MICROBIOLOGICAL REVIEWS, June 1978, p. 357-3840146-0749/78/0042-0357$02.00/0Copyright i 1978 American Society for Microbiology

Vol. 42, No. 2

Genetics of RhodospirillaceaeVENETIA A. SAUNDERSt

Department of Biochemistry, University of Liverpool, Liverpool L69 3BX, United Kingdom

TR ODU TION 357PHYSIOLOGICAL ASPECTS 358

EVOLUTIONARY CONSIDERATIONS ........................................ 358

STUDIES WITH MUTANTS ............................................ 359Survey of Mutants of the Rhodospillae 359

Resistant mutants 359

Auxotrophs 359

Pigment mutants 359

Electron transfer mutants 361

Temperature-sensitive mutants 362

Morphological mutants ....................................... ..... 362Miscellaneous mutants 362

Bacteriochlorophyll Biosynthesis and Related Phenomena .. ........ 362Towards Elucidation of Electron Transport Systems ... ............. 364

GENETIC ORGANIZATION 366EXTRACHROMOSOMAL DEOXYRIBONUCLEIC ACID.367

Occurrence.367Functions Evaluated 368

BACTERIOPHAGE AND BACTERIOCINS.369Isolation and Characterization of Bacteriophage and Cyanophage .. 369Bacteriocinogeny.370

"GENE TRANSFER AGENT"' OF RHODOPSEUDOMONAS CAPSULATA 371

Discovery and Properties 371

Mapping Genes for Bacteriochlorophyll and Carotenoid Biosynthesis ... 372TRANSFORMATION 372

CONJUGATION 373

CONCLUDING REMARKS.374LITERATURE CTED.375

INTRODUCTIONPhotosynthetic bacteria have been effectively

used for many years as model systems for inves-tigating photosynthesis and related metabolicphenomena. Although this has led to a consid-erable accumulation of biochemical and bio-physical data concerning the mechanics of bac-terial photosynthesis (for example, 26, 27, 58, 73,101, 173, 174), there has, until recently, been a

conspicuous lack of complementary genetic in-formation. A profound analysis of the geneticsof the photosynthetic'bacteria would certainlyenhance their present status as research tools.This would, in turn, afford unique opportunitiesfor exploring the development and function ofenergy-conserving systems.One of the primary aims in studying the mo-

lecular biology of photosynthetic bacteria is todetermine the nature, arrangement, and activityof those genes specifying the photosynthetic ap-

paratus. Accordingly, this paper considers pro-gess made towards this goal and outlines poten-tial avenues of inquiry. Some emphasis will be

t Present address: Department of Biology, Liverpool Po-lytechnic, Liverpool L3 3AF, United Kingdom.

laid on physiological processes of the photosyn-thetic bacteria which are amenable to investi-gation via the construction and subsequent anal-ysis of specific mutants. Indeed, this kind ofapproach has contributed substantially to thecurrent volume of literature regarding thesephotosynthetic organisms.The photosynthetic procaryotes presently

comprise the cyanobacteria (blue-green algae),the prochlorophyta, and the green and purplebacteria (179). (For the purposes of this review,the photosynthetic bacteria refer to the greenand purple bacteria.) Four families are recog-nized within the green and purple groups: theChlorobiaceae (green and brown sulfur bacte-ria), the Chloroflexaceae (filamentous glidinggreen bacteria), the Chromatiaceae (purple sul-fur bacteria), and the Rhodospirillaceae (purplenonsulfur bacteria) (179, 180). This review ofnecessity focuses on the latter family, reflectingthe limits of existing knowledge. An attempt isalso made to integrate pertinent aspects of thecyanobacteria in line with reports of a closeaffinity between these and other procaryotes(for example, 36, 43, 53, 59, 227, 229, 253).For more extensive coverage of the genetics of

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the cyanobacteria refer to recent reviews ofWolk (273) and Delaney et al. (43).

PHYSIOLOGICAL ASPECTSPhotosynthetic bacteria are unique amongst

phototrophs in respect of the anaerobic natureof bacterial photosynthesis. No oxygen isevolved in the process, and oxidizable substratesother than water serve as electron donors (70,177, 224, 259). On the other hand, the cyanobac-teria typically exhibit oxygenic photosynthesisin a process comparable to that operative inphotosynthetic eucaryotes (65, 112). Most pho-tosynthetic bacteria and certain cyanobacteriaare capable of utilizing atmospheric nitrogen astheir sole nitrogen source for photosyntheticgrowth (65, 69, 72, 103, 170, 232-234, 273).

Typical representatives of the Rhodospirilla-ceae (purple nonsulfur photosynthetic bacteria)are facultative anaerobes possessing the adap-tive capacity to grow anaerobically in the light(photosynthetically) and aerobically in darkness(by oxidative phosphorylation). Certain mem-bers of this family are also capable of growinganaerobically in the dark (251, 277). Rhodopseu-domonas sphaeroides,, Rhodopseudomonaspalustris, and Rhodospirillum rubrum can fer-ment pyruvate under strictly anaerobic condi-tions in darkness (251). However, growth ofRho-dopseudomonas capsulata under such condi-tions necessitates addition of dimethyl sulfoxideto the growth medium (277). By virtue of theirmetabolic versatility, these photosynthetic bac-teria are particularly well suited to the study ofprocesses involved in the formation and differ-entiation of energy-conserving membranes.Moreover, mutant strains can be readily isolatedthat are either photosynthetically or aerobicallyincompetent, but capable of growing in the al-ternative energy conversion mode. Such mu-tants have been effectively exploited in examin-ing the electron transport systems of these or-ganisms as discussed below (see Towards Elu-cidation of Electron Transfer Systems). Thegeneral physiologies of the photosynthetic bac-teria and the cyanobacteria have been detailedelsewhere (for example, 23, 60, 71, 73, 106, 110,170, 177, 224, 228, 258, 273).

EVOLUTIONARY CONSIDERATIONSThere is considerable speculation about the

evolutionary significance of the photosyntheticprocaryotes (18, 21, 35, 49, 168, 185, 197, 225,226, 244). On the one hand, ancestors of thephotosynthetic bacteria are presumed to beamongst the earliest of organisms utilizing ra-diant energy in an anaerobic environment (168,197). On the other, ancestors of cyanobacteria

were supposedly responsible for early biologicaloxygen production and hence for the dramaticevolutionary consequences stemming from thistransition in the gaseous environment (18, 30,65, 168). Cyanobacteria have for some time beencited as likely candidates for endosymbioticprecedents to photosynthetic plastids of certaineucaryotic cells (136, 146, 226, 237, 244). Morerecently, it has been suggested that ancestors ofsome contemporary respiring bacteria may haveevolved from some purple nonsulfur photosyn-thetic bacteria by atrophy of their photosyn-thetic capacity. Likewise, certain of the glidingbacteria may have derived from cyanobacteria(49).Data concerning the structure and sequence

of electron transfer proteins which have beenamassed in recent years (for example, 5-8, 49,214, 240-242, 245, 255, 260, 275) may providesome clues to evolutionary connections betweenprocaryotes and eucaryotes. Close structural andsequence similarities are apparent for cyto-chrome c2 from purple nonsulfur bacteria (8, 56,198), cytochrome c50 from Paracoccus denitri-ficans (247-249), and mitochondrial cytochromec (41, 47, 48). These findings, together with thesimilarities in respiratory electron transfer prop-erties of P. denitrificans, the purple nonsulfurbacterium R. sphaeroides, and the mitochon-drion (58, 99, 204), encouraged speculation aboutthe evolution of bacterial energy metabolism.Dickerson and colleagues (49) have suggestedthat "the point of divergence between photosyn-thesis and respiration occurred in the ancestorsof purple nonsulfur photosynthetic bacteria."However, phylogenetic relationships betweenorganisms may well have been blurred via theagency of genetic exchange (cf. reference 6);thus, interpretations ofevolutionary occurrencesbased on such molecular methodology mayprove to be oversimplifications of actual events.Comparison of amino acid sequence similari-

ties between f-type cytochromes from certaincyanobacteria and eucaryotic algae indicate acloser sequence correlation between the cyto-chrome f of the cyanobacteria and that of thered algae than with that of any other algae (5,7). Aitken (5) points out that, although genetictransfer may have occurred, obscuring the inter-relatedness ofsuch photosynthetic proteins, pro-tein sequence studies have produced much in-formation in keeping with the hypothesis of acommon origin of oxygenic photosynthesis inprocaryotes and eucaryotes. Indeed, patterns ofhomologies from ribosomal ribonucleic acid(RNA) sequence studies with cyanobacteria andchloroplasts (16, 17, 52, 175) lend credence tothis notion.

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GENETICS OF RHODOSPIRILLACEAE 359

Recently, certain "procaryotic green algae"have been observed and studied which possessa unique combination of characteristics, sometypically procaryotic and others eucaryotic(127). Significantly, these organisms containboth chlorophyll a and chlorophyll b and per-form oxygen-evolving photosynthesis. Whetherthese algae or relatives are progenitors of greenplant chloroplasts remains open to question.

STUDIEES WITH MUTANTSMutant strains of various microorganisms

have proven invaluable in the elucidation of anumber of metabolic pathways. Of particularrelevance here are those mutants of photosyn-thetic procaryotes that facilitate studies on elec-tron transfer processes, nitrogen fixation, pig-ment biosynthesis, membrane development anddifferentiation, and related biological phenom-ena.Typical mutants of the Rhodospirillaceae are

obtained by ultraviolet irradiation or N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis essen-tially by the method of Adelberg et al. (3), withor without subsequent penicillin screening (124).Less frequently, mutants have been isolatedafter spontaneous mutation. Mutations in thecyanobacteria are commonly induced with ni-trosamines. Fairly extensive surveys of the mu-tants of cyanobacteria have recently appeared(43, 254).This section is confined to a description of

mutants of the Rhodospirillaceae. These mu-tants not only represent valuable research toolsin biochemical investigations but also provide abank of genetically marked strains convenientfor gene transfer studies.

Survey ofMutants ofthe RhodospirillaceaeVarious classes of mutants of the Rhodospi-

rillaceae have been reported, most commonlyfor R. sphaeroides, R. capsulata, and R. rub-rum.Resistant mutants. A range of antibiotic-

resistant mutants has been obtained, includingstrains resistant to rifampin, streptomycin, nali-dixic acid, and kanamycin (142, 153, 206, 217,222, 262, 276, 277; V. A. Saunders, Ph.D. thesis,University of Bristol, Bristol, U.K.). Mutantstrains ofR. capsulata resistant to arsenate havealso been isolated (for example, 281). One suchmutant, strain Z-1, exhibits enhanced rates ofphotophosphorylation. After prolonged culturein the presence of arsenate, cells have elevatedcontents of cytochromes, reaction center bacter-iochlorophyll, and photophosphorylation cou-pling factor (129, 281). This class of mutantsshould prove useful for analyzimg mechanisms of

energy conservation in photosynthetic bacteria.Auxotrophs. Mutants requiring specific

amino acids have been described (for example,13, 14, 78, 126, 217, 222, 276, 277). Adenine-requiring (unpublished data) and uracil-requir-ing (125) mutants of R. sphaeroides have beenprepared and used in the radiolabeling of deoxy-ribonucleic acid (DNA) and RNA, respectively.Certain glycerol-requiring strains of R. capsu-lata have recently been isolated and character-ized (108). A series of mutants of R. capsulatahave been obtained that lack the capacity to fixnitrogen (Nif-) (264) presumably because of theabsence of nitrogenase activity or because ofdefects associated with the synthesis or metab-olism of glutamine or glutamate (262, 264).Pigment mutants. The Rhodospirillaceae

synthesize colored carotenoids belonging to the"spirilloxanthin series" (98). In R. capsulata andR. sphaeroides, spheroidene and hydroxyspher-oidene predominate under anaerobic conditionsin the light. On the other hand, R. rubrumsynthesizes mainly spirilloxanthin. Various mu-tants with altered carotenoid complement areknown. Classical "blue-green" mutants, lackingcolored carotenoids and accumulating phytoene,have been described for R. sphaeroides (forexample, 29, 80, 81, 216, 218, 219), R. capsulata(for example, 55, 267, 276), and R. rubrum (forexample: 39; R. K. Clayton, cited in references95 and 113). "Green" mutants, presumablyblocked at the neurosporene or chloroxanthinstages of carotenoid biosynthesis, have been ob-tained for R. sphaeroides (for example: 37, 38,80, 81, 145, 203; Saunders, Ph.D. thesis) and R.capsulata (267, 276). Yen and Marrs (276) re-cently described "yellow" mutants of R. capsu-lata that apparently were phenotypically indis-tinguishable from the so-called brown mutantsof R. sphaeroides isolated by Griffiths and Stan-ier (81). Further "brown" mutants, phenotypi-cally distinct from those of Griffiths and Stanier(81), have also been characterized (210). Fur-thermore, certain mutants of R. sphaeroideshave been isolated that combine the traits ofcarotenoid deficiency and a high catalase activ-ity (29).There is a spectrum of mutants with blocks at

specific stages in bacteriochlorophyll biosyn-thesis (Table 1). The propensity ofsuch mutantsfor accumulating various tetrapyrrole pigments,presumably bacteriochlorophyll precursors, hascontributed significantly to the elucidation ofreactions involved in bacteriochlorophyll bio-synthesis as outlined in the following section.The inability of "albino" mutants to form carot-enoid and bacteriochlorophyll is possibly a man-ifestation of loss or dysfunction of a genetic

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TABLE 1. Typical mutants ofphotosynthetic bacteria with lesions affecting bacteriochlorophyll biosynthesisSpecies Mutant strain Remarks Reference

R. sphaeroides H-4, H-5 Lack 8-aminolevulinate synthase ac- 121tivity, require aminolevulinate forgrowth; H-5 normalized by amino-levulinate

6-6 Excretes porphobilinogen, no mag- 120nesium tetrapyrroles formed

2-33 (Met-) Excretes coproporphyrin, methio- 117nine pathway blocked at homo-cysteine methylation level

M-17 (Met-) Excretes coproporphyrin, methio- 126nine pathway blocked at stage be-fore cysteine synthesis

2-731 Excretes magnesium divinylpheo- 116V-3 f porphyrin a,,, Saunders,

Ph.D. thesis

8-32 Excretes magnesium divinylpheo- 188porphyrin a.,, bacteriochlorophyl-lide, and heme

"Tan" Magnesium divinylpheoporphyrin 228a, accumulated by cells

"Griffiths mu- Magnesium divinylpheoporphyrin 79tants" a, (and, probably, later interme-

diates) accumulated by cells

2-21 Excretes 2-devinyl-2-hydroxyethyl- 116chlorophyllide a

8-29 Excretes 2-devinyl-2-hydroxyethyl- 188chlorophyllide a and some pheo-phorbide a

8-47, 8-53 Excrete 2-desacetyl-2-hydroxyeth- 188ylbacteriochlorophyllide and 2-devinyl-2-hydroxyethylchloro-phyllide a

8-17 Excretes bacteriochlorophyllide 188

8-13 Accumulates heme, no magnesium 120tetrapyrroles formed

"Albino" mutants, neither bacte-L-57, 3-1 riochlorophyll nor precursors 120V-2 formed, also fail to make carote- 204

noids

"Griffiths mu- "Albino" strains, neither bacterio- 81tants" chlorophyll nor carotenoids

formed

L-57 R, TA-R, Cells accumulate bacteriochloro- 123DW-R phyll aerobically in darkness

8 Excretes 2-desacetyl-2-vinylbacte- 184riopheophorbide

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GENETICS OF RHODOSPIRILLACEAE 361

TABLE 1-ContinuedSpecies Mutant strain Remarks Reference

O, Excretes pigments with absorbance 102maximum below 660 nm

Excrete magnesium divinylpheopor-phyrin a., monomethylester

Excrete 2-devinyl-2-hydroxyethyl-pheophorbide a, some pheophor-bide a

Excretes 2-devinyl-2-hydroxyethyl-pheophorbide a, some pheophor-bide a and bacteriochlorophyll

Excretes phytylated (fully esteri-fied) magnesium-2-vinylpheopor-phyrin a,, as protein complex

Accumulates precursor with absorb-ance maximum at 630 to 635 nm(presumably magnesium 2,4-divi-nylpheoporphyrin a,s)

165

163, 165

165

55

{276277

SB21, Y34, Y62,Y92, Y121,Y122, Y165,Y167, Y451

Y89}

HH 910, HH 911

Accumulate precursor with absorb-ance maximum 665-670 nm

Incapable of synthesizing bacter-iochlorophyll and carotenoids

Accumulate precursor with absorb-ance maximum at 730 nm

Magnesium divinylpheoporphyrina5 and a phytylated form ex-tracted from cells

element governing synthesis of the entire pho-topigment system. Alternatively, synthesis ofthe photosynthetic membrane components maybe dependent on the assembly process; thus, ifany one of the structural components was ab-sent, synthesis of the entire system would beswitched off. A photosynthetically incompetentstrain of R. rubrum has been isolated whichproduces a "pheophytin-protein-carbohydrate"complex. Some nonfinctional bacteriochloro-phyll is also formed. Failure of the pigmentcomplex to associate with the membrane may

reflect alteration or absence of requisite com-

ponents for its incorporation (207). In addition,a mutant of R. sphaeroides has recently beendescribed which accumulates 4-vinyl protochlo-rophyllide, presumably because of defective syn-thesis of membrane components required forincorporation of bacteriochlorophyllide into theintracytoplasmic membrane system (183).Electron transfer mutants. Mutants with

specific defects in the respiratory or photosyn-thetic electron transfer system have been de-scribed (for example, 44, 45, 115, 138, 139, 141,272). The biochemical lesions affecting some ofthem will be considered below (see TowardsElucidation of Electron Transport Systems).Certain strains of R. sphaeroides (216, 218, 239)and R. capsulata (277) lack functional reactioncenter bacteriochlorophyll (P870), whereas theysynthesize the light-harvesting (bulk) bacter-iochlorophyll. Accordingly, such mutants do notmanifest those activities associated with the pri-mary photochemistry of photosynthetic cells

(211, 218, 220). Mutants of R. rubrum have alsobeen reported with properties characteristic ofstrains with defective reaction centers (44, 181).

In addition, a strain of R. rubrum, F24.1, hasrecently been isolated with an altered reactioncenter (181). This mutant is a spontaneous pho-totrophic revertant derived from a photosyn-thetically incompetent strain with a nonfunc-

R. rubrum F3, F4, F6

F5, F8, F9

F12

R. capsulata Ala (Pho-)

R. palustris

276277

{263277

277

Green 1, 2, 3Yellow I

63, 111{223, 252

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362 SAUNDERS

tional reaction center. Strain F24.1 apparentlylacks bacteriochlorophyll P800, a constituent ofthe reaction center (27, 174), but is, nevertheless,capable of photosynthetic growth. Picorel andco-workers (181) suggest that this mutant maybe enriched in a second kind of reaction centerwhich does not contain P800 and which is pres-ent as a minor component in wild-type cells.Such an explanation would reinforce previousproposals (236, 256) that two different kinds ofreaction center coexist in membranes of R. rub-rum. Alternatively, the reaction center of strainF24.1 may be modified so as to render P800unnecessary. Indeed, if this is the case, studieswith such mutants should further resolve theprecise role of P800 in bacterial photosynthesis.Temperature-sensitive mutants. Temper-

ature-sensitive lesions of the photosynthetic ap-paratus of R. rubrum provide strains condition-ally unable to perform photosynthetic functionsassociated with electron flow. Such mutantswere selected by the phototactic enrichmenttechnique, assuming that the phototactic re-sponse of photosynthetic cells relies on aproperly functioning electron transport system(266). Other temperature-sensitive mutants ofR. sphaeroides have been isolated (unpublisheddata) which are unable to grow aerobically atthe nonpermissive temperature. The exact na-ture of the lesions affecting these strains is un-known.The use of temperature-sensitive mutants,

particularly in essential functions, has paid un-doubted dividends in studies of other biologicalsystems. It is perhaps surprising, therefore, thatthere has been a dearth of reports of investiga-tions involving similar mutations in the photo-synthetic bacteria. In particular, studies withtemperature-sensitive mutants of the Rhodo-spirillaceae should facilitate identification ofelectron transfer components which may becommon to both the respiratory and the photo-synthetic electron transfer systems. The notionofshared electron transport components in theseorganisms is supported by several workers (seeTowards Elucidation of Electron Transport Sys-tems). Mutations affecting such componentsmight reasonably be expected to be lethal duringdark aerobic growth or photosynthetic growth.(When cells are grown anaerobically in darknessthe components may be dispensable [277], andhence the mutations would not prove lethal.)Thus, the isolation of appropriate temperature-sensitive mutants should enable inroads to bemade towards defining interrelationships ofpho-tosynthesis and respiration in the purple non-sulfur bacteria.Morphological mutants. Vibrio (158) and

bacilliform (155) mutants of R. rubrum havebeen described which apparently result fromdefects in D-alanine metabolism (156, 157).The vibrio mutants were initially selected as

strains resistant to D-cycloserine (an analog ofD-alanine) (158). The phenotype of these mu-tants is evidently a consequence ofperturbationsin cell envelope biosynthesis.Miscellaneous mutants. Certain mutants of

R. rubrum have been selected on the basis ofpigmentation after prolonged growth anaerobi-cally in the dark (250). One mutant, strain C,synthesized bacteriochlorophyll a, altered mem-brane structures, and chromatophores duringdark growth. Furthermore, strain C was capableofgrowing anaerobically in the light. In contrast,a second mutant, strain Gi, was light sensitiveand produced only trace amounts of bacterio-chlorophyll.

Typically, wild-type strains of R. capsulata,unlike strains ofR. sphaeroides and R.palustris,are unable to utilize glycerol as a carbon source(257). However, a spontaneous variant of R.capsulata, strain Li, capable of using glycerolfor both anaerobic photosynthetic growth andaerobic dark growth, has been isolated (132).Two enzymes, glycerokinase and glycerophos-phate dehydrogenase, not detectable in the par-ent, were found to be synthesized constitutivelyin this mutant (131, 132). By contrast, suchenzymes are inducible, in the presence of glyc-erol, in R. sphaeroides (182). Constitutive syn-thesis of these enzymes in the mutant possiblyreflects derepression in strain Li of an operonnot normally expressed in the wild type.Mutants of R. palustris have been reported

that differ from wild-type strains in that theyare incapable of growing aerobically at the ex-pense ofcyclohexanecarboxylic acid or pimelate.Such mutants have been exploited in determin-ing reactions involved in the photometabolismof benzoate by R. palustris (83).

Bacteriochlorophyll Biosynthesis andRelated Phenomena

Elucidation of reactions involved in bacter-iochlorophyll biosynthesis has depended largelyupon the analysis of tetrapyrrole pigments ac-cumulated by strains of purple nonsulfur bacte-ria in which bacteriochlorophyll biosynthesis isderanged by mutation or metabolic inhibitors.Figure 1 outlines reactions involved in bacter-iochlorophyll biosynthesis and incorporates themutational blocks for a series of mutants of R.sphaeroides. It is noteworthy that the precursoraccumulated by R. sphaeroides strain 8 (seeTable 1) does not fit into the scheme per se andmay be indicative of an alternative pathway to

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GENETICS OF RHODOSPIRILLACEAE 363

Glycine + Succinyl CoA

6-aminolevulinic acid (bALA)

Porphobilinogen

UropoorrrinZgen

Coprophorpyrinogen

PROTOPORPHYRIN+ Mg I + Fe

Hagnesium protoporphyrin

Magnesium protoporphyrinmonomethyl ester

Magnesium divinylpheoporphiyrina5 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

Phytol

hyllide

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-ence 184). Mechanisms regulating bacteriochlo-rophyll biosynthesis have been proposed largelyon the basis of the behavior of appropriate mu-

tants under various environmental conditions. Abrief review has recently appeared (119). 8-Ami-nolevulinate synthase represents one control lo-cus, presumably regulated, at least in part, bythe intracellular heme concentration (122). Mag-nesium chelatase, which appears to be criticallysensitive to oxygen, is of particular importancein regulating the magnesium branch of the path-way (121). Certain of the biosynthetic enzymes

appear to be subject to repression in the pres-

ence of oxygen. Mutants of R. sphaeroides havebeen isolated that continue to synthesize bacter-iochlorophyll under conditions of high aeration

(123). Presumably, this response is attributableto a derepression of synthesis of certain enzymesof the magnesium path under conditions nor-

mally causing their repression. It has been spec-ulated that synthesis of all the enzymes of themagnesium branch of the pathway may be con-

trolled by a regulatory gene which is in turninfluenced directly or indirectly by oxygen (123).Cohen-Bazire et al. (37) advanced a hypothe-

sis, referred to as the "redox-governer" hypoth-esis, to account for the almost immediate cessa-

tion of bacteriochlorophyll biosynthesis in re-

sponse to oxygen. It was suggested that the rateof bacteriochlorophyll and carotenoid biosyn-thesis was governed by the state of oxidation ofa carrier in the electron transport system. Thisnotion has subsequently been superseded in

H-5; H-4

2-33; M-17

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light of observations with specific mutants of R.capsulata impaired in respiratory electrontransport (139). It is envisaged that an oxygen-sensitive factor regulates bacteriochlorophyllsynthesis. This factor, inactivated by oxygen,can be reactivated by a flow of electrons fromthe electron transport system, diverted possiblyat the level of cytochrome c. The factor mayrepresent one or more of the enzymes directlyinvolved in bacteriochlorophyll synthesis or aneffector molecule that interacts with them (139)(see Fig. 2). The involvement of cytochrome ccould be tested for by investigating regulation ofbacteriochlorophyll synthesis in mutantsblocked in electron transport between cyto-chromes b and c.An inverse correlation appears to exist be-

tween the intracellular concentration of adeno-sine 5'-triphosphate and the rate of bacterio-chlorophyll biosynthesis in certain photosyn-thetic bacteria (62, 209). Furthermore, it hasbeen proposed (62) that the amount ofadenosine5'-triphosphate within cells of R. sphaeroides isin itself decisive in modulating bacteriochloro-phyll synthesis.

Ultrastructure studies with mutants blockedat specific stages in bacteriochlorophyll biosyn-thesis reveal that synthesis of the entire bacter-iochlorophyll molecule is a prerequisite for as-sembly of the intracytoplasmic membrane sys-tem characteristic of pigmented cells (19, 165).

lighthl BCi

IightBfh < bchl 02 bchle nactive Fictive

SuccinMte dehyd."2 + yc : cytb

<UQNADH dehyd..5J2ctW °2

FIG. 2. Model ofelectron transportpathways pres-ent in membranes of R. capsulata, incorporating apossible scheme for the regulation of bacteriochloro-phyll synthesis by molecular oxygen (modified fromMarrs and Gest [139] and La Monica and Marrs[115]). Both respiratory and light-driven electronflow systems are indicated. The numbered arrowsrepresent steps in the respiratory electron transportpathways. The mutational blocks in respiration-de-ficient strains of R. capsulata are as follows: strainMl is blocked in step 1; M2 is blocked in steps 1 and2; M3 is blocked in step 2; M6 is blocked in step 5;M7 is blocked in step 4 by virtue of lacking theassociated cytochrome b. Abbreviations: BChl, bac-teriochlorophyll; UQ, ubiquinone; cyt, cytochrome; e,electrons; dehyd., dehydrogenase; NADH, reducednicotinamide adenine dinucleotide. Fi'h! represents apostulated 02-sensitive factor required for BChl syn-thesis (139).

In support of this are observations (28, 164, 165,238, 239) which indicate an obligatory couplingbetween bacteriochlorophyll biosynthesis andthe formation of specific chromatophore pro-teins. The role of the bacteriochlorophyll mole-cule in this process remains obscure. Possibly,the pigment exerts its effect at the level of tran-scription or translation. Alternatively, the bac-terichlorophyll molecule may be necessary forassembly of these specific proteins into the ar-chitecture of the membrane (119, 239).

Analysis of glycerol auxotrophs of R. capsu-lata indicated a dependence of bacteriochloro-phyll and carotenoid biosynthesis on phospho-lipid synthesis (108). An increased lipid contentis associated with pigmented cells. It is notknown whether lipid synthesis exerts direct reg-ulatory control on carotenoid synthesis orwhether a slow-down in carotenoid synthesis isa secondary effect of decreased bacteriochloro-phyll production.

Clearly, more investigations at the geneticlevel are required to clarify the regulatory mech-anisms involved in photopigment biosynthesis.

Towards Elucidation of ElectronTransport Systems

The intracytoplasmic membrane of purple'nonsulfur bacteria accommodates both the res-piratory and the photosynthetic electron trans-port systems. Resolution of the precise natureand arrangement of components of either ofthese systems is thus complicated by their dualoccurrence in the membrane. A promising ap-proach to the analysis of these systems involvesthe use of mutants with lesions specifically af-fecting photosynthetic or respiratory compe-tence. In addition, mutants with altered carote-noid patterns (notably, green, blue-green, andalbino strains) have been widely exploited inmonitoring electron transfer reactions (for ex-ample, 38, 57, 100, 204). These mutants have theadvantage of permitting spectroscopic observa-tion of electron transport components (for ex-ample, cytochromes) in the spectral region nor-mally masked by the absorbance of photopig-ments characteristic of wild-type strains.A series of respiration-deficient mutants of R.

capsulata, including strains with depressed nic-otinamide adenine dinucleotide, reduced form,and/or succinate dehydrogenase activities(strains Ml, M2, and M3 [138]) and those withdefects in the terminal portion of the electrontransport system (strains M4, M5, M6, and M7[138]), selected through the use of the "Nadireaction" (105), have been investigated (115, 138,139, 141, 278-280). It is inferred from the growthcharacteristics of such mutants that reduced

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nicotinamide adenine dinucleotide dehydrogen-ase activity is essential to the aerobic compe-tence of the organism, whereas succinate dehy-drogenase activity is not (138, 141). The re-sponses ofthese mutants, particularly to conven-tional inhibitors of electron flow, are compatiblewith a branched respiratory electron transportsystem for R. capsulata (Fig. 2), with two dis-tinct terminal oxidases (115, 138, 279, 280). Ahigh-potential, membrane-bound, b-type cyto-chrome (Eo' = +413 mV) found in aerobicallygrown cells of R. capsulata is apparently in-volved in cytochrome c oxidase activity (278,279). A further high-potential b-type component(Eo' = +270 mV) which interacts strongly withcarbon monoxide has been proposed as the trueo-type oxidase of R. capsulata (280). b-Typecomponents of similar oxidation-reduction po-tential to these have also been identified inaerobically grown cells of an albino mutant(strain V-2) of R. sphaeroides (205). Moreover,the a-type cytochrome which develops in R.sphaeroides during aerobic growth (107) hasbeen partially characterized. Potentiometric ti-tration at 607 nm revealed two components withoxidation-reduction midpoint potentials similarto those of eucaryotic cytochrome oxidase (Eo'= +375 mV and Eo' = +200 mV) (204) in mem-branes from strain V-2 of R. sphaeroides. Of allthe members of the Rhodospirillaceae, the a-type cytochrome remains exclusive to R. sphae-roides.There is speculation that certain electron

transfer components are common to both therespiratory and the photosynthetic electrontransport systems of R. capsulata (67, 115, 138,279). The possibility of shared components haspreviously been suggested by the studies of Con-nelly et al. (38) and Jones and Plewis (102) withmutants of R. sphaeroides. Interrelationshipsbetween photosynthetic and respiratory electrontransfer systems will be further resolved withthe isolation and analysis of more mutants. Inthis connection, a mutant of R. capsulatablocked in electron transport between cyto-chromes b and c is currently being investigated(B. L. Marrs, private communication).Other lesions responsible for respiratory defi-

ciency in purple nonsulfur bacteria have beenreported. Wittenberg and Sistrom (272) haveisolated a mutant of R. sphaeroides, strain 37,incapable of growing aerobically in the darkexcept when the oxygen tension is low enoughto permit photopigment synthesis. This is anal-ogous to the behavior of certain reputedly "ob-ligately anaerobic" photosynthetic bacteria(178). Possibly, the biochemical defect in suchnaturally occurring obligate phototrophs may beidentical with that of R. sphaeroides strain 37.

GENETICS OF RHODOSPIRILLACEAE 365

It has been proposed (272) that heme synthesisin strain 37 is obligatorily coupled to bacterio-chlorophyll synthesis, implying some alterationin the normal regulatory mechanisms for syn-thesis of electron transport components. Studieswith additional aerobically incompetent mu-tants may well provide a molecular explanationfor the differential response of photosyntheticbacteria to molecular oxygen.

del Valle-Tasc6n et al. (44) have isolated mu-tants of R. rubrum which are incapable of pho-totrophic growth. One such mutant, strain Fll,exhibits normal rates of endogenous cyclic pho-tophosphorylation but is defective in photooxi-dase activity. Such observations suggest that theconstituent(s) altered by mutation does not be-long to the cyclic photophosphorylation system.The electron transfer step deranged in strainFll is supposedly located on the low-potentialside of the photosynthetic reaction center, be-tween the primary electron acceptor and oxygen(45). Results from studies on the photooxidationof exogenous electron donors and of reactioncenter bacteriochlorophyll by chromatophoresfrom mutant Fll are consistent with a branchedmodel for light-driven electron transfer in chro-matophores of R. rubrum (Fig. 3) (45). It istentatively proposed that specific constituentsof the photooxidase system are located in a sidechain which connects a pool of cyclic electronacceptors with oxygen. Strain Fll is blocked inthis side chain and is consequently deficient inphotooxidase activity. The photooxidase systemis apparently essential for the normal photosyn-thetic metabolism of R. rubrum, although itsprecise physiological role in vivo requires verifi-cation.Mutants blocked in bacteriochlorophyll bio-

synthesis provide appropriate test systems for

(Fll)

A

ligt_T HQNO

P870 CDCIPH2DAD

FIG. 3. Model of light-driven electron transport inisolated chromatophores of R. rubrum (after delValle-Tasc6n et al. [451). Strain Fll is blocked in aside chain which connects a pool ofsecondary accep-tors and oxygen. The dashed arrow represents analternative site for oxygen reduction in this mutant.Abbreviations: HQNO, 2-heptyl-4-hydroxyquinoline-N-oxide; DAD, 2,3,5,6-tetramethyl-p-phenylenedi-amine; DCIP, 2,6-dichlorophenolindophenol; A, pri-mary acceptor; P870, reaction center bacteriochloro-phyll.

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monitoring the contributions of specific compo-nents to the reconstitution of photosyntheticactivity and hence provide information concern-

ing the assembly of the photosynthetic appara-

tus. Reconstitution of photosynthetic reactionshas been demonstrated with the bacteriochlo-rophyll-lacking membranes of aerobically growncells of mutants of R. sphaeroides (102) and R.capsulata (67, 68) when supplemented with re-

action center bacteriochlorophyll. The addi-tional effect of light-harvesting chlorophyll on

the "reconstituted" system has been studied inR. sphaeroides (96). It has been suggested, atleast for R. sphaeroides, that the respiratoryelectron transport system is of similar composi-tion and orientation to the photosynthetic elec-tron transport system. Terminal oxidases or re-

action center complexes may thus be incorpo-rated into a preexisting photosynthetic or res-

piratory electron transport chain, depending on

the mode of growth (101, 102). Other mutants(and, in particular, those specifically lacking re-

action center bacteriochlorophyll, but not theantenna chlorophyll) should add a further re-

finement to the reconstitution system.Recently, anaerobic dark growth of R. capsu-

lata has been demonstrated with glucose as thecarbon source, but only in the presence of di-methyl sulfoxide (277). In this respect R. cap-sulata apparently differs from other members ofthe Rhodospirillaceae, namely, R. palustris, R.sphaeroides, and R. rubrum, which do not re-

quire dimnethyl sulfoxide for fermentative anaer-obic growth in darkness (251). A series of mu-tants of R. capsulata, including photosyntheti-cally or aerobically incompetent strains, were

examined for the ability to grow under anaerobicconditions in the dark (277). Results indicatedthat the bacteriochlorophyll-mediated energyconversion system of R. capsulata is unneces-

sary for anaerobic dark growth. Furthermore,neither reduced nicotinamide adenine dinucleo-tide dehydrogenase activity nor either of theterminal respiratory oxidases is essential for an-

aerobic growth in darkness. However, althoughthe entire respiratory chain is apparently notrequired, certain cytochromes may be neededfor reduction of the dimethyl sulfoxide. It isproposed that R. capsulata requires a terminalelectron acceptor for anaerobic dark growth andthat dimethyl sulfoxide can serve the purpose.The preceding sections amply illustrate the

importance of a combined biochemical and ge-netic approach in the elucidations of electrontransfer systems and of reactions involved inbacteriochlorophyll biosynthesis. Providing thatthe effects of mutation are strictly localized anddo not excessively perturb the system under

investigation, valid extrapolation can be madeto the situation appertaining in wild-type cells.Theoretically, appropriate classes of mutantscoupled to efficient gene transfer systems shouldpermit a thorough analysis of photosyntheticprocesses in the Rhodospirillaceae.

GENETIC ORGANIZATIONThe base composition of DNA from members

of the Rhodospirillaceae ranges from 60 to 67mol% guanine plus cytosine (G+C) for Rhodo-spirillum species, 64 to 70 mol% G+C for Rho-dopseudomonas species, and 62 to 65 mol% G+Cfor Rhodomicrobium vannielii, as estimatedfrom thermal denaturation profiles, density gra-dient centrifugation, and ultraviolet spectropho-tometry (93, 177, 215). The G+C content ofDNA from purple sulfur bacteria (Chromatiumspecies) is from 48 to 70 mol%, whereas the valuefor green sulfur bacteria ( Chlorobium species) islower (from 50 to 58 mol% G+C) (93, 177) (Table2). By contrast, there is considerable variationin the G+C content ofDNA in the cyanobacteria(from 35 to 70 mol%) (273).The genome size of the purple nonsulfur bac-

terium R. sphaeroides is about 1.6 x 109 daltons(75), a value comparable to that of other bacte-ria. On the other hand, the genome sizes of anumber of cyanobacteria, determined from re-naturation kinetics, range from-approximately 2x 109 to 7 x 109 daltons and appear to fall intofour distinct groups (90). In fact, the largest ofthese genomes are amongst the most complexreported for procaryotes. Further, the increasein genetic complexity coincides with increasingmorphological and biochemical complexitywithin the cyanobacteria (90). Certain cyano-bacteria apparently contain multiple copies oftheir genome per cell (192, 273). Moreover, atleast some photosynthetic procaryotes containextrachromosomal (covalently closed circular[CCC]) DNA in addition to the chromosomeTABLE 2. Base composition (moles percent G+0C) of

DNA of some photosynthetic bacteria

Species mol% G+C of to- Referencetal bases'

Chlorobium spp. 51-58 93, 177Chromatium okenii 48-50 133Chromatium grac- 68-70 133

ileR. vannielii 62-65 177, 215R. capsulata 65-67 177, 215R. sphaeroides 66-70 215Rhodopseudomo- 67-68 215nas viridis

R. rubrum 62-65 215

aA range of values for moles percent G+C is givenresulting from the different methods used (see text).

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GENETICS OF RHODOSPIRILLACEAE 367

(see Extrachromosomal Deoxyribonucleic Acid).It has been reported for certain purple non-

sulfur bacteria (78, 125, 140, 194) and for thecyanobacterium Anacystis nidulans (51) thatthe ribosomal RNA (rRNA) complement isatypical. There is apparently no stable 23SrRNA component. The 23S rRNA is consideredto be a precursor molecule which is subsequentlycleaved into two smaller stable rRNA species(51, 78, 140, 194). This has implications for therole of rRNA and raises questions as to therelationship between rRNA cleavage and ribo-some function in these organisms.

In passing, not much is known about DNAreplication in relation to the cell cycle in theRhodospirillaceae. Studies of Westmacott andPrimrose (268) on the effect of nalidixic acid onthe cell cycle of R. palustris indicate a depend-ence of cell division and flagellum and holdfastsyntheses on the completion ofchromosome rep-lication. However, photosynthetic membraneformation and wall extension are apparently in-dependent of chromosome replication. In addi-tion, pre- and postsynthetic gaps accompany around of DNA replication in R. palustris. Suchcorrelation between cell division and DNA rep-lication has previously been reported for otherorganisms, notably, Escherichia coli (for exam-ple, 22, 25, 50). However, coupling between thetermination ofDNA replication and cell divisionis not a universal occurrence in bacteria. Indeed,in the cyanobacterium A. nidulans initiation ofDNA replication appears to be a necessary eventfor cell division to occur (134, 135).

EXTRACHROMOSOMALDEOXYRIBONUCLEIC

ACIDOccurrence

Extrachromosomal (plasmid) DNA is com-monly found in bacteria and determines a diver-sity of biological functions (for reviews, see ref-erences 31, 61, 147, 160-162, 186, 269). There arereports of the presence of plasmid DNA in bothphotosynthetic bacteria (74, 75, 206, 235) andcyanobacteria (10, 187, 193; D. Heaton and E.W. Frampton, Abstr. Annu. Meet. Am. Soc.Microbiol. 1976, I117, p. 131).Suyama and Gibson (235) reported the pres-

ence of plasmid DNA in R. sphaeroides. Despitethe failure of other workers (152) to detect suchDNA in this organism, these initial observationswere later substantiated by Gibson and Nieder-man (75), who, furthermore, detected not one,but two, species of plasmid DNA of similarmolecular weights (70 x 106 to 75 x 106) but ofdifferent buoyant densities (1.718 and 1.724 g/cm:3) in R. sphaeroides strain NCIB 8327. Re-

cent work of Saunders and colleagues (206) sup-ported and extended these observations. ThreeCCC species of extrachromosomal DNA wereidentified by electron microscopic analyses (Fig.4) in both aerobically and photosyntheticallygrown cells of R. sphaeroides strain 2.4.1. Mo-lecular weights of these plasmids, as determinedfrom contour length, were 75 x 106,66 x 106, and28 x 106 for R. sphaeroides strain 2.4.1. Plasmidsweighing 28 x 106 and 66 x 106 daltons were ofa buoyant density of 1.717 g/cm3, and thoseweighing 75 x 106 daltons were of a buoyantdensity of 1.724 g/cm3. A photosynthetically in-competent strain of R. sphaeroides, SLS I, se-lected after treatment of cells of strain 2.4.1 withsodium lauryl sulfate, also contained three spe-cies of plasmid DNA. The molecular weights ofthe two larger plasmids were identical with thoseof strain 2.4.1. The molecular weight of the thirdplasmid of strain SLS I was significantly larger(34 x 106). It has been tentatively proposed (206)that the increased size of this plasmid derivesfrom some kind of gene duplication and/or rear-rangement. Such a modification could arise byintegration of an insertion sequence(s) with con-comitant loss of gene function (154, 230). Thisincreased size of a plasmid species is not a gen-eral property of photosynthetically incompetent(Pho-) strains of R. sphaeroides, since the plas-mid complement of another Pho- strain, ob-tained after nitrosoguanidine mutagenesis, wasidentical with that of strain 2.4.1 (206).Plasmid DNA has been detected in other pho-

tosynthetic bacteria, including R. capsulata (Huand Marrs, private communication; V. A. Saun-ders, unpublished data) and Chromatium D(235). R. capsulata apparently contains speciesof CCC DNA of 70 x 106 and 100 x 106 daltons,as estimated from sucrose gradient sedimenta-tion analysis (Hu and Marrs, personal commu-nication).Of the cyanobacteria, A. nidulans has been

reported to contain plasmid DNA of about 28X 106 to 33 x 106 daltons, which is present as atleast seven to eight copies per genome equiva-lent (Heaton and Frampton, Abstr. Annu. Meet.Am. Soc. Microbiol. 1976, I117, p. 131; E. W.Frampton, private communication). Analyses ofthe plasmid DNA of Agmenellum quadrupli-catum by electron microscopy and agarose gelelectrophoresis have revealed a number of dis-crete classes of DNA circles, ranging in molecu-lar weight between 3 x 106 and 65 x 106 to 80x 106 (193). The CCC DNA in this organism wasequivalent to about 5% ofthe total cellular DNA.The presence of multiple classes of CCC DNAwithin a procaryotic cell is not without precedent(see, for example, reference 149). It has yet to be

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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 ubiquitouswithin the photosynthetic procaryotes.

Functions EvaluatedThe nature of the information encoded by

plasmid DNA in photosynthetic procaryotes re-mains largely a matter for conjecture. By anal-ogy with the plastid DNA of eucaryotic plantcells, it has been suggested that the plasmidDNA may have a role in specifying the photo-synthetic apparatus (75). However, naturally oc-curring plasmids are generally dispensable inprocaryotes (160, 162). Thus, essential biologicalfunctions, presumably including photosyntheticactivity in this case, are unlikely to be plasmiddetermined. Furthermore, no transcriptionalspecificity between the RNA from aerobically orphotosynthetically grown cells has been dem-onstrated in R. sphaeroides and R. rubrum byusing both chromosomal and extrachromosomalDNA as DNA-RNA hybridization probes (24,77, 271, 274). Irrespective of the gross structuraldifferences and the variation in enzyme patterns(118) associated with growth of these organismsin different atmospheric milieus, no qualitativedifferences are obvious between the RNA spe-cies of aerobic and photosynthetic cells (24, 64,271, 274). However, there does appear to besome difference in the stability of an RNA com-ponent under aerobic and anaerobic conditions

(270). These observations led to the idea that atranslational mode of regulation of protein syn-thesis may exist for these bacteria (24, 270, 271,274).

Possible genetic functions for the plasmidDNA of R. sphaeroides have been evaluated bySaunders et al. (206). Part of the plasmid DNAcomplement may be composed of temperatephage. Indeed, several temperate bacteriophageshave been isolated from strains of R. sphae-roides (153), though no infective phage particleshave been detected in cultures of strain 2.4.1.Recently, putative viral R plasmids have beenreported for a number of R. sphaeroides isolates(176). These strains were resistant to penicillin,by virtue of a diffusible penicillinase, and carriedbetween one and three prophages. Bacterio-phages released by such strains were activeagainst derivatives of these strains which hadbeen "cured" of prophage. Furthermore, resist-ance to penicillin could be transferred at highfrequency to susceptible recipients. However,generalized transduction mediated by thesephages could not be demonstrated. The penicil-linase-producing strain R. sphaeroides RS601released the bacteriophage designated R06Pspontaneously. The DNA extracted from bac-teriophage R46P was circular DNA of 33 (+2)x 106 daltons (176), a value close to that of thesmallest plasmid of R. sphaeroides strain 2.4.1

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GENETICS OF RHODOSPIRILLACEAE 369

(cf. reference 206). It has been suggested (176)that a gene for penicillinase production is carriedas part of the wild-type bacteriophage genomeand that this genome exists as an extrachromo-somal element in these lysogenic strains. Thecarriage of transposable resistance genes by bac-teriophages has been demonstrated in the labo-ratory in the Enterobacteriaceae (15, 76). How-ever, this appears to be the first report of abacteriophage isolated from the wild which ap-parently carries antibiotic resistance genes.An isolate of R. capsulata, strain SP108, sim-

ilarly produces a diffusible penicillinase whichconfers considerable resistance to penicillin onthis strain (267). This contrasts with the extremesusceptibility of classical laboratory strains of R.capsulata to this antibiotic. Indeed, the greatsusceptibility of many strains of R. capsulata topenicillin G is a feature which distinguishes thisspecies from closely related members ofthe Rho-dospirillaceae (267). The penicillin-inactivatingenzyme of strain SP108 is apparently an induc-ible f?-lactamase, with a marked preference forbenzylpenicillin as a substrate (V. A. Saunders,manuscript in preparation). Furthermore, resist-ance to penicillin in strain SP108 is lost at arelatively high frequency (267; unpublisheddata), which would be consistent with a plasmid-specified character (160).

It is perhaps noteworthy at this juncture thatphotosynthetic bacteria are sometimes isolatedfrom stagnant ponds which have been contami-nated with farm effluents (see, for example, 1,142, 153, 267). This may provide localized con-centrations of antibiotics derived from, for ex-ample, animal feed and a reservoir of bacteriacapable of acting as donors of antibiotic resist-ance genes.

Penicillinase (f8-lactamase) production hasalso recently been reported for certain cyano-bacteria, in particular Anabaena sp. (strain7120) and Coccochloris elabens (strain 7003)(114). Penicillin did not appear to induce peni-cillinase production in these organisms. Further-more, the enzymes from these strains were moreactive on penicillins than on cephalosporins,thereby resembling the "type II' enzymes ofgram-negative bacteria (190).The production of penicillinases by photosyn-

thetic procaryotes poses fundamental questionsas to the nature and origin of the penicillinasegene(s). For instance, do these enzymes corre-spond to those widely distributed amongst theenteric bacteria, Pseudomonas and Haemophi-lus (87, 202)? Of particular interest in this re-spect is the type IIIa (TEM) ,8-lactamase deter-mined by transposon A (86). Transposon A iscapable of translocation from one replicon toanother and thus may be a significant contribu-

tor to the prevalence of antibiotic resistanceamongst bacteria (33, 200). It is likely that somedisparity in genetic composition will exist be-tween common laboratory strains isolated some20 or more years ago and those recently ob-tained. Increased levels of pollutants, includingantibiotics and heavy-metal ions, have undoubt-edly had dramatic effects on the genetic consti-tution of bacterial populations in general (see,for example, references 130, 186, 189). Thus,photosynthetic procaryotes will, in all certainty,acquire resistance genes to protect themselvesagainst such pollutants. In turn, this should con-veniently provide naturally marked derivativesof these organisms for use in genetic experi-ments.

BACTERIOPHAGE AND BACTERIOCINS

Isolation and Characterization ofBacteriophage and Cyanophage

The value of transduction in the provision offine-structure genetic maps and the constructionof specific mutant strains of bacteria is indisput-able (see, for example, references 12, 94, 199, 231,243, 246). The quest for corresponding phage-mediated gene transfer systems within the pho-tosynthetic procaryotes has promoted studies onthe virology of these organisms.The first report of a virulent phage specific for

a member of the Rhodospirillaceae describedphage Rpl of R. palustris (66). Both virulentand temperate phages specific for R. sphae-roides (1, 153) and R. capsulata (208, 263) havesubsequently been isolated and characterized.Their properties have recently been documentedin detail (143). In addition, temperature-sensi-tive mutants of the phage RC1 of R. capsulatahave been obtained (261). Thus far, however, nobona fide transduction has been reported involv-ing any of them. More promising are the resultswith certain phages of R. sphaeroides recentlyisolated by Kaplan and colleagues (S. Kaplan,private communication). Phage-mediated trans-fers of antibiotic resistance and nutritionalmarkers have been achieved between strains ofR. sphaeroides, albeit at fairly low transfer fre-quencies. Although these results are prelimi-nary, it is conceivable that with further refine-ments such a transducing system will prove suit-able for exploring the genome of R. sphaeroides.

First reports of viruses attacking and lysingspecies of the cyanobacteria were those of Saf-ferman and Morris (196). Several viruses (cyano-phages) have since been characterized, and thereis considerable documentation of their morphol-ogy, physiology, and ecology (for example, 172,195, 212, 273). However, their role as mediatorsof genetic exchange remains unproven. Temper-

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ature-sensitive mutants of the cyanophageLPP2-SP1, which lysogenizes Plectonema bor-yanum, have been isolated, and a linkage mapof the phage has been constructed based onrecombination between mutants in two-pointcrosses (191).

In addition to genetic considerations, host-bacteriophage interactions necessarily relate tohost cell physiology and, in turn, provide infor-mation on fundamental aspects of bacteriophagereplication and assembly (2, 34, 231). In thisregard, photosynthetic procaryotes undoubtedlyoffer distinct advantages over other procaryotes,primarily because the energy status of the hostcell can be conveniently manipulated merely byadjusting such parameters as light intensity.Schmidt et al. (208), investigating the bioener-getics of bacteriophage RC1 replication in R.capsulata, concluded that the energy require-ment for the replication of this phage is morecritical than that for uninfected host cell growth.In photosynthetically grown host cells, phageRC1 replication could be supported either byphotophosphorylation or oxidative phosphoryl-ation. However, in aerobically grown host cells,phage multiplication was supported by oxidativephosphorylation; the anaerobic photophosphor-ylation capacity of such cells would not suffice.Clearly, in aerobically grown cells developmentof the photosynthetic pigment system is drasti-cally suppressed (118). Therefore, such cells areseverely limited in photosynthetic energy con-version capacity. The required photophosphor-ylation capacity for phage development can onlybe achieved in aerobically grown cells whichcontain a sufficient quantity of bacteriochloro-phyll (0.6 ug/mg of dry weight) before phageinfection. Once phage has infected aerobic cells,subsequent synthesis of the photophosphoryla-tion system is prevented when cells are incu-bated under anaerobic conditions in the light.Phage RC1 infection apparently interferes withsynthesis of both bacteriochlorophyll and pro-tein in R. capsulata. It is proposed (208) thatthe infecting phage is entirely dependent on thetemporal energy conversion activity of the hostand that a relatively high rate of adenosine 5'-triphosphate regeneration is required for properexpression of the viral genome.The characteristics of infection of R. sphae-

roides by phage RS1 indicate that some form ofphysiological specificity exists (1). Anaerobicallygrown cells of R. sphaeroides strain 2.4.1 areapparently less susceptible to such infectionthan are aerobically grown cells (the adsorptionrate constants of RS1 are 1.2 x 10-9 ml/min toaerobic cells and 0.58 x 10-9 ml/min to anaerobiccells). This could reflect differences betweenthese two cell types in cell surface properties

and/or the intracytoplasmic membrane systemsuch that the adsorption and/or penetrationprocess is hampered in anaerobically grown cells(1). Once effective penetration of such cells hasoccurred, the burst size is similar to that ob-served during aerobic infection (15 to 20 plaque-forming units per cell).Bacteriophage R,0-1 is a temperate phage spe-

cific for R. sphaeroides (153). It was isolatedfrom the prophage state by induction with mi-tomycin C. R. sphaeroides strain 2.4.1 is notsusceptible to infection by R4-1. However, mu-tant derivatives of the phage have been isolatediwhich can form plaques on strain 2.4.1. Interest-ingly, the original phage RO-1 is chloroform sus-ceptible, whereas the mutant is chloroform re-sistant. The growth pattern of the phage is sim-ilar whether it begins its life cycle by inductionor infection. Furthermore, R4-1 forms plaqueswith equal efficiency on susceptible host cellswhether grown aerobically in darkness or anaer-obically in the light.The requirements for optimal replication of

phage particles appear to vary. For example, inboth R. capsulata (208) and the cyanobacteriumNostoc muscorum W4), optimal phage replicationnecessitates illumination throughout the latentperiod, whereas reproduction of cyanophageLPPI-G in P. boryanum requires illuminationsolely through the eclipse period (171). Thesedifferent requirements may reflect intrinsic dif-ferences in metabolism of the particular hostspecies.

BacteriocinogenyRecent surveys (82, 263) have revealed that

representatives of the Rhodospirillaceae pro-duce bacteriocins. R. sphaeroides and R. pal-ustris exhibit few intraspecies-specific inhibitoryinteractions. Greatest inhibitory activity, bothinterspecies and intraspecies specific, was ex-hibited by strains of R. capsulata. In addition,it is noteworthy that purple nonsulfur bacteriaproduce antimicrobial substances which are notakin to bacteriocins but are metabolites extract-able with organic solvents (104). Such antibioticeffects produced by R. sphaeroides (strain 1c7)are restricted to gram-positive bacteria, for ex-ample, Bacillus subtilis, whereas those pro-duced by R. capsulata (strain FC101) appear tobe unspecific. The precise natures of these anti-biotic substances require elucidation. No phe-nomenon analogous to that of bacteriocinogenyhas been reported in the cyanobacteria.

Bacteriocins are in themselves of interest fromstructural, functional, and evolutionary stand-points (85). Furthermore, their production isoften determined by transmissible plasmids (85).Therefore, the possibility exists that if compa-

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rable plasmids reside in members of the Rho-dospirillaceae, they could be exploited as vehi-cles of genetic exchange. However, the locationof the genetic determinants for bacteriocinogenyin these organisms remains obscure, and no as-sociated gene transfer has so far been reported.

"GENE TRANSFER AGENT" OFRHODOPSEUDOMONAS CAPSULATA

Discovery and PropertiesThe first report of a genetic exchange system

for a photosynthetic bacterium was that ofMarrs (142) for R. capsulata. Various isolates ofR. capsulata were screened for recombinationof antibiotic resistance markers. Genetic ex-change appeared to be mediated by a ribonucle-ase- and deoxyribonuclease-resistant vector pro-duced specifically by strains of R. capsulata andthereafter designated the "gene transfer agent"(GTA) (142). Different isolates vary in theirability to donate and receive the GTA (263).Furthermore, strains receiving genetic informa-tion via the GTA do not themselves becomeGTA producers (143). Genetic transfer mediatedby the GTA is limited to R. capsulata and doesnot extend to other members of the Rhodospi-rillaceae (263). Moreover, no comparable ge-netic exchange system has, to date, been discov-ered for other purple nonsulfur bacteria (263;unpublished data). Morphologically, the GTAparticle resembles a small bacterial virus, withan icosahedral head, short spikes, and a tail(143). The nucleic acid of the GTA is lineardouble-stranded DNA of 3.6 x 106 daltons (143,221), and the ultraviolet inactivation spectrumof the GTA is similar to that of bacterial viruses(222). However, the physical size of the GTAparticle (70S [142]) is much smaller than that ofany known transducing bacteriophage. Further-more, no plaque-forming activity appears to beassociated with the system, and there is no ob-vious correlation between the capacity of strainsof R. capsulata to produce GTAs and theirsusceptibility to known bacteriophages (263).Kinetics ofGTA release by a donor culture differfrom those normally associated with phage pro-duction (cf. references 97, 144). GTAs are typi-cally released in one or two abrupt waves to-wards the end ofthe exponential phase ofgrowth(222). Whether cell lysis always accompaniesthis process has yet to be fully ascertained.The gene transfer process in R. capsulata

seemingly resembles generalized transduction.All regions of the bacterial genome thus farexamined can be transferred, and transfer fre-quencies comparable to those for generalizedtransducing systems (4 x 1O-4 "transferants" perrecipient) can be achieved (222). Moreover, the

transferred genetic markers appear to be stablyinherited. Recombination is apparently accom-panied by displacement of the correspondingresident marker (143). Analyses of the kineticsof renaturation (Cot analysis) of the DNA con-tained in the GTA (Hu and Marrs, manuscriptin preparation) indicate that more than 95% ofthis DNA is from the bacterial genome. Se-quences complementary to the chromosomaland plasmid DNA of R. capsulata are found inthe GTA, and it appears that all portions of thegenome of R. capsulata are equally representedin the DNA of a population of GTA particles.The precise nature ofthis gene transfer system

remains enigmatic. Marrs and co-workers spec-ulate that it may represent a "prephage" system(143, 222), in which case it is envisaged that aGTA-like system evolved in response to theselective advantage which the capacity for ge-netic exchange might confer. Thus, the GTAsystem could represent a precursor of the bac-terial virus, rather than a derivative of a preex-isting phage. Alternatively, the GTA may be adefective or cryptic phage capable of generalizedtransduction (222). This permits an equallyplausible explanation to be inferred from theobservations that the GTA has a limited abilityto transfer 3 x 106 to 4 x 106 daltons of DNA(221) (presumably equivalent to approximatelyfive to seven genes, if transcription of the genesis nonoverlapping). Phage particles of similarcomplexity to, albeit of larger size than, the GTA(for example, those of the T-even group) containa genome of about 108 daltons (265). If, as seemslikely, the genome size of the GTA itself were toexceed five to seven gene equivalents, then thehead of the GTA would be unable to accommo-date all the genetic information necessary tospecify a mature GTA particle. Hence, GTAparticles would, at best, carry only fragments oftheir own genetic complement. Accordingly,such particles would be nonlytic and lack overtviral activity. Furthermore, recipients of geneticmaterial via GTAs would never become GTAproducers unless, by chance, through multipleinfections they simultaneously acquired all thegenes necessary to specify GTA production.Even if the GTA particle could carry its genomein its entirety, failure to transmit GTA produc-tion to recipient cells could be explained if thegenetic determinants for the GTA were scat-tered at various loci on the replicons resident inR. capsulata. Consequently, chances of incor-poration of a complete GTA genome into a singleparticle during the encapsidation process wouldbe drastically reduced. Possibly, this gene trans-fer system could be dissected by isolating fromGTA-producing parent strains mutants im-paired in GTA production. Such "GTA defec-

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tive" strains could be used as recipients in ge-netic crosses with other GTA producers as do-nors. Restoration of the capacity to produce theGTA in transferants may allow the extent andmap positions of the GTA determinants to beestablished. Certain mutants of R. capsulatahave recently been isolated that are "overpro-ducers" of the GTA (143). These strains mayfurther resolve the nature of this genetic ex-change system and, in particular, enable an es-timate of how many genes are involved in spec-ifying the GTA and their location in R. capsu-lata. However, there still remains the problemof screening for a characteristic for which thereis no direct selection procedure and for which abiological assay is the sole means of detection.The GTA system of R. capsulata has been

used in manipulating genes specifying the pho-topigment system (54, 143, 263, 276) and thenitrogen fixation machinery (264). Moreover,specific mutant strains have been constructed,and the lesions characterizing others have beeninvestigated with this genetic vehicle.

Transfer of genes for nitrogen fixation, via theGTA, to certain Nif- mutants of R. capsulataresults in acquisition by recipients of the dualability to fix nitrogen and produce hydrogen(264). These findings support the proposal (170)that nitrogenase and hydrogen-evolving hydro-genase activities of purple nonsulfur bacteria arecatalyzed by the same enzyme complex. Fur-thermore, this hydrogenase activity apparentlydiffers from that associated with the utilizationof hydrogen as an electron donor for photoau-totrophic growth (264).

Restoration of photosynthetic competence toPho- mutants of R. capsulata has been effectedwith the GTA (54, 263). Drews and co-workers(54) demonstrated concomitant restoration ofthe abilities to synthesize bacteriochlorophylland to form reaction center and light-harvestingproteins to R. capsulata mutants defective inthese abilities. Analyses of various transferantssuggest that reaction center proteins and light-harvesting complex 1 (128) form a structural unitin the intracytoplasmic membranes of R. cap-sulata (54). It has yet to be ascertained whetherthe genes specifying synthesis of these specificproteins of the photosynthetic apparatus and ofbacteriochlorophyll are closely aligned on thegenome of R. capsulata.Mapping Genes for Bacteriochlorophyll

and Carotenoid BiosynthesisThe GTA has been successfully exploited in

conjunction with a series of mutants of R. cap-sulata in the construction of a map for genesdetermining bacteriochlorophyll and carotenoidproduction (276). One-, two-, and three-point

and ratio test crosses were performed betweenvarious strains, and a new mapping function wasderived to convert cotransfer data into map dis-tances. Lacking cis-trans complementation datafor this organism, it was assumed that a clusterof mutations giving the same phenotype repre-sented a gene. Accordingly, clusters ofmutationsdelineating seven genes, five affecting carotenoidbiosynthesis and two affecting bacteriochloro-phyll biosynthesis, have been arranged in onelinkage group (Fig. 5). Mutations in either thecrtB or crtE gene can give rise to the blue-greenphenotype, whereas mutations in the crtD orcrtC gene cause the green phenotype and thosein the crtA gene cause a yellow phenotype. Theloci bchA and bchB specify products necessaryfor reactions in bacteriochlorophyll biosynthesis.Linkage between these genes has possible impli-cations for their coordinate expression at thetranscriptional level. An interesting observationfrom the mapping studies was the apparentlyobligatory requirement for two specific muta-tions to obtain viable blue-green strains of R.capsulata: one lesion results in loss of coloredcarotenoids; the other (as yet of undefined maplocation) results in an alteration of the absorp-tion spectrum of bacteriochlorophyll (54, 137,143, 276). It has been suggested (137) that themutation(s) responsible for alteration in the ab-sorption spectrum of bacteriochlorophyll in factblocks formation of light-harvesting bacterio-chlorophyll complex II (128). If these mutationsperforce accompany each other in blue-greenstrains, this could help to clarify the role(s) ofcarotenoids in photosynthetic bacteria.

TRANSFORMATIONSo far, there has been no unequivocal dem-

onstration of genetic transformation in the pho-tosynthetic bacteria. By contrast, there are sev-eral reports of gene transfer by transformationin the cyanobacteria (see reference 43). Trans-formation in A. nidulans was first reported byShestakov and Khyen (213). The process wasmediated by chemically extracted DNA and wasdeoxyribonuclease sensitive. Subsequently,Herdman and Carr (91) described a transfor-mation system for A. nidulans effected by anextracellular DNA:RNA complex. More exten-sive genetic linkage was observed in this processthan in that mediated by chemically pure DNA.However, a mutagenic phenomenon appeared tobe associated with the transformation process(whether with extracellular or chemically ex-tracted donor DNA) (88, 89) which presumablyinterfered with accurate determination of link-age values in genetic mapping studies with A.nidulans. Nevertheless, it has been possible toexploit the mutagenic process per se in aligning

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62A71 e#/B16 ed"C112 cr/D150 eiE9 A4AA92FIG. 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 ofeach gene. Distances obtained by subtraction are given in parentheses.

certain genetic markers, and recombinationmaps of A. nidulans have been constructed (43,88, 89).

In addition to the intraspecies-specific trans-formation systems for A. nidulans (88, 89, 92,148, 169, 213) and Aphanocapsa 6714 (11), anintergeneric transformation system has recentlybeen reported for cyanobacteria (46). Interge-neric transfer of antibiotic resistance markershas been demonstrated from A. nidulans toGloeocapsa alpicola and vice versa, in a processsensitive to both deoxyribonuclease and ribo-nuclease. Such genetic transfer across genericboundaries offers considerable scope for mobiliz-ing genes (for example, those specifying nitrogenfixation) within the cyanobacteria.

Investigators' inability to develop a genetictransformation system for photosynthetic bac-teria may be due to one or a number of contrib-utory factors. First, the possibility exists thatthe donor nucleic acid may be inactivated byextracellular nucleases. This explanation doesnot, however, apply to R. sphaeroides strain2.4.1, which apparently produces no extracellu-lar deoxyribonuclease (unpublished data). Alter-natively, the cell walls of purple nonsulfur bac-teria may never achieve a competent state fortaking up the DNA. Even if donor DNA couldtraverse the cell envelope, successful transfor-mation with linear DNA would, presumably, stilldepend on a functional recombination system.The success of GTA-mediated gene transfer inR. capsulata and of R plasmid-directed genetransfer in R. sphaeroides (see below) impliesthat these organisms are recombination profi-cient. However, it is noteworthy that transfer ofchromosomal markers by transformation in E.coli was initially hampered by the activities ofthe recombination exonuclease system (recB/

recC exonuclease V) (166), which degraded theincoming linear "transforming" DNA. In con-trast, linear DNA entering the cell by conjuga-tion or transduction in E. coli is apparentlyrefractory to such attack. It was only with sub-sequent mutational blocks in the recB/recC ex-onuclease, coupled with a suppressing mutationopening up a further minor recombination path-way, that effective transformation with chro-mosomal DNA was achieved in E. coli (166).Thus, the portal used in transformation mayresult in the genetic information being particu-larly vulnerable to nuclease attack. By analogy,whereas a recombination system is operative incertain members of the Rhodospirillaceae, itcould equally act as a specific barrier to trans-formation in these organisms. Only when agreater understanding of the general geneticsand, particularly, of recombination is availablein the Rhodospirillaceae can such problems beresolved. Clearly, therefore, a useful approach inthe development of a transformation system forthe purple nonsulfur bacteria may be to usesuitably marked plasmid (CCC) DNA as a probein deriving potential competence regimes. Thiscould circumvent any requirement for, or dam-age done by, recombination nucleases.

CONJUGATIONTransfer of genetic material by cell-to-cell

contact has thus far not been observed to beindigenous to members of the Rhodospirilla-ceae. The presence im certain members of thisgroup of plasmid DNA, which is of comparablesize to the F factor of E. coli, prompted specu-lation that such plasmids may specify sex factoractivity (75). However, genetic conjugation me-diated by native plasmid species has not beenobserved in R. sphaeroides (206). Of course, this

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may be merely a manifestation of intrinsicallylow transfer frequencies for chromosomalmarkers coupled with such phenomena as sur-face exclusion and incompatibility (201), if allstrains used in the genetic crosses contain ho-mologous sex factors.On the other hand, R. sphaeroides and R.

rubrum have been shown to act as recipients forthe resistance (R) plasmid R1822 derived fromPseudomonas aeruginosa. However, the plas-mid was unstable in these recipients in the ab-sence of the appropriate selection pressures(167). Subsequently, W. R. Sistrom (privatecommunication) has demonstrated transfer ofthe same plasmid to strains of Rhodopseudo-monas gelatinosa. However, cotransfer of chro-mosomal genes has not yet been observed.By contrast, current attempts to introduce

other sex factors and, notably, further plasmidsof the P incompatibility (IncP) group (20, 40,162) into these organisms are more encouraging.Kaplan (private communication) and co-workerscan demonstrate stable transfer of several Rplasmids from E. coli to R. sphaeroides andsubsequently between strains of R. sphaeroides.Concomitant intraspecies transfer of chromo-somal markers, for example, antibiotic resistanceand nutritional markers, has been achieved forR. sphaeroides.

Recently, Sistrom (217) has accomplished sta-ble transfer of the R plasmid R68.45 (84, 109)from P. aeruginosa (strain PA025 [R68.45]) tostrains of R. gelatinosa and R. sphaeroides. Thefrequency of transfer of the plasmid-determinedneomycin resistance was of the order of l0' to10`6 transconjugants per recipient cell (217).Subsequent transfer of neomycin resistanceamongst strains of R. sphaeroides occurred at ahigh frequency (about 10-2 transconjugants perdonor cell). Interestingly, strains of R. gelati-nosa receiving R68.45 manifest resistance toboth neomycin and carbenicillin. In contrast,strains of R. sphaeroides acquiring the plasmidare neomycin resistant but remain susceptibleto carbenicillin. Transfer of chromosomalmarkers (notably, antibiotic resistance determi-nants and restoration of prototrophy to specificauxotrophs) occurred at frequencies of around10-6 to 10-7 recombinants per recipient cell forR. sphaeroides. Apparently, transfer of R68.45itself or of chromosomal genes occurs only onsolid media (217; cf. reference 84). The prelimi-nary cotransfer data suggest that R68.45 will bea useful genetic tool in the construction of link-age maps of R. sphaeroides. Indeed, there isevery likelihood that the aforementioned plas-mids or relatives will ultimately provide conven-ient vehicles for manipulating genes withinmembers of the Rhodospirillaceae.

The precise mechanism of chromosome mo-bilization in these cases remains to be elucidated.By analogy with processes of conjugation in E.coli, gene transfer may involve some form ofcovalent association with the chromosome, as inthe formation of R-prime plasmids or Hfr do-nors. Alternatively, the acquired plasmid maydirect transfer of chromosomal genes by a mech-anism not unlike that postulated for mobiliza-tion of non-self-transmissible plasmids and thechromosome by sex factors in E. coli. This kindof activity, which is poorly understood, appar-ently does not involve covalent linkage betweenplasmid and chromosome (61, 147, 150).

Clearly, a useful objective would be the isola-tion ofstable Hfr donor strains ofphotosyntheticbacteria for the construction of large-scale ge-netic maps. The integration of plasmids into thechromosome to form stable Hfr strains is a rareoccurrence, the most notable exception beingthe F factor of E. coli (150). However, it may bepossible to obtain such strains by exploiting theability of self-transmissible plasmids to suppressdefects in initiation of chromosome replication.This process of integrative suppression (159) hasbeen used in E. coli to generate plasmid-me-diated Hfr-type strains (147, 151). By construct-ing mutants of the photosynthetic bacteriawhich are temperature sensitive for the initia-tion ofDNA replication and by using the capac-ity of transmissible plasmids to suppress thismutation at the restrictive temperature by in-sertion into the chromosome, Hfr-type strainsmay be produced. Moreover, it may be possibleto force the selection of Hfr-type strains me-diated by IncP plasmids, of which some arealready known to be capable of infecting mem-bers of the Rhodospirillaceae.

CONCLUDING REMARKSInterest in the genetics of photosynthetic pro-

caryotes has burgeoned considerably over thepast decade. The recent advances in geneticmanipulation, which now permit transfer andrecombination of genetic material within thisbiological group, justify great expectations forelucidation of the hitherto intransigent geneticsystems of these organisms. Indeed, the GTA ofR. capsulata represents a landmark in the mo-lecular biology of the photosynthetic bacteria.The applicability of the GTA system to fine-structure genetic mapping of R. capsulata isclearly demonstrable. However, the limitationsof this genetic vector emphasize an urgency forfurther genetic exchange systems, especially forthose capable of transferring longer stretches ofthe genome and for those capable of establishingstable partial diploids for performance of cis-trans complementation analyses. At this time,

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transduction and conjugation appear the morepromising areas for future developments. Theassignment of genetic determinants to plasmidsnative to photosynthetic procaryotes should fa-cilitate identification of their role, if any, ingenetic interplay. Recently, Reanney (186) hasexpounded the virtues of extrachromosomal ele-ments as executors of evolution in both procar-yotes and eucaryotes. He contends that extra-chromosomal DNA may have had a dominantrather than peripheral role in the processes ofdevelopment, adaptation, and speciation. Thevery presence of plasmids, phage, and the GTAwithin photosynthetic procaryotes affords scopefor such evolutionary mechanisms to have em-braced this biological group.An alternative approach to genetic mapping

in the Rhodospirillaceae, and one which doesnot rely directly on any gene transfer system,would be to study the change in mutation fre-quency of genes at replication. This techniquehas been successfully used in the construction oftemporal genetic maps of the cyanobacteriumA. nidulans (9,42,43,92). Such temporal geneticmaps ofA. nidulans show good correlation witha conventional genetic map derived from trans-formation studies (88).

It is conceivable that the sophisticated meth-odology used in engineering genes within theEnterobacteriaceae (see, for example, reference32) will ultimately be applicable to the photo-synthetic bacteria. Genes specifying photopig-ment production or the ability to fix nitrogen,for instance, could thereby be cloned eitherwithin the photosynthetic bacteria themselvesor in organisms of more fully defined geneticbackground, such as E. coli. Acquisition of thesegenes by diverse organisms may facilitate molec-ular studies on their expression. However, ap-propriate transformation regimes for the intro-duction of recombinant DNA into photosyn-thetic bacteria are wanting at present.

In essence, representatives of the Rhodospi-rillaceae provide attractive experimental sys-tems for the integration of biochemical, bio-physical, and genetic technology. Recent trendsin the molecular biology of photosynthetic bac-teria make it more likely that the full potentialof these organisms for ascertaining mechanismsunderlying development and function of thephotosynthetic apparatus will be realized. Aclearer understanding of energy-conserving sys-tems in general should thus ensue.

ACKNOWLEDGMENTSI thank J. R. Saunders (University of Liverpool,

Liverpool, U.K.) for much helpful discussion and con-structive criticism of this review, B. L. Marrs (St.Louis University, St. Louis, Mo.), W. R. Sistrom (Uni-versity of Oregon, Eugene), S. Kaplan (University of

Illinois, Urbana), E. W. Frampton (Northern IllinoisUniversity, De Kalb), J. D. Wall (Indiana University,Bloomington), and others for making data availableprior to publication and for discussion; and T. W.Goodwin (University of Liverpool) for providing re-search facilities within the Department of Biochemis-try during the preparation of this article.

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