Analysis of transfer genes and gene products within the traB-traC region of the Escherichia coli fertility factor, F

Abstract

A series of plasmids that carry overlapping segments of F DNA encoding the genes in the traB-traC interval was constructed, and a restriction enzyme map of the region was derived. Plasmids carrying deletions that had been introduced at an HpaI site within this interval were also isolated. The ability of these plasmids to complement transfer of F lac plasmids carrying mutations in trail, traV, traW, and traC was analyzed. The protein products of the plasmids were labeled in UV-irradiated cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. These analyses showed that the product of traV is a polypeptide that migrates with an apparent molecular weight of 21,000. It was not detected when [35S]methionine was used to label plasmid products, but was readily detected in '4C-amino acid labeling experiments. A 21,500-dalton product appeared to stem from the region assigned to traP. A 9,000-dalton product was found to stem from a locus, named traR, that is located between traV and traC. No traW activity could be detected from the region of tra DNA examined. Our data also indicated that traC is located in a more promoter-proximal position than suggested on earlier maps. The plasmids constructed are expected to be useful in studies designed to identify the specific functions of the traB, -P, -V, -R, and -C products.

Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2

“…2). By using transducing phages carrying the traP gene, a 21.5-kDa polypeptide has been identified as the gene product of traP (18). The N-terminal residues of R100-1 TraP are significantly different from those of either the F or ColB2 proteins, while the major changes in ColB2 TraP occur in a stretch of amino acids at positions 81 to 96, with several single substitutions occurring at intervals throughout the distal end of the protein (Fig.…”

“…The product of the F traB gene contains 475 aa and is predicted to be a 50.5-kDa, predominantly hydrophilic protein exposed to the periplasm and anchored in the inner membrane via a short transmembrane segment from aa 13 to 33 (7). It has been visualized on SDS-polyacrylamide gels as a 60-kDa protein (18). Amino acid sequence comparisons reveal that the TraB protein of ColB2 is also 475 aa in length and has 100% sequence identity to F TraB while the R100-1 TraB contains 486 aa, with an additional 11 aa at the carboxy terminus (Fig.…”

“…The product of the traK gene is predicted to be a 242-aa periplasmic protein which is processed to a soluble product of 23.3 kDa (221 aa) (19). The traK gene product has been visualized on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 24-kDa protein by [ 35 S]methionine labelling of transducing phages carrying the traB-traC region (18). Sequence differences between the F and ColB2 TraK proteins occur at three positions: M-333I, L-503M, and E-1093K.…”

“…Two recombinant plasmids carrying the kan gene oriented either in the direction opposite of traP (pKI470) or in the same direction as traP (pKI474) were isolated. To obtain pOX38 derivatives, triparental matings were performed by mixing the donor strain, E. coli RD-17/pOX38 (18), with a derivative of strain XK5456 (18) carrying either pKI470 or pKI474 and with a final recipient strain, XK1200. After allowing in vivo recombination to occur, transconjugants were selected for kanamycin and nalidixic acid resistance and ampicillin sensitivity.…”

Membrane insertion of the F-pilin subunit is Sec independent but requires leader peptidase B and the proton motive force

“…F pili are assembled from a pool of F-pilin subunits localized in the cytoplasmic membrane of F ϩ strains (16,(26)(27)(28). Subunits are derived from the 121-amino-acid precursor polypeptide, propilin, encoded by the F transfer region gene, traA (9).…”