Genetic-Physical Mapping of a Photosynthetic Gene Cluster in Rhodopseudomonas Capsulata

摘要

The photosynthesis genes of Rhodopseudomonas capsulata are investigated using transposon mutagenesis, phenotypic characterization of transposon induced mutants, and complementation analysis. The genes investigated are located on an R-prime plasmid, pRPS404, containing 50 kilobase pairs of R. capsulata DNA. A gene replacement scheme is described utilizing the antibiotic resistance markers from Tn7 and the instability of pRPS404 in a recombination proficient background. A transposon, Tn5.7, has been constructed incorporating the antibiotic resistance markers from Tn7 and the transposition functions from Tn5. It was used to mutagenize the plasmid pRPS404. In conjunction with the mutagenesis, physical mapping of the restriction endonuclease recognition sites for XhoI, Bg1II, KpnI, and SstI on pRPS404 has been accomplished. Sixty insertions of Tn5.7 into the R. capsulata DNA on the plasmid were isolated and thirty-one of these were crossed into the R. capsulata chromosome with subsequent deletion of wild type alleles. Mutants were characterized by absorption spectroscopy. SDS-PAGE, and determination of capability for photosynthetic growth. Many mutations in the bacteriochlorophyll and the carotenoid biosynthesis genes were isolated. A regulatory mutation was isolated affecting reaction centers as well as a 44kd heme containing polypeptide. Mature bacteriochlorophyll, carotenoids, and light harvesting complexes are made in this mutant. Complementation analysis using various pRPS404::Tn5.7 plasmids has led to the postulation of transcriptional units including bacteriochlorophyll and carotenoid biosynthetic genes as well as reaction center structural genes. 97 references, 34 figures, 10 tables. (ERA citation 09:029326)