Analysis of transfer genes in plasmid R64

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Conjugal transfer is an important process to exchange genetic material among bacterial cells. Forty-nine genes were found in the 54-kb transfer region of plasmid R64. To test the requirement of these genes for R64 conjugation, deletion and insertion mutations were introduced into all genes. For R64 surface mating, 24 transfer genes were found to be essential, while for liquid mating, additional 12 genes were required. The pnd gene was responsible for plasmid stability. Many gene products were detected by the maxicell method. The 12 genes required for liquid mating were responsible for the formation of R64 thin pilus. The purified R64 thin pilus consists of the pils and pilV products. The pils product was first synthesized as 22-kDa protein, and then processed to 19-kDa mature pilin mediated by the pilU product. To reveal the relationship between structure and function of the pilS gene, many pils mutants were isolated. DNA rearrangement of the shufflon selects one of the seven pilV genes with different C-terminal segments and determined the recipient specificity in liquid mating. Lipopolysaccharides in the recipient cells are suggested to function as receptors for pilV proteins from mating experiments using various rfa mutants of E.coli k-12 and S.typhimurium LT2 as recipient strains. The rci gene encoding shufflon-specific recombinase was overxpressed and the Rci protein was purified. An in vitro recombination system was constructed using the purified Rci protein. A plasmid cyrrying two tandemly repeated oriT sequences was constructed. A recombination event to lose a DNA segment between two oriT sequences was observed after mobilization. A recombination depending nikAB genes was also observed in the donor cells.