Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.

No abstract

The traW gene of the Escherichia coli K-12 sex factor, F, encodes one of the numerous proteins required for conjugative transfer of this plasmid. We have found that the nucleotide sequence of traW encodes a 210-amino-acid, 23,610-Da polypeptide with a characteristic amino-terminal signal peptide sequence; in DNA from the F lac traW546 amber mutant, the traW open reading frame is interrupted at codon 141. Studies oftraW expression in maxicells in the presence and absence of ethanol demonstrate that the traW product does undergo signal sequence processing. Cell fractionation experiments additionally demonstrated that mature TraW is a periplasmic protein. Electron microscopy also showed that F lac traWS46 hosts do not express F pili, confirming that TraW is required for F-pilus assembly. Our nucleotide sequence also revealed the existence of an additional gene, trbI, located between traC and traW. The trbI gene encodes a 128-amino-acid polypeptide which could be identified as a 14-kDa protein product. Fractionation experiments demonstrated that TrbI is an intrinsic inner-membrane protein. Hosts carrying the pOX38-trbI::kan insertion mutant plasmids that we constructed remained quite transfer proficient but exhibited increased resistance to F-pilus-specific phages. Mutant plasmids pOX38-trbI472 and pOX38-trbI473 expressed very long F pili, suggestive of a pilus retraction deficiency. Expression of an excess of TrbI in hosts carrying a wild-type pOX38 plasmid also caused F-pilus-specific phage resistance. The possibility that TrbI influences the kinetics of pilus outgrowth and/or retraction is discussed.

We characterized the traU gene of the Escherichia coli K-12 conjugative plasmid F. Plasmids carrying segments of the F transfer operon were tested for their capacity to complement F lac traU526. The protein products of TraU+ clones were identified, and the nucleotide sequence of traU was determined. traU mapped between traW and trbC. It encodes a 330-amino-acid, Mr36,786 polypeptide that is processed. Ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing of the protein to occur. Because F lac traU526 strains appear to be resistant to F-pilus-specific phages, traU has been considered an F-pilus assembly gene. However, electron microscopic analysis indicated that the traU526 amber mutation caused only a 50% reduction in F-piliation. Since F lac traU526 strains also retain considerable transfer proficiency, new traU mutations were constructed by replacing a segment of traU with a kanamycin resistance gene. Introduction of these mutations into a transfer-proficient plasmid caused a drastic reduction in transfer proficiency, but pilus filaments remained visible at approximately 20% of the wild-type frequency. Like traU526 strains, such mutants were unable to plaque F-pilus-specific phages but exhibited a slight sensitivity on spot tests. Complementation with a TraU+ plasmid restored the wild-type transfer and phage sensitivity phenotypes. Thus, an intact traU product appears to be more essential to conjugal DNA transfer than to assembly of pilus filaments.

We constructed derivatives of the Escherichia coli conjugative plasmid F that carry altered sequences in place of the major transfer operon promoter, PY. Replacement of PY with a promoter-deficient sequence resulted in a transfer-deficient, F-pilus-specific phage-resistant plasmid (pOX38-tra701) that could still express TraJ and TraT; TraY, F-pilin, TraD, and TraI were not detectable on Western blots. On a second plasmid (pOX38-tra715) we replaced PY with a phage T7 late promoter sequence. In hosts carrying a lacUV5-promoter-regulated T7 RNA polymerase gene, all transfer-associated properties of pOX38-tra715 could be regulated with IPTG. After induction, pOX38-tra715 transferred at the wild-type frequency, expressed normal numbers of F-pili and conferred sensitivity to pilus-specific phages. No adverse effects on cell viability were apparent, and additional mutations could easily be crossed onto pOX38-tra715. A traJ deletion (pOX38-tra716) had no effect on the IPTG-induced transfer phenotype. Insertion of cam into trbC, resulted in a mutant (pOX38-tra715trbC33) which, after induction, exhibited the same phenotype associated with other trbC mutants; it could also be complemented by expression of trbC in trans. With pOX38-tra715 or its derivatives, we were able to label specifically the products of tra genes located throughout the long tra operon, by using rifampicin. This feature can be used to investigate transfer protein interactions and to follow changes in these proteins that are associated with conjugal mating events.