Mechanism of conjugal transfer in IncI plasmids

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The 54-kb transfer region of IncI1 plasmid R64 contains 49 genes, in which 24 genes are essential for matings in general. Additional 12 pil genes encoding type IV pili are required only for liquid matings to reveal the relationship between the structure and function of PilU. Multiple DNA inversions of R64 shufflon select one of seven PilV adhesins with different C-terminal segments.1) Forty-two mutants of R64 pilU gene encoding prepilin peptidase were isolated. The PilU protein was shown to belong to the aspartic acid peptidase family.2) TraC, a putative positive regulator for transfer gene expression, was shown to inhibit shufflon inversions by interacting with a C-terminal segment of Rci, the shufflon-specific recombinase.3) The sfx recombination sequences of R64 shufflon have been shown to be asymmetric. Frequencies of seven pilV genes were not equal in equilibrium. When in vivo inversion of an artificial shufflon segment carrying two sfx sequence with different left and right arms at both ends was induced by concomitant presence of rci plasmid, the frequencies of shufflon segments with normal or reverse orientation were not equal after inversions reached equilibrium. Theses results indicate that the asymmetry of the sfx sequences results in the unequal frequency of the seven pilV genes in equilibrium in R64 shufflon.4) DNA transfer in R64 conjugation is initiated by the introduction of a site-and strand-specific nick at the oriT site by NikAB relaxase. NMR analysis indicated that two molecules of NikA N-terminal segment (NikA1-51) form a ribbon-helix-helix fold. SELEX analysis indicated that NikA binds to ACGGTA sequence present in the repeat A sequence of R64 oriT.