The effect of tra mutations on the synthesis of the F-pilin membrane polypeptide.

We had previously demonstrated that several F specific polypeptide bands could be detected in the membranes of Flac, but not F- strains of Escherichia coli K12, (Moore et al. 1981). One of these polypeptides co-migrated with F-pilin protein on polyacrylamide gels. We have now analyzed 35[S]methionine labelled membrane preparations from a series of strains containing Flac tra mutant plasmids. The F-pilin polypeptide was absent from preparations of strains containing all traA mutants tested, confirming the importance of the traA gene on F-pilin biosynthesis. A polypeptide which migrate in the F-pilin position was still present, however, in membranes prepared from Flac strains carrying mutations in traL, traE, traK, traB, traV, traW, traC, traU, traF, traH or traG despite the inability of these mutants to elaborate F-pili filaments. Thus, all of these gene products may be concerned with F-pilus assembly and outgrowth rather than biosynthesis of the F-pilin subunit. The polar mutation tra-4 did, however, prevent the appearance of pilin polypeptide, indicating that at least one unidentified gene in the region between traE and traG must also be required in F-pilin biosynthesis. Our analysis also permitted the identification of a 100,000 dalton membrane protein as the product of traG. The appearance of an F specific 12,000 dalton protein was prevented by traD amber mutants. As expected, traJ mutants prevented the expression of all the tra operon products detected except the product of traT. The traT product band was reduced only to 50 - 60% of its normal intensity.