Analysis of thetraLEKBPSequence and the TraP Protein from Three F-Like Plasmids: F, R100-1, and ColB2

The sequence of a region of the F plasmid containing thetraLEKBPgenes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2. ThetraLEKgene products of all three plasmids were virtually identical, with the most changes occurring in TraE. ThetraBgenes were also nearly identical except for an 11-codon extension at the 3*end of the R100-1traBgene. The TraP protein of R100-1 differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoAmutagen- esis. The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2. The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer. The transfer ability of pOX38-traP474was shown to be affected by mutations in therfa(lipopolysaccharide) locus and inompAin the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with thetraPanalogs from R100-1 and ColB2togiveanF-likephenotype.Thus,theTraPproteinappearstoplayaminorroleinconjugationandmay interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmem- brane complex formed by thetraoperon products. The genes involved in establishing an effective mating pair duringFplasmid-mediatedconjugationareencodedwithinthe 33-kb transfer (tra) operon. Fifteen of the nearly 40 known genes in the tra operon are involved in F pilus synthesis and assembly (for a review, see reference 7). This filamentous ex- tracellular organelle is thought to identify suitable recipient cells by binding to a receptor on the cell surface and drawing the recipient cell to the donor cell surface via pilus retraction. The process of retraction is controversial and is based on electron microscopic examination of cells during mating pair formation andfilamentous phage (f1, M13, or fd) infection. In addition, a second role for the F pilus involving the actual transferofDNAhasbeenproposedonthebasisofevidenceof DNA transfer between cells separated physically from one another but connected by a pilus (11). This latter function for the F pilus is in agreement with genetic analysis of the F tra region, since mutations in genes that affect pilus synthesis blockDNAtransferwhilemutationsintherecipientcellwhich