Twelve pil genes are required for biogenesis of the R64 thin pilus

The IncI1 plasmid R64 produces two kinds of sex pill: a thin pilus and a thick pilus, The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes, pill to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594-3603, 1997), In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis, From the analyses of extracellular secretion of thin pill and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus, Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes, traBC, were previously shown to be required for the expression of the pil genes. In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function.