Mechanism of conjugal transfer in plasmid R64

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The 54-kb transfer region of IncI1 plasmid R64 contains 49 genes, in which 24 genes are essential for surface matings. Additional 12 pil genes encoding thin pili are required for liquid matings, located at the tip of thin pili. Multiple DNA inversions of R64 shufflon select one of seven PilV adhesins with different C-terminal segments. 1)Forty-three mutants of pilU gene encoding prepilin peptidase were isolated, suggesting that PilU protein belongs to the aspartic acid peptidase family. Complicated membrane topology of PilU was suggested by constructing pilU-phoA and pilU-lacZ fusion genes. 2)R64 sfx shufflon recombination sequences have been shown to be asymmetric. Recombination between artificial symmetric sfx sequences in a direct orientation was detected. The Rci C-terminal segment was not required for the recombination using symmetric sfx sequences. Specific binding of lipopolysaccharide of recipient cell envelope to C-terminal segments of various PilV adhesins was demonstrated by blotting experiments and Biacore analyses. 3)R64 trbAC genes were shown to be not required for the mobilization of CloDF13 by R64, suggesting that trbAC products act the function similar to F TraD. 4)The R64 oriT operon consists of oriT sequence and nikAB genes. Introduction of oriT-specific nick was demonstrated for oriT-NikAB complex constructed from oriT DNA and purified proteins. 5)Transfer region of IncIγ plasmid R621a was found to be similar to that of R64, although the structures of their traY and exc genes are different each other. R621a and R64 were found to belong to distinct exclusion groups. Many traY mutants were constructed to identify the region determining exclusion specificity.