Gene transfer mechanisms among members of the genus Rhodopseudomonas

Ann. Microbiol. (Inst. Pasteur) 1983, 134 B, 195-204

GENE

TRANSFER OF

THE

MECHANISMS

GENUS

AMONG

MEMBERS

RHODOPSEUDOMONAS

by J. M. Pemberton, S. Cooke and A. R. St. G. Bowen

Department o/Microbiology, University o/Queensland, St. Lucia (Australia) 4067

SUMMARY Recent studies on species of the genus Rhodopseudomonas, particularly R. capsulata and R. sphaeroides, have resulted in the development of a range of systems of genetic exchange without peer among the photosynthetic prokaryotes. In

R. eapsulala, systems of generalized transduction and R-prime formation have provided a detailed map of the arrangement of photosynthesis genes, while systems of conjugation and chromosome transfer in R. sphaeroides have provided a map of the location of genes involved in amino acid biosynthesis, antibiotic resistance and photosynthesis. A recent report of plasmid transformation in R. sphaeroides provides another important avenue for the analysis of genes such as those involved in photosynthesis and photochemical nitrogen fixation, through the application of DNA cloning technology. That plasmid transformation, generalized and specialized transduction, conjugation, chromosome transfer and R-prime formation do occur in Rhodopseudomonas indicates the rapid emergence of genetic and molecular biological techniques applicable to studies of these bacteria. KEY-WORDS: Rhodopseudomonas, Genetic transfer, Photosynthesis; Gene cloning

[,

--

INTRODUCTION.

Studies of photosynthesis, arguably the most important biological process on earth, rate highly in terms of basic research. The mechanism of photosynthesis is a complex one, and for this reason relatively simple photosynthetic organisms, such as members of the genus Rhodopseudomonas, seem a logical choice for a rapid and detailed analysis of this process. Work with photosynthetic bacteria such as R. sphaeroides has led to the accumulation of extensive data on the biochemical and biophysical aspects of bacterial photosynthesis [7, 29, 22]. Notwithstanding the success of biochemical and biophysical studies, until recently, few if any means of genetic analysis were available for photosynthetic bacteria. Ideally, the development of transformation and specialized and general transduetion, as well as conjugation and ehromosome transfer systems in species of the genus Rhodopseudomonus, would provide the basis for a successful genetic analysis of bacterial Manuscrit recu le 23 mars 1983.

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photosynthesis, photochemical nitrogen fixation and the photoevolution of hydrogen. Additional techniques employing transposable genetic elements and in vitro DNA cloning should provide an intimate insight into these biologically important processes at the molecular level, enabling a fuller understanding of the biochemical and biophysical data already obtained. As I hope this short review will show, many of the techniques which have proven so successful in the genetic analysis of Escherichia coli are available in the genus Rhodopseadomonas, particularly in the two species R. sphaeroides and R. capsulata. I [.

- TBANSFOBMATION.

Under appropriate conditions, a small number of bacterial species have the ability to take up DNA from solution. This process is known as transfornlation, and cells able to take up DNA are described as competent [27]. Physiological competence occurs at a particular point in the growth of a bacterial cell and is characteristic of such Gram-positive bacteria as Bacillus subtilis and Streptococcus pneumoniae. Gram-negative organisms such as Escherichia coli and Pseudomonas aeruginosa do not exhibit physiological competence; instead competence can be induced in these organisms by exposure to high levels of calcium chloride [4, 5]. Regardless of whether competence is physiological or is chemically induced, the process of transformation does provide a means of introducing chromosomal, plasmid and phage DNA into living bacterial cells; the entry of phage DNA into bacterial cells from solution is called transfection. Although transformation has proven particularly useful for mapping certain bacterial genomes [27], it has achieved its greatest usage as a means for transferring genetically engineered plasmids into bacterial cells [3]. Because they form such an integral part of gene cloning technology, transformation systems have been developed in a number of Gram-positive and Gramnegative species of bacteria; recently, this technique was extended to R. sphaeroides. The first indication that a member of the genus Rhodopseudomonas could be transformed came from the report of Tucker and Pemberton [33] that DNA extracted from the bacteriophage R006P could be re-introduced to R. sphaeroides with the aid of another temperate phage 1-1009. Since R006P encoded a }-lactamase, cells lysogenised with R006P became penicillin resistant; the uptake of R006P DNA from solution was followed by assaying for the acquisition of penicillin resistance. Since H006P exists as a plasmid in its prophage state, there is the possibility of using it or a mini-derivative as a cloning vector. The phenomenon of <~helped ,, transformation or transfection was originally reported by Kaiser and Hogness [12] using a wild-type lambda (X) bacteriophage to enhance the transformation of Xdg DNA into E. coll. This discovery of transfection in E. coli led to the development of a more general method of transformation which allowed for the uptake of both chromosomal and plasinid DNA from solution. The recent demonstration of plasmid transformation in 1{. sphaeroides by Fornari and Kaplan (personal communication) provides a major contribution to the development of molecular biological techniques for this group of organisms. Using the broad host range cloning vector pUIS1, constructed in vitro from plasmids RSFI010 and pSL25 (a pBB322 derivative), Fornari and Kaplan were able to demonstrate transformation in R. sphaeroides. Their technique of washing cells with 500 mM hydroxymethyl aminomethane (Tris) induced competence for DNA uptake. Transformation frequencies as high as 1 • 10 ~ (transformants per viable cell) were achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaC1, and 20 % polyethylene glycol 6000. Maximal frequencies of transformation were obtained after plasmid genes were allowed to express for 6.5 h before plating on appropriate selective media. The availability of a transformation procedure for plasmid DNA into B. sphaeroides provides a means of direct selection of genes cloned into suitable cloning vectors.

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IN BHODOPSEUDOMONAS

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III. - - TnANSDUCTION.

Phages. A variety of phages, both temperate and virulent, which plaque on strains of Rhodopseudomonas, particularly R. sphaeroides and R. capsulata, were isolated by a number of workers [1, 14, 17, 34]. The first such phage, RS1, was virulent and formed plaques on R. sphaeroides strain 2.4.1 [1]. A second phage, the temperate R ~ I , was shown to plaque on a number of strains of R. sphaeroides [17]. A more recent study by Tucker and Pemberton [31] revealed t h a t all seventeen natural isolates of R. sphaeroides isolated from stagnant ponds in and around Brisbane, Australia, possessed at least one and anything up to three prophages. All three morphological phage types were capable of plaque formation on the wild-type isolate RS901. The most commonly encountered morphological type of bacteriophage isolated possessed a 50-60 nm icosahedral head and a 250 nm long flexuous tail with 2-4 tail fibres; RqO6P fell into this class [18]. The second class possessed a thick 20 nm wide tail with a distinct base plate and a 65 nm hexagonal head. The third class appeared to be a tailless phage with a 65 nm hexagonal head, similar in morphology to RS1. Although none of the phages appeared to be capable of general transduction, it is encouraging to note t h a t temperate phages are widespread among strains of R. sphaeroides. The failure to detect a suitable transducing phage may reflect the lack of an exhaustive isolation program, incorrect conditions for growing such phages or the lack of restrictionless (Res-) mutants of R. sphaeroides which would enhance our ability to screen for such phages. By way of contrast, all 95 phages isolated by Wall el al. [34] and active against strains of R. eapsulata were virulent. On the basis of their host range, the 95 independent isolates were allocated to sixteen groups. Additional attempts to isolate temperate phages from 33 strains of R. capsulala proved unsuccessful, perhaps because of the lack of a suitable indicator strain. Ideally, such a strain would need to lack restriction enzymes (Res-) and be free of prophages.

Specialized lransduction. Apart from their prophages, 16 of the 17 strains of R. sphaeroides isolated by Tucker and Pemberton [31], were resistant to penicillin G. In all these strains, the penicillin resistance phenotype could be cured by growth in the presence of mitomycin C [31]. In 7 of these 16 strains, the ~-lactamase gene was shown to be encoded by a phage; one of these ~-lactamase-encoding phages, R(I)6P, was chosen for further study. In the prophage state, Rqb6P appears to occur as a plasmid and in this respect has much in common with the ~-lactamase encoding phages, P7, which lysogenises certain strains of E. eoli [24]. Phage RO6P is an unusual biological entity, combining the genetic and biophysical properties of a virus and a drug resistance plasmid, possessing as it does a supercoiled circular genome both in the viral particle and as a plasmid in the prophage state. Although the remaining penicillin-resistant strains of R. sphaeroides (nine isolates in all) could be cured of their resistance phenotype and produced a phage of similar morphology to Rqb6P, none of the ~-lactamase genes appeared to be associated with a phage. The presumptive penicillinase plasmids were not transferable to cured derivatives of the same strain, nor could they be mobilised by a range of P and W group plasmids. In view of the widespread occurrence of plasmids in strains of R. sphaeroides, it is likely t h a t at least some will encode the ~-laetamases detected in these strains.

General lransduction. Although temperate phages appear to be widespread among strains of R. sphaeroides, few if any such phages have been detected in R. capsulala. Nevertheless, a form of general transduction does occur in R. capsulata [13, 14, 15, 25, 26] which is mediated by a non-infective, small phage-like particle described as a gene transfer

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agent or GTA. GTA particles resemble small bacterial phages possessing a 30-nm icosahedral head and a short tail of variable length. These particles package random segments of genomic DNA some 2-3 kilobases in size, sufficient DNA to encode for 5-6 non-overlapping genes. The GTA system of genetic exchange appears to be widespread among strains of R. capsulata, and this system was used to produce the first fine structure map of the photosynthetic region of the R. capsulata genome [35, 36]. GTA provide an ideal system for fine structure mapping of DNA in R. capsulata, and it is disappointing to note t h a t none of the strains of R. sphaeroides so far examined produces and exchanges such particles [34]. IV.

--

CONJUGATION.

Naturally-occurring plasmids. Most strains of both R. spbaeroides and R. capsulala possess one or more large plasmids [8, 11, 20, 21]. For both R. sphaeroides and R. capsulata, the most common plasmids have sizes of 90 and 150 kilobases [11, 21]. Apart from the ~-lactamase gene encoded by the Rq~6P plasmid prophage, no other characteristics have been ascribed to these plasmids. There has been a temptation to suggest that genes involved in photosynthesis and related metabolic activities occur on these plasmids; as yet no positive evidence exists to support this view.

P-1 and W incompatibility group plasmids. Because of their broad host range, multiple antibiotic resistance plasmids of the incompatibility (Inc) groups P-l, W, N and X were likely candidates for setting up conjugation systems in species of the genus Rhodopseudomonas [20]. In 1977, Sistrom [23] reported the transfer of the Inc P-1 plasmid R68.45 into strains of R. sphaeroides. Since t h a t time, a variety of Inc P, W and Q plasmids have been transferred both into R. sphaeroides and R. capsulata [16, 20, 31]. Such plasmids transfer freely between various strains of R. sphaeroides and R. capsulata, and from there into a range of other organisms such as E. coli, Alcaligenes eutrophus, A. paradoxus, Pseudomonas putida, P. aeruginosa, Agrobaclerium tume/aciens and Cellvibrio sp., to name but a few examples (Pemberton, unpublished data). Such plasmids are relatively stable in such strains of Rhodopseudomonas, and their ready transfer by conjugation has provided the basis for the development of systems of chromosome transfer, for the introduction of transposable genetic elements and for the generation of R prime elements. V.

--

CHROMOSOME

TRANSFER.

Plasmids R68.45 and RP1 : :Tn501. The plasmid R68.45, isolated by Haas and Holloway [9] has been shown to promote chromosome transfer in a wide range of bacterial species and genera [10]. Transfer of R68.45 into R. sphaeroides by Sistrom [23] enabled the detection of chromosome transfer; although a small number of auxotrophic mutants were used in this study, recovery of recombinants occurred at a sufficiently high level to suggest t h a t this plasmid might be useful in mapping the genome of this organism. Following this initial success with R68.45, Pemberton and Bowen [19] reported high levels of chromosome transfer in R. sphaeroides promoted by the lncP-1 plasmid RP1 carrying the mercury transposon Tn501. Using multiply-marked recipient strains carrying a variety of auxotrophic, antibiotic resistance and photopigment mutations, a map of the genome of R. spbaeroides consisting of two linkage groups was compiled ([19] and fig. 1).

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199

-str-14 -gly-1

-leu-3

-arg-2 - met- 5

phe- 1 aro-2 phe-2

aro - 1 phe-4 arg- 4 -bcr-7 -bcr-t9

crt - 6 -trp-5 - his-3

-crt-18, crt-29

crt -8 crt -5-

-crt-1

-bch-7 -bch-12

FI6. 1. -- Linkage maps of the genome of It. sphaeroides from Pemberlon and Bowen [19].

The interesting feature of the linkage map is that all of the carotenoid and bacteriochlorophyll genes so far examined map together in a single region of the genome and are linked to genes involved in biosynthesis of the amino acids phenylalanine, arginine, leucine and glycine, and in resistance to streptomycin. Since the genes encoding photopigment biosynthesis were located in a single region of the genome easily accessible to conjugal mapping techniques, further mapping was undertaken using over 200 independently-isolated mutants in carotenoid and bacteriochlorophyll biosynthesis. The blue-green mutations fell into two groups on the basis of their linkage to phe-2; crt-B showed 50 % linkage while crt-E showed 33 % linkage. Similarly, the green mutations fell into two groups; crt-C was 40 % and crt-D was 36 % linked to phe-2. Two additional mutations, yellow showing 48 % linkage and brown-tan showing 35 % linkage to phe-2 were located. Tile bacteriochlorophyll mutations mapped in a tightly-linked group which showed 12-15 % linkage to phe-2. The allocation of cistronic designations crtB, crtC, etc. was purely arbitrary, and followed the convention for allocating these designations to similar groups of mutants in R. capsulata [35]. Although conjugational mapping involving such closely linked genes is much less accurate than the same analysis performed by general transduction, the arrangement of carotenoid and bacterioehlorophyll genes in R. sphaeroides (crlB, crtA, crtC, crlD, crtF, crtE, bchAB) is similar to that in R. capsulala (crtA, crlB, crtC, CrtD, crtE, bchAB) [35]. The recent report [15] of the isolation of an R-prime element carrying

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the carotenoid and bacteriochlorophyll biosynthesis genes might provide the opportunity to test the relationship between the photosynthetic operons carried by R. capsulata and R. sphaeroides.

A possible mechanism o[ chromosome mobilisalion by RP1 : :Tn501. Insertion of the mercury resistance transposon Tn501 into the broad host range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in R. sphaeroides [19]. Compared with the wild type RP1, which produced less than 10 8 recombinants per donor cell, RPI::Tn501 produced between 10 -3 and 10 7 recombinants per donor cell, depending upon the marker selected. The question arises as to the mechanism by which Tn501 confers chromosome mobilizing ability on RP1 while other transposons such as Tnl, Tn5, Tn402 and Tn7 have no parallel effect. One interesting possibility presents itself to explain this phenomenon. The transposon Tn501 undergoes transposition from one replicon to another at high frequency [30]. This transposon belongs to a group of transposons including Tn3, Tn1771, Tn1721 and Tn21, which form co-integrates as intermediates in the transposition process. For most of these transposons, the co-integrates are resolved with great efficiency, resulting in the separation of the donor and recipient replicons each now containing a copy of the transposon. The ability to resolve such co-integrates is vested in a resolvase (lnpB) gene carried by the transposon. As De I.a Cruz and Grinsted [6] have pointed out, the resolvase activity of Tn501 is very weak compared with other transposons in this group, indicating that once co-integrates have formed during the process of transposition, the two replicons involved remain stably attached to one another. In the case of BPI::Tn501, the plasmid becomes physically attached to the ft. sphaeroides chromosome during transposition and remains attached to it because of the low activity of the resolvase; when this RPI::Tn501 transfers to another cell, it takes along with it the attached R. sphaeroides chromosome. If this is the mechanism by which Tn50l enhances chromosome transfer in R. sphaeroides, then alterations in the resolvase activity of this and other transposons, which have co-integrate formation as part of the transposition event, should provide not only a range of transposon-plasmid combinations with chromosome mobilising abilities, but also transposon-plasmid combinations which promote chromosome transfer from a variety of origins on the It. sphaeroides chromosome. VI. --- GENE CLONING, Although E. coli K12 has proven an ideal host for the cloning, amplification and structural analysis of DNA from a wide range of prokaryotic and eukaryotic organisms, it should be remembered that many interesting properties of bacteria such as hydrogen oxidation, symbiotic nitrogen fixation and photosynthesis are not expressed in this bacterium. Differences in gene expression of ~ foreign 7) gene DNA in E. coli have been attributed to differences in ribosome binding sites, in DNA-dependent RNA polymerase binding sites and in the stability of ~ foreign )) proteins and the lack of complementary biochemical pathways in E. coli compared with the original host from which the cloned DNA was derived. Bearing these difficulties in mind, it is difficult to envision such a complex process as photosynthesis, encoded as it is by a large number of genes, as being successfully expressed in E. coll. For this reason it is necessary to set up systems of gene cloning in the photosynthetic organisms themselves. At least two strategies seem feasible, given the present state of sophistication of photosynthetic prokaryotic genetics. On the one hand, systems of gene cloning can be set up in the bacterium of interest, e. g. R. sphaeroides or R. capsulata. Such an approach requires the development of an efficient system of transformation for plasmid DNA into the bac-

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terial strain possessing the characteristic under study; this has already been achieved for It. sphaeroides, with the likelihood that the method can be extended to It. capsulata and other photosynthetic prokaryotes. As the photosynthetic genes to be cloned are chromosomally located in It. sphaeroides, recombination deficient (Rec-) mutants will be required to prevent recombination between the cloned DNA and the homologous sequence on the main chromosome. In It. sphaeroides, the high level of recovery of His+ recombinants should provide an adequate screening method for such mutants. Finally, it is desirable not only to develop a host strain free of endogenous plasmids but also to construct a range of suitable cloning vectors. Such a system has the advantage of allowing direct selection of a desired phenotypic characteristic in its original host. On the other hand, photosynthetic genes can be cloned in E. coli, followed by transfer of the cloned DNA back into their original host to detect expression. Such experiments make use of small, broad host-range cloning vectors and the well-defined genetic systems of this, the most intensively studied of prokaryotes.

Broad host range cloning vectors. Although there are a wide variety of cloning vectors available for E. coli, few have the broad host range required if the cloned DNA are to be transferred back into their original bacterial hosts to allow for expression. To date, the most useful cloning vectors developed for use in a range of Gram-negative bacteria are derivatives of the IncQ plasmids, RSF1010, R300B and Rl162 which are 8-9 kilobases in length and specify resistance to streptomycin and sulphonamides (for review, see [2]). Such cloning vectors have a number of advantages for the cloning of photosynthetic genes. First, these small, non-conjugative plasmids can be mobilised with high efficiency by the IncP-1 group of conjugative plasmids, e. g. RP4, into strains of R. sphaeroides. Second, these plasmids have high copy nmnbers, allowing for the ready isolation of plasmid DNA. Third, they can be transformed in R. sphaeroides. In addition, they provide an ideal tool for the detection of transformation in bacterial species, particularly the photosynthetic bacteria, which have had little genetic characterization. Finally, being broad host range plasmids, they have evolved strategies for the maintenance, replication and expression of their DNA in a variety of genetic backgrounds, and these properties can be exploited to allow for the maintenance, replication and expression of any cloned DNA that are added to them. Cloning o/~-lactamase genes. One of the most significant features of It. sphaeroides strains isolated in our laboratory was that almost all were resistant to penicillin G. The parental strain of It. sphaeroides, RS630, used to generate multiple auxotrophie and antibioticresistant strains for genetic studies, has been cured of the Rqb6P prophage and hence is penicillin-sensitive [32]; RS640 is an R@6P lysogen of RS630, and hence is penicillin-resistant. Both RS630 and RS640 produce a ~-lactamase, pI 5.15; RS640 produces an additional ~-lactamase, pI 7.2. This is presumably the ~-laetamase encoded by Rq~6P (M. Matthew, personal communication). Using Z 1059 and an in vitro packaging technique, a gene bank of RS630 was generated; screening of this bank revealed that the ~-lactamase of RS630 not only had been cloned, but also was expressed in E. coli (Bowen and Pemberton, unpublished data). At present, attempts are being made to clone the ~-lactamase gene of RO6P. These ~-lactamase genes may prove useful in constructing cloning vectors which allow expression of It. sphaeroides genes in E. coll. VII. - - CONCLUSION. Extension of a variety of genetic and molecular biological techniques to members of the genus Rhodopseudomonas angers well for combined biochemical and

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genetic analyses directed towards elucidation of the mechanism of bacterial photosynthesis. That all photosynthetic genes so far located map in a single region of the genomes of both R. sphaeroides and R. capsulata raises the possibility that a photosynthetic operon exists, t h a t is, a self-contained segment of DNA encodes all photosynthetic functions. Such a presumptive photosynthetic operon opens up a number of interesting possibilities for future research. First, can such an operon be transferred to a non-photosynthetic bacterium and be expressed in this alternate genetic background? The work of Woese and his colleagues [28] suggests t h a t the distinctions between bacterial groups based on photosynthetic capability are perhaps arbitrary. It seems t h a t such a predilection has obscured the true relationships between photosynthetic and non-photosynthetic organisms, and it appears t h a t many non-photosynthetic bacteria are phylogenetically related to photosynthetic bacteria. This is exemplified by the relationship between R. sphaeroides, R. capsulata and Paracoccus denilrificans, while plant pathogenic Agrobacterium lume/aciens is related to non-photosynthetic Rhizobium leguminosarum and Pseudomonas diminutia and the photosynthetic R. viridis, R. paluslris and Rhodomicrobium vaneilli. Such projected relationships indicate the potential range of non-photosynthetic microorganisms which could be used in studies involving the transfer and expression of photosynthetic genes. Second, the occurrence of a photosynthetic operon opens up the possibility of cloning all the photosynthetic genes into a single plasmid; alternatively, conventional genetic manipulations involving the generation of R-prime elements carrying photosynthetic genes has proven successful in R. capsulata and would be successful in R. sphaeroides where these genes are close to an origin of transfer. Finally, the possibility t h a t this photosynthetic operon could occur on plasmids In other species of photosynthetic bacteria cannot be ruled out. Many taxonomically important properties of bacteria, such as nodulation and nitrogen fixation among species of the genus Rhizobium, tumour formation among species of the genus Agrobaclerium and degradative pathways among species of the genus Pseudomonas, occur as plasmid-borne characters in one species and chromosomally located operons in another species. The occurrence and spread of plasmids encoding photosynthetic functions would be expected to have a profound effect on the evolution not only of bacteria but also of eukaryotic photosynthetic organisms.

RESUME MECANISMES DE TRANSFERT GENETIQUE P A R M I (( I ~ H O D O P S E U D O M O N A S ))

Les 6tudes r6centes sur des esp~ces des genres Rhodopseudomonas. en particulier R. capsulala et R. spheroides, ont permis le d6veloppement de divers syst6mes d'6ehanges g~n6tiques sans 6gaux parmi les procaryotes photosynth~tiques. Chez

R. capsulata, la transduction g6n6ralis6e et la formation de R-primes ont permis d'6tablir une carte d6taill6e de l'arrangement des g~nes impliqu6s dans la synth~se de l'appareil photosynth6tique. Les syst~mes de transfert du chromosome par eonjugaison chez R. sphaeroides ont permis d'6tablir la cartographie des g~nes impliqu6s dans la biosynth6se des acides amin6s, dans la rSsistance aux antibiotiques et dans la photosynth6se. L'annonce r6cente de transformation plasmid!que chez R. sphaeroides laisse prSvoir des progr6s rapides dans l'analyse des genes impliqu6s dans la photosynth~se et la fixation de l'azote par application des techniques de clonage de I'ADN. L'occurrence de transformation plasmidique, de transduction gen6ralis6e et sp6cialis6e et de transfert de chromosome par conjugaison ou par formation de

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R-primes dans le genre Rhodopseudomonas montre l'essor rapide des techniques de g~n~tique et de biologie mol6culaire applicable ~ l'~tude de ces bact~ries. MOTS-CL~'S : Rhodopseudomonas, Transfert gdndtique, Photosynth6se; Clonage de g6nes.

ACKNOWLEDGEMENTS The research carried out in my laboratory is supported by Grant D2-77/15086 from the Australian Research Grants Scheme.

BIBLIOGRAPHY

[1] ABELIOVICH, A. & KAPLAN, S., Bacteriophages of Rhodopseudomonas sphaeroides: isolation and characterisation of a Rhodopseudomonas sphaeroides bacteriophage. J. Virol., 1974, 13, 1392. [2] BAGD,~SARIAN,M. & T~MMIS, K. N., Host: vector systems for gene cloning in Pseudomonas, in (( Current Topics in Microbiology and immunology )), vol. 96 (47). Springer-Verlag, Berlin, 1982. [3] CHAKRABARTY, A. M., Genetic engineering. CRC Press Inc., West Pahn Beach, Florida, 1978. [4] CosLoY, S. D. & 0ISHI, M., Genetic transformation in Escherichia coll. Proc. nat. Acad. Sci. (Wash.), 1973, 70, 84. [5] DAGERT, M. & EHRLICH, S. D., Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene, 1979, 6, 23. [6] DE LA Cnuz, F. & GRINSTED, J., Genetic and molecular characterization of Tn21, a multiple-resistance transposon from R100-1. J. Bact., 1982, 151, 222. [7] GEST, H., Comparative biochemistry of photosynthetic processes. Nalure (Lond.), 1966, 209, 879. [8] GIBSON,K. D. & NIEDERMAN, R. A., Characterization of two circular satellite species of deoxyribonucleic acid in Rhodopsendomonas sphaeroides. Arch. Biochem. Biophgs., 1970, 141, 694. [9] HAAS, D. & HOLLOVVAY,B. W., R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol. gen. Genetics, 1976, 144, 243. [10] HOLLOWAY, B. W., Plasmids that mobilize bacterial chromosome. Plasmid, 1978, 2, 1. [11] Hu, N. T. & MARRS, B. L., Characterization of the plasmid DNAs of Rhodopseudomonas capsulata. Arch. Microbiol., 1979, 121, 61. [12] KAISER, A. D. & HOGNESS, D. S., Transformation of Escherichia coli with deoxyribonucleic acid isolated from bacteriophage Z dg. J. tool. Biol., 1960, 2, 392. [13] MABRS, B. L., Genetic recombination in Rhodopseudomonas capsulata. Proc. nat. Acad. Sci. (Wash.), 1974, 71, 971. [14] MARRS, B. L., Mutations and genetic manipulations as probes of bacterial photosynthesis. Curr. Top. Bioenerg., 1978, 8, 261. [15] MABRS,B. L., Mobilization of the genes for photosynthesis from Rhodopseudomonas capsnlata by a promiscuous plasmid. J. Bact., 1981, 146, 1003. [16] MILLER, L. & KAPLAN, S., Plasmid transfer and expression in Rhodopseudomonas sphaeroides. Arch. Biochem. Biophgs., 1978, 187, 229. [17] MURAL,R. J. & FRIEDMAN, D. I., Isolation and characterization of a temperate bacteriophage specific for Rhodopseudomonas sphaeroides. J. Virol., 1974, 14, 1288.

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[18] PEMBERTON,J. M. & TUCKER, W. T., Naturally occurring viral R plasmid with a circular supercoiled genome in the extracellular state. Nature (Lond.), 1977, 266, 50. [19] PEMBERTON, J. M. & BOWEN, A. R. St. O., High frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted bv broad host range plasmid RP1 carrying mercury transposon Tn501. ~J. Bact., 1981, 147, 110. [20] SAUNDERS, V. A., Genetics of Rhodospirillaceae. Microbiol. Rev., 1978, 42, 357. [211 SAUNDERS, V. A., SAUNDERS, J. R. ~2 BENNETT, P. M., Extrachromosomal deoxyribonucleic acid in wild type and photosynthetically incompetent strains of Rhodopseudomonas sphaeroides. J. Bact., 1976, 125, 1180. [22] SISTHOM, W. R., The spectrum of bacterioehlorophyll in vivo: observations on mutants of Rhodopseudomonas sphaeroicles unable to grow photosynthetically. Photobiologg, 1966, 5, 845. [23] SISTROM,W. R., Transfer of chromosomal genes mediated by plasmid R68.45 in Rhodopseudomonas sphaeroides. J. Bact., 1977, 131, 526. [24] SMITH, H. W., Ampicillin resistance in Escherichia coli by phage infection. Nature New Biol. (Lond.), 1972, 238, 205. [25] SOLIOZ,M. & ~V[ARRS,B., The gene transfer agent of Rhodopseudomonas capsulala: Purification and characterization of its nucleic acid. Arch. Biochem. Biophgs., 1977, 181, 300. [26] Socioz, M., YE~, H. C. & MateRs, B., Release and uptake of gene transfer agent by Rhodopseudomouas eapsulala. J. Bacl., 1975, 123, 651. [27] SPIZIZEN, J., RILEY, B. E. & EVANS, A. H., Microbial transformation and transfection. Ann. Rev. Microbiol., 1966, 20, 371. [28] STACKEBRANDT,E. & WOESE, C. R., in ~ Molecular and Cellular Aspects of Microbial Evolution ~. Society for Gen. Microbiol. Symposia. Pitman Medical Publ. Co. Ltd., Tunbudge Wells (Kent.), 1981. [29] STANIE~, R. Y., Photosynthetic mechanisms in bacteria and plants; development of a unitary concept. Bacl. Rev., 1961, 25, 1. [30] NTANISICH,V. A., BENN~ETT, P. M. & tZlICHMOND,M. H., Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a non-conjugative plasmid in Pseudomonas aeruginosa. J. Bacl, 1977, 129, 1227. [31] TUCKEn, W. T., Initial investigations of some gene transfer mechanisms in Rhodopseudomonas sphaeroides. Ph.D. Thesis. University of Queensland, St. Lucia, Australia, 1980. [32] TUCKER, W. T. & PEMBERTON,J. M., Viral R plasmid Rqb6P: properties of the penicillinase plasmid prophage and the supercoiled circular encapsidated genome. J. Bact., 1978, 135, 207. [33] TUKER, W. T. & PEMBEnTON, J. M., Transformation of Rhodopseudomonas sphaeroides with deoxyribonucleic acid isolated from bacteriophage Rqb6P. J. Bacl., 1980, 143, 43. [34] WALL, J. D., WEAVER, P. F. c~r GEST, H., Gene transfer agents, bacteriophages and bacteriocins of Rhodopseudomonas capsulala. Arch. Microbiol., 1975, 105, 217. [35] YEN, H.-C. & MARRS, B., Map of the genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. J. Bacl., 1976, 126, 619. [36] YEN, H.-C., Hu, N. T. & MARRS, B., Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsalata. J. tool. Biol., 1979, 131, 157.