A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10-2) thus far demonstrated in purple bacteria. It Photosynthetic reaction centers are complexes of integral membrane proteins and pigments which together catalyze light-driven electron transport through the photosynthetic membrane. The reaction center of the nonsulfur purple bacterium Rhodopseiudomonas viridis was crystallized (40), and the three-dimensional structure of the pigment-protein complex was determined by X-ray diffraction (13). Elucidation of the primary structure of the four subunits of the reaction center (41, 42, 57) makes investigations of the structure-function relationship in the complex, specifically amino acid side chains participating in the electron transfer process, by site-specific mutagenesis extremely interesting. A prerequisite for such experiments, however, is the availability of a gene transfer system. For such a system to be effective, transfer of DNA into cells at high frequency should be possible and the transferred DNA should be stable in the cells; i.e., it should replicate without deletions or rearrangements. Several techniques could, in principle, be applied.Bacteriophages RS1 (1), RF1 (44), RP6P (46), and I)RsGl (20) were isolated from Rhodobacter sphaeroides and characterized. In Rhodobacter capsulatus, a gene transfer agent, which presumably is a phagelike system (62), was characterized in more detail (37, 52). Bacteriophages RC1 (48) and RC2 (56) were also isolated from Rhodobacter capslulatus.In contrast to these two purple bacterial species, there are no known phages in R. viridis that could be used for transduction. Because of the size of R. viridis cells (1.5 iLm in length), a mechanical injection system seems experimentally infeasible. On the basis of optimized conditions for transformation by permeabilization of the bacterial cell wall, gene transfer systems were developed for Rhodobacter sphaeroides (22) with the broad-host-range vector pUI81; when applied to R. viridis, however, these systems were found to be ineffective under a variety of conditions. A transformation system for Rhodobacter capsulatus (P. Jasper, N. T. Hu, and B. L. Microbiol. 1978), that has a very low transformation frequency of 10-9 was not investigated for applicability in R. viridis.At the beginning of this work, donor cells that could undergo sexual conjugation with R. viridis were not known. Furthermore, no endogenous plasmids of R. viridis whose replicons could be used for replication of extrachromosomal DNA in R. viridis were available; in contrast, several such plasmids exist in Rhodobacter sphaeroides (3,45,55) and Rhodobacter c apsulatius (29). The work described here therefor...
