Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R.sphaeroides. Transfer frequencies can be as high as 10-2 to 10-3 per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS24le, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed.Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer.Rhodobacter sphaeroides is an a-purple nonsulfur photosynthetic eubacterium able to grow aerobically as a chemoheterotroph, by anaerobic respiration, fermentation, and anoxygenic photosynthesis, the latter either photoheterotrophically or photoautotrophically. In addition, this bacterium is also able to grow diazotrophically. The remarkable metabolic diversity of this organism and its ability to synthesize photosynthetic membrane invaginations (intracytoplasmic membranes) when grown anaerobically have made it and related bacteria, such as R. capsulatus, excellent model systems for the study of complex biological and biophysical phenomena. For example, photosynthetic membrane biogenesis (18), carbon dioxide fixation (42), nitrogen fixation (20), biophysical and structural studies of the light reactions of photosynthesis (5,19,35), pigment biosynthesis (1, 31), and hydrogen metabolism (49), to mention only a few, are fertile areas of research employing these facultative photosynthetic bacteria.All natural strains of R. sphaeroides which have been examined harbor at least one endogenous plasmid, and some strains carry as many as six different plasmid species, with sizes ranging from 42 to 140 kb (10). Despite their stable maintenance and relatively large sizes, these plasmids (as well as the endogenous plasmids of several other species of anoxygenic photosynthetic bacteria) are cryptic. However, given the above-described physiological diversity, it is logical to attempt to relate phenotypes to some of these plasmids.R. sphaeroides 2.4.1 harbors five endogenous plasmids (originally designated pRS241a,...