gliding tosynthetic bacteria and certain cyanobacteria bacteria may have derived from cyanobacteria are capable of utilizing atmospheric as (49). their sole nitrogen source for photosynthetic Data concerning the structure and sequence growth (65, 69, 72, 103, 170, 232-234, 273). of electron transfer proteins which have been Typical representatives of the Rhodospirilla- amassed in recent years (for example, 5-8, 49, ceae (purple nonsulfur photosynthetic bacteria) 214, 240-242, 245, 255, 260, 275) may provide are facultative anaerobes possessing the adap- some clues to evolutionary connections between tive capacity to grow anaerobically in the light procaryotes and eucaryotes. Close structural and (photosynthetically) and aerobically in darkness sequence similarities are apparent for cyto- (by oxidative phosphorylation). Certain mem- chrome c2 from purple nonsulfur bacteria (8, 56, bers of this family are also capable of growing 198), cytochrome c50 from Paracoccus denitri- anaerobically in the dark (251, 277). Rhodopseu- ficans (247-249), and mitochondrial cytochrome domonas sphaeroides,, Rhodopseudomonas c (41, 47, 48). These findings, together with the palustris, and Rhodospirillum rubrum can fer- similarities in respiratory electron transfer prop- ment pyruvate under strictly anaerobic condi- erties of P. denitrificans, the purple nonsulfur tions in darkness (251). However, growth ofRho- bacterium R. sphaeroides, and the mitochon- dopseudomonas capsulata under such condi- drion (58, 99, 204), encouraged speculation about tions necessitates addition of dimethyl sulfoxide the evolution of bacterial energy metabolism. to the growth medium (277). By virtue of their Dickerson and colleagues (49) have suggested metabolic versatility, these photosynthetic bac- that "the point ofdivergence between photosyn- teria are particularly well suited to the study of thesis and respiration occurred in the ancestors processes involved in the formation and differ- of purple nonsulfur photosynthetic bacteria." entiation of energy-conserving membranes. However, phylogenetic relationships between Moreover, mutant strains can be readily isolated organisms may well have been blurred via the that are either photosynthetically or aerobically agency of genetic exchange (cf. reference 6); incompetent, but capable of growing in the al- thus, interpretations ofevolutionary occurrences ternative energy conversion mode. Such mu- based on such molecular methodology may tants have been effectively exploited in examin- prove to be oversimplifications of actual events. ing the electron transport systems of these or- Comparison of amino acid sequence similari- ganisms as discussed below (see Towards Elu- ties between f-type cytochromes from certain cidation of Electron Transfer Systems). The cyanobacteria and eucaryotic algae indicate a general physiologies of the photosynthetic bac- closer sequence correlation between the cyto- teria and the cyanobacteria have been detailed chrome f of the cyanobacteria and that of the elsewhere (for example, 23, 60, 71, 73, 106, 110, red algae than with that of any other algae (5, 170, 177, 224, 228, 258, 273). 7). Aitken (5) points out that, although genetic transfer may have occurred, obscuring the inter- EVOLUTIONARY CONSIDERATIONS relatedness ofsuch photosynthetic proteins, pro- There is considerable speculation about the tein sequence studies have produced much in- evolutionary significance of the photosynthetic formation in keeping with the hypothesis of a procaryotes (18, 21, 35, 49, 168, 185, 197, 225, common origin of oxygenic photosynthesis in 226, 244). On the one hand, ancestors of the procaryotes and eucaryotes. Indeed, patterns of photosynthetic bacteria are presumed to be homologies from ribosomal ribonucleic acid amongst the earliest of organisms utilizing ra- (RNA) sequence studies with cyanobacteria and diant energy in an anaerobic environment (168, chloroplasts (16, 17, 52, 175) lend credence to 197). On the other, ancestors of cyanobacteria this notion. VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 359 Recently, certain "procaryotic green algae" energy conservation in photosynthetic bacteria. have been observed and studied which possess Auxotrophs. Mutants requiring specific a unique combination of characteristics, some amino acids have been described (for example, typically procaryotic and others eucaryotic 13, 14, 78, 126, 217, 222, 276, 277). Adenine- (127). Significantly, these organisms contain requiring (unpublished data) and uracil-requir- both and chlorophyll b and per- ing (125) mutants of R. sphaeroides have been form oxygen-evolving photosynthesis. Whether prepared and used in the radiolabeling of deoxy- these algae or relatives are progenitors of green ribonucleic acid (DNA) and RNA, respectively. plant chloroplasts remains open to question. Certain glycerol-requiring strains of R. capsu- lata have recently been isolated and character- STUDIEES WITH MUTANTS ized (108). A series of mutants of R. capsulata Mutant strains of various microorganisms have been obtained that lack the capacity to fix have proven invaluable in the elucidation of a nitrogen (Nif-) (264) presumably because of the number of metabolic pathways. Of particular absence of activity or because of relevance here are those mutants of photosyn- defects associated with the synthesis or metab- thetic procaryotes that facilitate studies on elec- olism of or glutamate (262, 264). tron transfer processes, , pig- Pigment mutants. The Rhodospirillaceae ment biosynthesis, membrane development and synthesize colored belonging to the differentiation, and related biological phenom- "spirilloxanthin series" (98). In R. capsulata and ena. R. sphaeroides, spheroidene and hydroxyspher- Typical mutants of the Rhodospirillaceae are oidene predominate under anaerobic conditions obtained by ultraviolet irradiation or N-methyl- in the light. On the other hand, R. rubrum N'-nitro-N-nitrosoguanidine mutagenesis essen- synthesizes mainly spirilloxanthin. Various mu- tially by the method of Adelberg et al. (3), with tants with altered carotenoid complement are or without subsequent penicillin screening (124). known. Classical "blue-green" mutants, lacking Less frequently, mutants have been isolated colored carotenoids and accumulating phytoene, after spontaneous mutation. Mutations in the have been described for R. sphaeroides (for cyanobacteria are commonly induced with ni- example, 29, 80, 81, 216, 218, 219), R. capsulata trosamines. Fairly extensive surveys of the mu- (for example, 55, 267, 276), and R. rubrum (for tants of cyanobacteria have recently appeared example: 39; R. K. Clayton, cited in references (43, 254). 95 and 113). "Green" mutants, presumably This section is confined to a description of blocked at the neurosporene or chloroxanthin mutants of the Rhodospirillaceae. These mu- stages of carotenoid biosynthesis, have been ob- tants not only represent valuable research tools tained for R. sphaeroides (for example: 37, 38, in biochemical investigations but also provide a 80, 81, 145, 203; Saunders, Ph.D. thesis) and R. bank of genetically marked strains convenient capsulata (267, 276). Yen and Marrs (276) re- for gene transfer studies. cently described "yellow" mutants of R. capsu- of lata that apparently were phenotypically indis- Survey Mutants ofthe Rhodospirillaceae tinguishable from the so-called brown mutants Various classes of mutants of the Rhodospi- ofR. sphaeroides isolated by Griffiths and Stan- rillaceae have been reported, most commonly ier (81). Further "brown" mutants, phenotypi- for R. sphaeroides, R. capsulata, and R. rub- cally distinct from those of Griffiths and Stanier rum. (81), have also been characterized (210). Fur- Resistant mutants. A range of antibiotic- thermore, certain mutants of R. sphaeroides resistant mutants has been obtained, including have been isolated that combine the traits of strains resistant to rifampin, streptomycin, nali- carotenoid deficiency and a high catalase activ- dixic acid, and kanamycin (142, 153, 206, 217, ity (29). 222, 262, 276, 277; V. A. Saunders, Ph.D. thesis, There is a spectrum of mutants with blocks at University of Bristol, Bristol, U.K.). Mutant specific stages in bacteriochlorophyll biosyn- strains ofR. capsulata resistant to arsenate have thesis (Table 1). The propensity ofsuch mutants also been isolated (for example, 281). One such for accumulating various tetrapyrrole pigments, mutant, strain Z-1, exhibits enhanced rates of presumably bacteriochlorophyll precursors, has photophosphorylation. After prolonged culture contributed significantly to the elucidation of in the presence of arsenate, cells have elevated reactions involved in bacteriochlorophyll bio- contents ofcytochromes, reaction center bacter- synthesis as outlined in the following section. iochlorophyll, and photophosphorylation cou- The inability of "albino" mutants to form carot- pling factor (129, 281). This class of mutants enoid and bacteriochlorophyll is possibly a man- should prove useful for analyzimg mechanisms of ifestation of loss or dysfunction of a genetic 360 SAUNDERS MICROBIOL. RE:V. TABLE 1. Typical mutants ofphotosynthetic bacteria with lesions affecting bacteriochlorophyll biosynthesis Species Mutant strain Remarks Reference R. sphaeroides H-4, H-5 Lack 8-aminolevulinate synthase ac- 121 tivity, require aminolevulinate for growth; H-5 normalized by amino- levulinate 6-6 Excretes porphobilinogen, no mag- 120 nesium tetrapyrroles formed 2-33 (Met-) Excretes coproporphyrin, methio- 117 nine pathway blocked at homo- cysteine methylation level M-17 (Met-) Excretes coproporphyrin, methio- 126 nine pathway blocked at stage be- fore cysteine synthesis 2-731 Excretes magnesium divinylpheo- 116 V-3 f porphyrin a,,, Saunders, Ph.D. thesis 8-32 Excretes magnesium divinylpheo- 188 porphyrin a.,, bacteriochlorophyl- lide, and heme "Tan" Magnesium divinylpheoporphyrin 228 a, accumulated by cells "Griffiths mu- Magnesium divinylpheoporphyrin 79 tants" a, (and, probably, later interme- diates) accumulated by cells 2-21 Excretes 2-devinyl-2-hydroxyethyl- 116 chlorophyllide a 8-29 Excretes 2-devinyl-2-hydroxyethyl- 188 chlorophyllide a and some pheo- phorbide a 8-47, 8-53 Excrete 2-desacetyl-2-hydroxyeth- 188 ylbacteriochlorophyllide and 2- devinyl-2-hydroxyethylchloro- phyllide a 8-17 Excretes bacteriochlorophyllide 188 8-13 Accumulates heme, no magnesium 120 tetrapyrroles formed "Albino" mutants, neither bacte- L-57, 3-1 riochlorophyll nor precursors 120 V-2 formed, also fail to make carote- 204 noids "Griffiths mu- "Albino" strains, neither bacterio- 81 tants" chlorophyll nor carotenoids formed L-57 R, TA-R, Cells accumulate bacteriochloro- 123 DW-R phyll aerobically in darkness 8 Excretes 2-desacetyl-2-vinylbacte- 184 riopheophorbide VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 361 TABLE 1-Continued Species Mutant strain Remarks Reference O, Excretes pigments with absorbance 102 maximum below 660 nm R. rubrum F3, F4, F6 Excrete magnesium divinylpheopor- 165 phyrin a., monomethylester F5, F8, F9 Excrete 2-devinyl-2-hydroxyethyl- 163, 165 pheophorbide a, some pheophor- bide a F12 Excretes 2-devinyl-2-hydroxyethyl- 165 pheophorbide a, some pheophor- bide a and bacteriochlorophyll R. capsulata Ala (Pho-) Excretes phytylated (fully esteri- 55 fied) magnesium-2-vinylpheopor- phyrin a,, as protein complex Accumulates precursor with absorb- Y80 ance maximum at 630 to 635 nm {276 Y4911 (presumably magnesium 2,4-divi- 277 nylpheoporphyrin a,s) SB21, Y34, Y62, Accumulate precursor with absorb- 276 Y92, Y121, ance maximum 665-670 nm 277 Y122, Y165, Y167, Y451 Incapable of synthesizing bacter- {263 Y89} iochlorophyll and carotenoids 277 HH 910, HH 911 Accumulate precursor with absorb- 277 ance maximum at 730 nm R. palustris Green 1, 2, 3 Magnesium divinylpheoporphyrin 63, 111 Yellow I a5 and a phytylated form ex- {223, 252 tracted from cells

element governing synthesis of the entire pho- specific defects in the respiratory or photosyn- topigment system. Alternatively, synthesis of thetic electron transfer system have been de- the photosynthetic membrane components may scribed (for example, 44, 45, 115, 138, 139, 141, be dependent on the assembly process; thus, if 272). The biochemical lesions affecting some of any one of the structural components was ab- them will be considered below (see Towards sent, synthesis of the entire system would be Elucidation of Electron Transport Systems). switched off. A photosynthetically incompetent Certain strains of R. sphaeroides (216, 218, 239) strain of R. rubrum has been isolated which and R. capsulata (277) lack functional reaction produces a "pheophytin-protein-carbohydrate" center bacteriochlorophyll (P870), whereas they complex. Some nonfinctional bacteriochloro- synthesize the light-harvesting (bulk) bacter- phyll is also formed. Failure of the pigment iochlorophyll. Accordingly, such mutants do not complex to associate with the membrane may manifest those activities associated with the pri- reflect alteration or absence of requisite com- mary photochemistry of photosynthetic cells ponents for its incorporation (207). In addition, (211, 218, 220). Mutants of R. rubrum have also a mutant of R. sphaeroides has recently been been reported with properties characteristic of described which accumulates 4-vinyl protochlo- strains with defective reaction centers (44, 181). rophyllide, presumably because of defective syn- In addition, a strain of R. rubrum, F24.1, has thesis of membrane components required for recently been isolated with an altered reaction incorporation of bacteriochlorophyllide into the center (181). This mutant is a spontaneous pho- intracytoplasmic membrane system (183). totrophic revertant derived from a photosyn- Electron transfer mutants. Mutants with thetically incompetent strain with a nonfunc- 362 SAUNDERS MICROBIOL. REV. tional reaction center. Strain F24.1 apparently bacilliform (155) mutants of R. rubrum have lacks bacteriochlorophyll P800, a constituent of been described which apparently result from the reaction center (27, 174), but is, nevertheless, defects in D-alanine metabolism (156, 157). capable of photosynthetic growth. Picorel and The vibrio mutants were initially selected as co-workers (181) suggest that this mutant may strains resistant to D-cycloserine (an analog of be enriched in a second kind of reaction center D-alanine) (158). The phenotype of these mu- which does not contain P800 and which is pres- tants is evidently a consequence ofperturbations ent as a minor component in wild-type cells. in cell envelope biosynthesis. Such an explanation would reinforce previous Miscellaneous mutants. Certain mutants of proposals (236, 256) that two different kinds of R. rubrum have been selected on the basis of reaction center coexist in membranes of R. rub- pigmentation after prolonged growth anaerobi- rum. Alternatively, the reaction center of strain cally in the dark (250). One mutant, strain C, F24.1 may be modified so as to render P800 synthesized bacteriochlorophyll a, altered mem- unnecessary. Indeed, if this is the case, studies brane structures, and chromatophores during with such mutants should further resolve the dark growth. Furthermore, strain C was capable precise role of P800 in bacterial photosynthesis. ofgrowing anaerobically in the light. In contrast, Temperature-sensitive mutants. Temper- a second mutant, strain Gi, was light sensitive ature-sensitive lesions of the photosynthetic ap- and produced only trace amounts of bacterio- paratus of R. rubrum provide strains condition- chlorophyll. ally unable to perform photosynthetic functions Typically, wild-type strains of R. capsulata, associated with electron flow. Such mutants unlike strains ofR. sphaeroides and R.palustris, were selected by the phototactic enrichment are unable to utilize glycerol as a carbon source technique, assuming that the phototactic re- (257). However, a spontaneous variant of R. sponse of photosynthetic cells relies on a capsulata, strain Li, capable of using glycerol properly functioning electron transport system for both anaerobic photosynthetic growth and (266). Other temperature-sensitive mutants of aerobic dark growth, has been isolated (132). R. sphaeroides have been isolated (unpublished Two enzymes, glycerokinase and glycerophos- data) which are unable to grow aerobically at phate dehydrogenase, not detectable in the par- the nonpermissive temperature. The exact na- ent, were found to be synthesized constitutively ture of the lesions affecting these strains is un- in this mutant (131, 132). By contrast, such known. enzymes are inducible, in the presence of glyc- The use of temperature-sensitive mutants, erol, in R. sphaeroides (182). Constitutive syn- particularly in essential functions, has paid un- thesis of these enzymes in the mutant possibly doubted dividends in studies of other biological reflects derepression in strain Li of an operon systems. It is perhaps surprising, therefore, that not normally expressed in the wild type. there has been a dearth of reports of investiga- Mutants of R. palustris have been reported tions involving similar mutations in the photo- that differ from wild-type strains in that they synthetic bacteria. In particular, studies with are incapable of growing aerobically at the ex- temperature-sensitive mutants of the Rhodo- pense ofcyclohexanecarboxylic acid or pimelate. spirillaceae should facilitate identification of Such mutants have been exploited in determin- electron transfer components which may be ing reactions involved in the photometabolism common to both the respiratory and the photo- of benzoate by R. palustris (83). synthetic electron transfer systems. The notion ofshared electron transport components in these Bacteriochlorophyll Biosynthesis and organisms is supported by several workers (see Related Phenomena Towards Elucidation ofElectron Transport Sys- Elucidation of reactions involved in bacter- tems). Mutations affecting such components iochlorophyll biosynthesis has depended largely might reasonably be expected to be lethal during upon the analysis of tetrapyrrole pigments ac- dark aerobic growth or photosynthetic growth. cumulated by strains of purple nonsulfur bacte- (When cells are grown anaerobically in darkness ria in which bacteriochlorophyll biosynthesis is the components may be dispensable [277], and deranged by mutation or metabolic inhibitors. hence the mutations would not prove lethal.) Figure 1 outlines reactions involved in bacter- Thus, the isolation of appropriate temperature- iochlorophyll biosynthesis and incorporates the sensitive mutants should enable inroads to be mutational blocks for a series of mutants of R. made towards defining interrelationships ofpho- sphaeroides. It is noteworthy that the precursor tosynthesis and respiration in the purple non- accumulated by R. sphaeroides strain 8 (see sulfur bacteria. Table 1) does not fit into the scheme per se and Morphological mutants. Vibrio (158) and may be indicative of an alternative pathway to VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 363 Glycine + Succinyl CoA H-5; H-4 6-aminolevulinic acid (bALA) Porphobilinogen UropoorrrinZgen

Coprophorpyrinogen 2-33; M-17 PROTOPORPHYRIN + Mg I + Fe Hagnesium protoporphyrin

Magnesium protoporphyrin monomethyl ester

Magnesium divinylpheoporphiyrin a5 monomethyl ester +2H l_ 273; 8-32

Magnesium vinyl pheoporphyrin a

Chloroph llide a +H2

2-devinyl 2a-hydroxyethyl chlorophyllide a 2-21; 8-29

2-desacetyl 2-a-hydroxyethyl bacteriochlorophyllide -2H 8-47 Bac teriochlorop hyllide Phytol 8-17

BACTERIOCHWROPHYLL FIG. 1. Scheme for heme and bacteriochlorophyll biosynthesis in R. sphaeroides (modified from Lascelles [119]). The mutational blocks in a number of strains of R. sphaeroides are indicated. bacteriochlorophyll in this organism (see refer- (123). Presumably, this response is attributable ence 184). Mechanisms regulating bacteriochlo- to a derepression of synthesis of certain enzymes rophyll biosynthesis have been proposed largely of the magnesium path under conditions nor- on the basis of the behavior of appropriate mu- mally causing their repression. It has been spec- tants under various environmental conditions. A ulated that synthesis of all the enzymes of the brief review has recently appeared (119). 8-Ami- magnesium branch of the pathway may be con- nolevulinate synthase represents one control lo- trolled by a regulatory gene which is in turn cus, presumably regulated, at least in part, by influenced directly or indirectly by oxygen (123). the intracellular heme concentration (122). Mag- Cohen-Bazire et al. (37) advanced a hypothe- nesium chelatase, which appears to be critically sis, referred to as the "redox-governer" hypoth- sensitive to oxygen, is of particular importance esis, to account for the almost immediate cessa- in regulating the magnesium branch of the path- tion of bacteriochlorophyll biosynthesis in re- way (121). Certain of the biosynthetic enzymes sponse to oxygen. It was suggested that the rate appear to be subject to repression in the pres- of bacteriochlorophyll and carotenoid biosyn- ence of oxygen. Mutants of R. sphaeroides have thesis was governed by the state of oxidation of been isolated that continue to synthesize bacter- a carrier in the electron transport system. This iochlorophyll under conditions of high aeration notion has subsequently been superseded in 364 SAUNDERS MICROBIOI.. REV. light of observations with specific mutants of R. In support of this are observations (28, 164, 165, capsulata impaired in respiratory electron 238, 239) which indicate an obligatory coupling transport (139). It is envisaged that an oxygen- between bacteriochlorophyll biosynthesis and sensitive factor regulates bacteriochlorophyll the formation of specific chromatophore pro- synthesis. This factor, inactivated by oxygen, teins. The role of the bacteriochlorophyll mole- can be reactivated by a flow of electrons from cule in this process remains obscure. Possibly, the electron transport system, diverted possibly the pigment exerts its effect at the level of tran- at the level of cytochrome c. The factor may scription or translation. Alternatively, the bac- represent one or more of the enzymes directly terichlorophyll molecule may be necessary for involved in bacteriochlorophyll synthesis or an assembly of these specific proteins into the ar- effector molecule that interacts with them (139) chitecture of the membrane (119, 239). (see Fig. 2). The involvement of cytochrome c Analysis of glycerol auxotrophs of R. capsu- could be tested for by investigating regulation of lata indicated a dependence of bacteriochloro- bacteriochlorophyll synthesis in mutants phyll and carotenoid biosynthesis on phospho- blocked in electron transport between cyto- lipid synthesis (108). An increased lipid content chromes b and c. is associated with pigmented cells. It is not An inverse correlation appears to exist be- known whether lipid synthesis exerts direct reg- tween the intracellular concentration of adeno- ulatory control on carotenoid synthesis or sine 5'-triphosphate and the rate of bacterio- whether a slow-down in carotenoid synthesis is chlorophyll biosynthesis in certain photosyn- a secondary effect of decreased bacteriochloro- thetic bacteria (62, 209). Furthermore, it has phyll production. been proposed (62) that the amount ofadenosine Clearly, more investigations at the genetic 5'-triphosphate within cells of R. sphaeroides is level are required to clarify the regulatory mech- in itself decisive in modulating bacteriochloro- anisms involved in photopigment biosynthesis. phyll synthesis. Ultrastructure studies with mutants blocked Towards Elucidation of Electron at specific stages in bacteriochlorophyll biosyn- Transport Systems thesis reveal that synthesis of the entire bacter- The intracytoplasmic membrane of purple' iochlorophyll molecule is a prerequisite for as- nonsulfur bacteria accommodates both the res- sembly of the intracytoplasmic membrane sys- piratory and the photosynthetic electron trans- tem characteristic of pigmented cells (19, 165). port systems. Resolution of the precise nature and arrangement of components of either of light hl BCi these systems is thus complicated by their dual IightBfh < bchl 02 bchl occurrence in the membrane. A promising ap- e nactive Fictive proach to the analysis of these systems involves the use of mutants with lesions specifically af- fecting photosynthetic or respiratory compe- SuccinMte dehyd."2 + yc : cytb tence. In addition, mutants with altered carote-

FIG. 4. Electron micrograph of extrachromosomal DNA from R. sphaeroides (from Saunders et al. [206]). Circular DNA of28 x 10' daltons isolated from strain 2.4.1 is shown. Bar = I ,Lm. established whether plasmids are ubiquitous (270). These observations led to the idea that a within the photosynthetic procaryotes. translational mode of regulation of protein syn- thesis may exist for these bacteria (24, 270, 271, Functions Evaluated 274). The nature of the information encoded by Possible genetic functions for the plasmid plasmid DNA in photosynthetic procaryotes re- DNA of R. sphaeroides have been evaluated by mains largely a matter for conjecture. By anal- Saunders et al. (206). Part of the plasmid DNA ogy with the plastid DNA of eucaryotic plant complement may be composed of temperate cells, it has been suggested that the plasmid phage. Indeed, several temperate bacteriophages DNA may have a role in specifying the photo- have been isolated from strains of R. sphae- synthetic apparatus (75). However, naturally oc- roides (153), though no infective phage particles curring plasmids are generally dispensable in have been detected in cultures of strain 2.4.1. procaryotes (160, 162). Thus, essential biological Recently, putative viral R plasmids have been functions, presumably including photosynthetic reported for a number ofR. sphaeroides isolates activity in this case, are unlikely to be plasmid (176). These strains were resistant to penicillin, determined. Furthermore, no transcriptional by virtue of a diffusible penicillinase, and carried specificity between the RNA from aerobically or between one and three prophages. Bacterio- photosynthetically grown cells has been dem- phages released by such strains were active onstrated in R. sphaeroides and R. rubrum by against derivatives of these strains which had using both chromosomal and extrachromosomal been "cured" of prophage. Furthermore, resist- DNA as DNA-RNA hybridization probes (24, ance to penicillin could be transferred at high 77, 271, 274). Irrespective of the gross structural frequency to susceptible recipients. However, differences and the variation in enzyme patterns generalized transduction mediated by these (118) associated with growth of these organisms phages could not be demonstrated. The penicil- in different atmospheric milieus, no qualitative linase-producing strain R. sphaeroides RS601 differences are obvious between the RNA spe- released the bacteriophage designated R06P cies of aerobic and photosynthetic cells (24, 64, spontaneously. The DNA extracted from bac- 271, 274). However, there does appear to be teriophage R46P was circular DNA of 33 (+2) some difference in the stability of an RNA com- x 106 daltons (176), a value close to that of the ponent under aerobic and anaerobic conditions smallest plasmid of R. sphaeroides strain 2.4.1 VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 369 (cf. reference 206). It has been suggested (176) tor to the prevalence of antibiotic resistance that a gene for penicillinase production is carried amongst bacteria (33, 200). It is likely that some as part of the wild-type bacteriophage genome disparity in genetic composition will exist be- and that this genome exists as an extrachromo- tween common laboratory strains isolated some somal element in these lysogenic strains. The 20 or more years ago and those recently ob- carriage of transposable resistance genes by bac- tained. Increased levels of pollutants, including teriophages has been demonstrated in the labo- antibiotics and heavy-metal ions, have undoubt- ratory in the Enterobacteriaceae (15, 76). How- edly had dramatic effects on the genetic consti- ever, this appears to be the first report of a tution of bacterial populations in general (see, bacteriophage isolated from the wild which ap- for example, references 130, 186, 189). Thus, parently carries antibiotic resistance genes. photosynthetic procaryotes will, in all certainty, An isolate of R. capsulata, strain SP108, sim- acquire resistance genes to protect themselves ilarly produces a diffusible penicillinase which against such pollutants. In turn, this should con- confers considerable resistance to penicillin on veniently provide naturally marked derivatives this strain (267). This contrasts with the extreme of these organisms for use in genetic experi- susceptibility of classical laboratory strains ofR. ments. capsulata to this antibiotic. Indeed, the great susceptibility of many strains of R. capsulata to BACTERIOPHAGE AND BACTERIOCINS penicillin G is a feature which distinguishes this species from closely related members ofthe Rho- Isolation and Characterization of dospirillaceae (267). The penicillin-inactivating Bacteriophage and Cyanophage enzyme of strain SP108 is apparently an induc- The value of transduction in the provision of ible f?-lactamase, with a marked preference for fine-structure genetic maps and the construction benzylpenicillin as a substrate (V. A. Saunders, of specific mutant strains of bacteria is indisput- manuscript in preparation). Furthermore, resist- able (see, for example, references 12, 94, 199, 231, ance to penicillin in strain SP108 is lost at a 243, 246). The quest for corresponding phage- relatively high frequency (267; unpublished mediated gene transfer systems within the pho- data), which would be consistent with a plasmid- tosynthetic procaryotes has promoted studies on specified character (160). the virology of these organisms. It is perhaps noteworthy at this juncture that The first report of a virulent phage specific for photosynthetic bacteria are sometimes isolated a member of the Rhodospirillaceae described from stagnant ponds which have been contami- phage Rpl of R. palustris (66). Both virulent nated with farm effluents (see, for example, 1, and temperate phages specific for R. sphae- 142, 153, 267). This may provide localized con- roides (1, 153) and R. capsulata (208, 263) have centrations of antibiotics derived from, for ex- subsequently been isolated and characterized. ample, animal feed and a reservoir of bacteria Their properties have recently been documented capable of acting as donors of antibiotic resist- in detail (143). In addition, temperature-sensi- ance genes. tive mutants of the phage RC1 of R. capsulata Penicillinase (f8-lactamase) production has have been obtained (261). Thus far, however, no also recently been reported for certain cyano- bona fide transduction has been reported involv- bacteria, in particular Anabaena sp. (strain ing any of them. More promising are the results 7120) and Coccochloris elabens (strain 7003) with certain phages of R. sphaeroides recently (114). Penicillin did not appear to induce peni- isolated by Kaplan and colleagues (S. Kaplan, cillinase production in these organisms. Further- private communication). Phage-mediated trans- more, the enzymes from these strains were more fers of antibiotic resistance and nutritional active on penicillins than on cephalosporins, markers have been achieved between strains of thereby resembling the "type II' enzymes of R. sphaeroides, albeit at fairly low transfer fre- gram-negative bacteria (190). quencies. Although these results are prelimi- The production of penicillinases by photosyn- nary, it is conceivable that with further refine- thetic procaryotes poses fundamental questions ments such a transducing system will prove suit- as to the nature and origin of the penicillinase able for exploring the genome ofR. sphaeroides. gene(s). For instance, do these enzymes corre- First reports of viruses attacking and lysing spond to those widely distributed amongst the species of the cyanobacteria were those of Saf- enteric bacteria, Pseudomonas and Haemophi- ferman and Morris (196). Several viruses (cyano- lus (87, 202)? Of particular interest in this re- phages) have since been characterized, and there spect is the type IIIa (TEM) ,8-lactamase deter- is considerable documentation of their morphol- mined by transposon A (86). Transposon A is ogy, physiology, and ecology (for example, 172, capable of translocation from one replicon to 195, 212, 273). However, their role as mediators another and thus may be a significant contribu- of genetic exchange remains unproven. Temper- 370 SAUNDERS MICROBIOL. REV. ature-sensitive mutants of the cyanophage and/or the intracytoplasmic membrane system LPP2-SP1, which lysogenizes Plectonema bor- such that the adsorption and/or penetration yanum, have been isolated, and a linkage map process is hampered in anaerobically grown cells of the phage has been constructed based on (1). Once effective penetration of such cells has recombination between mutants in two-point occurred, the burst size is similar to that ob- crosses (191). served during aerobic infection (15 to 20 plaque- In addition to genetic considerations, host- forming units per cell). bacteriophage interactions necessarily relate to Bacteriophage R,0-1 is a temperate phage spe- host cell physiology and, in turn, provide infor- cific for R. sphaeroides (153). It was isolated mation on fundamental aspects ofbacteriophage from the prophage state by induction with mi- replication and assembly (2, 34, 231). In this tomycin C. R. sphaeroides strain 2.4.1 is not regard, photosynthetic procaryotes undoubtedly susceptible to infection by R4-1. However, mu- offer distinct advantages over other procaryotes, tant derivatives of the phage have been isolatedi primarily because the energy status of the host which can form plaques on strain 2.4.1. Interest- cell can be conveniently manipulated merely by ingly, the original phage RO-1 is chloroform sus- adjusting such parameters as light intensity. ceptible, whereas the mutant is chloroform re- Schmidt et al. (208), investigating the bioener- sistant. The growth pattern of the phage is sim- getics of bacteriophage RC1 replication in R. ilar whether it begins its life cycle by induction capsulata, concluded that the energy require- or infection. Furthermore, R4-1 forms plaques ment for the replication of this phage is more with equal efficiency on susceptible host cells critical than that for uninfected host cell growth. whether grown aerobically in darkness or anaer- In photosynthetically grown host cells, phage obically in the light. RC1 replication could be supported either by The requirements for optimal replication of photophosphorylation or oxidative phosphoryl- phage particles appear to vary. For example, in ation. However, in aerobically grown host cells, both R. capsulata (208) and the cyanobacterium phage multiplication was supported by oxidative Nostoc muscorum W4), optimal phage replication phosphorylation; the anaerobic photophosphor- necessitates illumination throughout the latent ylation capacity of such cells would not suffice. period, whereas reproduction of cyanophage Clearly, in aerobically grown cells development LPPI-G in P. boryanum requires illumination of the photosynthetic pigment system is drasti- solely through the eclipse period (171). These cally suppressed (118). Therefore, such cells are different requirements may reflect intrinsic dif- severely limited in photosynthetic energy con- ferences in metabolism of the particular host version capacity. The required photophosphor- species. ylation capacity for phage development can only be achieved in aerobically grown cells which Bacteriocinogeny contain a sufficient quantity of bacteriochloro- Recent surveys (82, 263) have revealed that phyll (0.6 ug/mg of dry weight) before phage representatives of the Rhodospirillaceae pro- infection. Once phage has infected aerobic cells, duce bacteriocins. R. sphaeroides and R. pal- subsequent synthesis of the photophosphoryla- ustris exhibit few intraspecies-specific inhibitory tion system is prevented when cells are incu- interactions. Greatest inhibitory activity, both bated under anaerobic conditions in the light. interspecies and intraspecies specific, was ex- Phage RC1 infection apparently interferes with hibited by strains of R. capsulata. In addition, synthesis of both bacteriochlorophyll and pro- it is noteworthy that purple nonsulfur bacteria tein in R. capsulata. It is proposed (208) that produce antimicrobial substances which are not the infecting phage is entirely dependent on the akin to bacteriocins but are metabolites extract- temporal energy conversion activity of the host able with organic solvents (104). Such antibiotic and that a relatively high rate of adenosine 5'- effects produced by R. sphaeroides (strain 1c7) triphosphate regeneration is required for proper are restricted to gram-positive bacteria, for ex- expression of the viral genome. ample, Bacillus subtilis, whereas those pro- The characteristics of infection of R. sphae- duced by R. capsulata (strain FC101) appear to roides by phage RS1 indicate that some form of be unspecific. The precise natures of these anti- physiological specificity exists (1). Anaerobically biotic substances require elucidation. No phe- grown cells of R. sphaeroides strain 2.4.1 are nomenon analogous to that of bacteriocinogeny apparently less susceptible to such infection has been reported in the cyanobacteria. than are aerobically grown cells (the adsorption Bacteriocins are in themselves ofinterest from rate constants of RS1 are 1.2 x 10-9 ml/min to structural, functional, and evolutionary stand- aerobic cells and 0.58 x 10-9 ml/min to anaerobic points (85). Furthermore, their production is cells). This could reflect differences between often determined by transmissible plasmids (85). these two cell types in cell surface properties Therefore, the possibility exists that if compa- VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 371 rable plasmids reside in members of the Rho- transferred genetic markers appear to be stably dospirillaceae, they could be exploited as vehi- inherited. Recombination is apparently accom- cles of genetic exchange. However, the location panied by displacement of the corresponding ofthe genetic determinants for bacteriocinogeny resident marker (143). Analyses of the kinetics in these organisms remains obscure, and no as- of renaturation (Cot analysis) of the DNA con- sociated gene transfer has so far been reported. tained in the GTA (Hu and Marrs, manuscript in preparation) indicate that more than 95% of "GENE TRANSFER AGENT" OF this DNA is from the bacterial genome. Se- RHODOPSEUDOMONAS CAPSULATA quences complementary to the chromosomal and plasmid DNA of R. capsulata are found in Discovery and Properties the GTA, and it appears that all portions of the The first report of a genetic exchange system genome of R. capsulata are equally represented for a photosynthetic bacterium was that of in the DNA of a population of GTA particles. Marrs (142) for R. capsulata. Various isolates of The precise nature ofthis gene transfer system R. capsulata were screened for recombination remains enigmatic. Marrs and co-workers spec- of antibiotic resistance markers. Genetic ex- ulate that it may represent a "prephage" system change appeared to be mediated by a ribonucle- (143, 222), in which case it is envisaged that a ase- and deoxyribonuclease-resistant vector pro- GTA-like system evolved in response to the duced specifically by strains ofR. capsulata and selective advantage which the capacity for ge- thereafter designated the "gene transfer agent" netic exchange might confer. Thus, the GTA (GTA) (142). Different isolates vary in their system could represent a precursor of the bac- ability to donate and receive the GTA (263). terial virus, rather than a derivative of a preex- Furthermore, strains receiving genetic informa- isting phage. Alternatively, the GTA may be a tion via the GTA do not themselves become defective or cryptic phage capable of generalized GTA producers (143). Genetic transfer mediated transduction (222). This permits an equally by the GTA is limited to R. capsulata and does plausible explanation to be inferred from the not extend to other members of the Rhodospi- observations that the GTA has a limited ability rillaceae (263). Moreover, no comparable ge- to transfer 3 x 106 to 4 x 106 daltons of DNA netic exchange system has, to date, been discov- (221) (presumably equivalent to approximately ered for other purple nonsulfur bacteria (263; five to seven genes, if transcription of the genes unpublished data). Morphologically, the GTA is nonoverlapping). Phage particles of similar particle resembles a small bacterial virus, with complexity to, albeit oflarger size than, the GTA an icosahedral head, short spikes, and a tail (for example, those of the T-even group) contain (143). The nucleic acid of the GTA is linear a genome of about 108 daltons (265). If, as seems double-stranded DNA of 3.6 x 106 daltons (143, likely, the genome size of the GTA itself were to 221), and the ultraviolet inactivation spectrum exceed five to seven gene equivalents, then the of the GTA is similar to that of bacterial viruses head of the GTA would be unable to accommo- (222). However, the physical size of the GTA date all the genetic information necessary to particle (70S [142]) is much smaller than that of specify a mature GTA particle. Hence, GTA any known transducing bacteriophage. Further- particles would, at best, carry only fragments of more, no plaque-forming activity appears to be their own genetic complement. Accordingly, associated with the system, and there is no ob- such particles would be nonlytic and lack overt vious correlation between the capacity of strains viral activity. Furthermore, recipients of genetic of R. capsulata to produce GTAs and their material via GTAs would never become GTA susceptibility to known bacteriophages (263). producers unless, by chance, through multiple Kinetics ofGTA release by a donor culture differ infections they simultaneously acquired all the from those normally associated with phage pro- genes necessary to specify GTA production. duction (cf. references 97, 144). GTAs are typi- Even if the GTA particle could carry its genome cally released in one or two abrupt waves to- in its entirety, failure to transmit GTA produc- wards the end ofthe exponential phase ofgrowth tion to recipient cells could be explained if the (222). Whether cell lysis always accompanies genetic determinants for the GTA were scat- this process has yet to be fully ascertained. tered at various loci on the replicons resident in The gene transfer process in R. capsulata R. capsulata. Consequently, chances of incor- seemingly resembles generalized transduction. poration ofa complete GTA genome into a single All regions of the bacterial genome thus far particle during the encapsidation process would examined can be transferred, and transfer fre- be drastically reduced. Possibly, this gene trans- quencies comparable to those for generalized fer system could be dissected by isolating from transducing systems (4 x 1O-4 "transferants" per GTA-producing parent strains mutants im- recipient) can be achieved (222). Moreover, the paired in GTA production. Such "GTA defec- 372 SAUNDERS MICROBioi,. REV. tive" strains could be used as recipients in ge- and ratio test crosses were performed between netic crosses with other GTA producers as do- various strains, and a new mapping function was nors. Restoration of the capacity to produce the derived to convert cotransfer data into map dis- GTA in transferants may allow the extent and tances. Lacking cis-trans complementation data map positions of the GTA determinants to be for this organism, it was assumed that a cluster established. Certain mutants of R. capsulata of mutations giving the same phenotype repre- have recently been isolated that are "overpro- sented a gene. Accordingly, clusters ofmutations ducers" of the GTA (143). These strains may delineating seven genes, five affecting carotenoid further resolve the nature of this genetic ex- biosynthesis and two affecting bacteriochloro- change system and, in particular, enable an es- phyll biosynthesis, have been arranged in one timate of how many genes are involved in spec- linkage group (Fig. 5). Mutations in either the ifying the GTA and their location in R. capsu- crtB or crtE gene can give rise to the blue-green lata. However, there still remains the problem phenotype, whereas mutations in the crtD or of screening for a characteristic for which there crtC gene cause the green phenotype and those is no direct selection procedure and for which a in the crtA gene cause a yellow phenotype. The biological assay is the sole means of detection. loci bchA and bchB specify products necessary The GTA system of R. capsulata has been for reactions in bacteriochlorophyll biosynthesis. used in manipulating genes specifying the pho- Linkage between these genes has possible impli- topigment system (54, 143, 263, 276) and the cations for their coordinate expression at the nitrogen fixation machinery (264). Moreover, transcriptional level. An interesting observation specific mutant strains have been constructed, from the mapping studies was the apparently and the lesions characterizing others have been obligatory requirement for two specific muta- investigated with this genetic vehicle. tions to obtain viable blue-green strains of R. Transfer of genes for nitrogen fixation, via the capsulata: one lesion results in loss of colored GTA, to certain Nif- mutants of R. capsulata carotenoids; the other (as yet of undefined map results in acquisition by recipients of the dual location) results in an alteration of the absorp- ability to fix nitrogen and produce hydrogen tion spectrum of bacteriochlorophyll (54, 137, (264). These findings support the proposal (170) 143, 276). It has been suggested (137) that the that nitrogenase and hydrogen-evolving hydro- mutation(s) responsible for alteration in the ab- genase activities of purple nonsulfur bacteria are sorption spectrum of bacteriochlorophyll in fact catalyzed by the same enzyme complex. Fur- blocks formation of light-harvesting bacterio- thermore, this hydrogenase activity apparently chlorophyll complex II (128). If these mutations differs from that associated with the utilization perforce accompany each other in blue-green of hydrogen as an electron donor for photoau- strains, this could help to clarify the role(s) of totrophic growth (264). carotenoids in photosynthetic bacteria. Restoration of photosynthetic competence to Pho- mutants of R. capsulata has been effected TRANSFORMATION with the GTA (54, 263). Drews and co-workers So far, there has been no unequivocal dem- (54) demonstrated concomitant restoration of onstration of genetic transformation in the pho- the abilities to synthesize bacteriochlorophyll tosynthetic bacteria. By contrast, there are sev- and to form reaction center and light-harvesting eral reports of gene transfer by transformation proteins to R. capsulata mutants defective in in the cyanobacteria (see reference 43). Trans- these abilities. Analyses of various transferants formation in A. nidulans was first reported by suggest that reaction center proteins and light- Shestakov and Khyen (213). The process was harvesting complex 1 (128) form a structural unit mediated by chemically extracted DNA and was in the intracytoplasmic membranes of R. cap- deoxyribonuclease sensitive. Subsequently, sulata (54). It has yet to be ascertained whether Herdman and Carr (91) described a transfor- the genes specifying synthesis of these specific mation system for A. nidulans effected by an proteins of the photosynthetic apparatus and of extracellular DNA:RNA complex. More exten- bacteriochlorophyll are closely aligned on the sive genetic linkage was observed in this process genome of R. capsulata. than in that mediated by chemically pure DNA. However, a mutagenic phenomenon appeared to Mapping Genes for Bacteriochlorophyll be associated with the transformation process and Carotenoid Biosynthesis (whether with extracellular or chemically ex- The GTA has been successfully exploited in tracted donor DNA) (88, 89) which presumably conjunction with a series of mutants of R. cap- interfered with accurate determination of link- sulata in the construction of a map for genes age values in genetic mapping studies with A. determining bacteriochlorophyll and carotenoid nidulans. Nevertheless, it has been possible to production (276). One-, two-, and three-point exploit the mutagenic process per se in aligning VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 373

62A71 e#/B16 ed"C112 cr/D150 eiE9 A4AA92 FIG. 5. Genetic map of R. capsulata, indicating loci concerned with carotenoid and bacteriochlorophyll biosynthesis (from Yen and Marrs [276]). The numbers above the map represent the distances, in map units, between specific markers in each gene. The numbers below the map are estimates of the minimum length of each gene. Distances obtained by subtraction are given in parentheses. certain genetic markers, and recombination recC exonuclease V) (166), which degraded the maps of A. nidulans have been constructed (43, incoming linear "transforming" DNA. In con- 88, 89). trast, linear DNA entering the cell by conjuga- In addition to the intraspecies-specific trans- tion or transduction in E. coli is apparently formation systems for A. nidulans (88, 89, 92, refractory to such attack. It was only with sub- 148, 169, 213) and Aphanocapsa 6714 (11), an sequent mutational blocks in the recB/recC ex- intergeneric transformation system has recently onuclease, coupled with a suppressing mutation been reported for cyanobacteria (46). Interge- opening up a further minor recombination path- neric transfer of antibiotic resistance markers way, that effective transformation with chro- has been demonstrated from A. nidulans to mosomal DNA was achieved in E. coli (166). Gloeocapsa alpicola and vice versa, in a process Thus, the portal used in transformation may sensitive to both deoxyribonuclease and ribo- result in the genetic information being particu- nuclease. Such genetic transfer across generic larly vulnerable to nuclease attack. By analogy, boundaries offers considerable scope for mobiliz- whereas a recombination system is operative in ing genes (for example, those specifying nitrogen certain members of the Rhodospirillaceae, it fixation) within the cyanobacteria. could equally act as a specific barrier to trans- Investigators' inability to develop a genetic formation in these organisms. Only when a transformation system for photosynthetic bac- greater understanding of the general genetics teria may be due to one or a number of contrib- and, particularly, of recombination is available utory factors. First, the possibility exists that in the Rhodospirillaceae can such problems be the donor nucleic acid may be inactivated by resolved. Clearly, therefore, a useful approach in extracellular nucleases. This explanation does the development of a transformation system for not, however, apply to R. sphaeroides strain the purple nonsulfur bacteria may be to use 2.4.1, which apparently produces no extracellu- suitably marked plasmid (CCC) DNA as a probe lar deoxyribonuclease (unpublished data). Alter- in deriving potential competence regimes. This natively, the cell walls of purple nonsulfur bac- could circumvent any requirement for, or dam- teria may never achieve a competent state for age done by, recombination nucleases. taking up the DNA. Even if donor DNA could traverse the cell envelope, successful transfor- CONJUGATION mation with linear DNA would, presumably, still Transfer of genetic material by cell-to-cell depend on a functional recombination system. contact has thus far not been observed to be The success of GTA-mediated gene transfer in indigenous to members of the Rhodospirilla- R. capsulata and of R plasmid-directed gene ceae. The presence im certain members of this transfer in R. sphaeroides (see below) implies group of plasmid DNA, which is of comparable that these organisms are recombination profi- size to the F factor of E. coli, prompted specu- cient. However, it is noteworthy that transfer of lation that such plasmids may specify sex factor chromosomal markers by transformation in E. activity (75). However, genetic conjugation me- coli was initially hampered by the activities of diated by native plasmid species has not been the recombination exonuclease system (recB/ observed in R. sphaeroides (206). Of course, this 374 SAUNDERS MICROBIOL. REV. may be merely a manifestation of intrinsically The precise mechanism of chromosome mo- low transfer frequencies for chromosomal bilization in these cases remains to be elucidated. markers coupled with such phenomena as sur- By analogy with processes of conjugation in E. face exclusion and incompatibility (201), if all coli, gene transfer may involve some form of strains used in the genetic crosses contain ho- covalent association with the chromosome, as in mologous sex factors. the formation of R-prime plasmids or Hfr do- On the other hand, R. sphaeroides and R. nors. Alternatively, the acquired plasmid may rubrum have been shown to act as recipients for direct transfer of chromosomal genes by a mech- the resistance (R) plasmid R1822 derived from anism not unlike that postulated for mobiliza- Pseudomonas aeruginosa. However, the plas- tion of non-self-transmissible plasmids and the mid was unstable in these recipients in the ab- chromosome by sex factors in E. coli. This kind sence of the appropriate selection pressures of activity, which is poorly understood, appar- (167). Subsequently, W. R. Sistrom (private ently does not involve covalent linkage between communication) has demonstrated transfer of plasmid and chromosome (61, 147, 150). the same plasmid to strains of Rhodopseudo- Clearly, a useful objective would be the isola- monas gelatinosa. However, cotransfer of chro- tion ofstable Hfr donor strains ofphotosynthetic mosomal genes has not yet been observed. bacteria for the construction of large-scale ge- By contrast, current attempts to introduce netic maps. The integration of plasmids into the other sex factors and, notably, further plasmids chromosome to form stable Hfr strains is a rare of the P incompatibility (IncP) group (20, 40, occurrence, the most notable exception being 162) into these organisms are more encouraging. the F factor of E. coli (150). However, it may be Kaplan (private communication) and co-workers possible to obtain such strains by exploiting the can demonstrate stable transfer of several R ability of self-transmissible plasmids to suppress plasmids from E. coli to R. sphaeroides and defects in initiation of chromosome replication. subsequently between strains of R. sphaeroides. This process of integrative suppression (159) has Concomitant intraspecies transfer of chromo- been used in E. coli to generate plasmid-me- somal markers, for example, antibiotic resistance diated Hfr-type strains (147, 151). By construct- and nutritional markers, has been achieved for ing mutants of the photosynthetic bacteria R. sphaeroides. which are temperature sensitive for the initia- Recently, Sistrom (217) has accomplished sta- tion of DNA replication and by using the capac- ble transfer of the R plasmid R68.45 (84, 109) ity of transmissible plasmids to suppress this from P. aeruginosa (strain PA025 [R68.45]) to mutation at the restrictive temperature by in- strains ofR. gelatinosa and R. sphaeroides. The sertion into the chromosome, Hfr-type strains frequency of transfer of the plasmid-determined may be produced. Moreover, it may be possible neomycin resistance was of the order of l0' to to force the selection of Hfr-type strains me- 10`6 transconjugants per recipient cell (217). diated by IncP plasmids, of which some are Subsequent transfer of neomycin resistance already known to be capable of infecting mem- amongst strains of R. sphaeroides occurred at a bers of the Rhodospirillaceae. high frequency (about 10-2 transconjugants per donor cell). Interestingly, strains of R. gelati- CONCLUDING REMARKS nosa receiving R68.45 manifest resistance to Interest in the genetics of photosynthetic pro- both neomycin and carbenicillin. In contrast, caryotes has burgeoned considerably over the strains of R. sphaeroides acquiring the plasmid past decade. The recent advances in genetic are neomycin resistant but remain susceptible manipulation, which now permit transfer and to carbenicillin. Transfer of chromosomal recombination of genetic material within this markers (notably, antibiotic resistance determi- biological group, justify great expectations for nants and restoration of prototrophy to specific elucidation of the hitherto intransigent genetic auxotrophs) occurred at frequencies of around systems of these organisms. Indeed, the GTA of 10-6 to 10-7 recombinants per recipient cell for R. capsulata represents a landmark in the mo- R. sphaeroides. Apparently, transfer of R68.45 lecular biology of the photosynthetic bacteria. itself or of chromosomal genes occurs only on The applicability of the GTA system to fine- solid media (217; cf. reference 84). The prelimi- structure genetic mapping of R. capsulata is nary cotransfer data suggest that R68.45 will be clearly demonstrable. However, the limitations a useful genetic tool in the construction of link- of this genetic vector emphasize an urgency for age maps of R. sphaeroides. Indeed, there is further genetic exchange systems, especially for every likelihood that the aforementioned plas- those capable of transferring longer stretches of mids or relatives will ultimately provide conven- the genome and for those capable of establishing ient vehicles for manipulating genes within stable partial diploids for performance of cis- members of the Rhodospirillaceae. trans complementation analyses. At this time, VOL. 42, 1978 GENETICS OF RHODOSPIRILLACEAE 375 transduction and conjugation appear the more Illinois, Urbana), E. W. Frampton (Northern Illinois promising areas for future developments. The University, De Kalb), J. D. Wall (Indiana University, assignment of genetic determinants to plasmids Bloomington), and others for making data available native to photosynthetic procaryotes should fa- prior to publication and for discussion; and T. W. Goodwin (University of Liverpool) for providing re- cilitate identification of their role, if any, in search facilities within the Department of Biochemis- genetic interplay. Recently, Reanney (186) has try during the preparation of this article. expounded the virtues of extrachromosomal ele- ments as executors of evolution in both procar- LITERATURE CITED yotes and eucaryotes. He contends that extra- 1. Abeliovich, A., and S. Kaplan. 1974. Bacteri- chromosomal DNA may have had a dominant ophages of Rhodopseudomonas spheroides: rather than peripheral role in the processes of isolation and characterization of a Rhodopseu- development, adaptation, and speciation. The domonas spheroides bacteriophage. J. Virol. very presence of plasmids, phage, and the GTA 13:1392-1399. 2. Adams, M. H. 1959. Bacteriophages. Intersci- within photosynthetic procaryotes affords scope ence Publishers, Inc., New York. for such evolutionary mechanisms to have em- 3. Adelberg, E. A., M. Mandel, and G. Chein braced this biological group. Ching Chen. 1965. Optimal conditons for mu- An alternative approach to genetic mapping tagenesis by N-methyl-N'-nitro-N-nitroso- in the Rhodospirillaceae, and one which does guanidine in Escherichia coli K 12. Biochem. not rely directly on any gene transfer system, Biophys. Res. Commun. 18:788-795. would be to study the change in mutation fre- 4. Adolph, K. W., and R. Haselkorn. 1972. Pho- quency of genes at replication. This technique tosynthesis and the development of blue-green has been successfully used in the construction of algal virus N-1. Virology 47:370-374. temporal genetic maps of the cyanobacterium 5. Aitken, A. 1976. Protein evolution in cyanobac- teria. Nature (London) 263:793-796. A. nidulans (9,42,43,92). Such temporal genetic 6. Ambler, R. P. 1973. Bacterial cytochromes c and maps ofA. nidulans show good correlation with molecular evolution. Syst. Zool. 22:554-565. a conventional genetic map derived from trans- 7. Ambler, R. 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