Twelve pil genes are required for biogenesis of the R64 thin pilus - PubMed (original) (raw)

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

T Yoshida et al. J Bacteriol. 1999 Apr.

Abstract

The IncI1 plasmid R64 produces two kinds of sex pili: 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, pilI 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 pili 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.

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Figures

FIG. 1

FIG. 1

Gene organization of the traA to -D and pilI to -V regions of pKK641-A′. The top horizontal line represents a restriction map. B, _Bgl_II; C, _Cla_I; E, _Eco_RI; H, _Hin_dIII; Hp, _Hpa_I; P, _Pst_I; S, _Sma_I; V, _Pvu_II. Locations of insertion or deletion mutations and their transfer capacity are indicated (open circles, transfer proficient; solid circles, transfer deficient). Below the map, open reading frames are represented by arrows. tra, transfer; pil, formation of thin pilus; Shf, shufflon; rci, shufflon-specific recombinase. The solid lines on the bottom indicate DNA portions present in complementing plasmids.

FIG. 2

FIG. 2

(A) Effects of various pil mutations on the expression and processing of the pilS product. Whole extracts of E. coli cells harboring pKK641A′ with the indicated pil mutations were separated by SDS-PAGE and subjected to Western blot analysis with antipilin antiserum. (B) Effects of pil mutations on the formation of thin pili. A thin pilus fraction was prepared from the culture media of E. coli cells harboring pKK641A′ with the indicated pil mutations. Proteins were separated by SDS-PAGE and subjected to Western blot analysis with antipilin antiserum. The positions of prepilin (22 kDa) and pilin (19 kDa) are indicated on the right.

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