Roles of internal cysteines in the function, localization, and reactivity of the TraV outer membrane lipoprotein...

We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutant traV genes: traV(C10S), traV(C18S), and traV(C10S/C18S). All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV(C18S) and, especially, traV(C10S/C18S) mutant strains were significantly less than those of the traV(+) and traV(C10S) strains. Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraV(C18S) mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraV(C10S) or TraV(C10S/C18S) proteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraV(C10S) and TraV(C18S) proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraV(C10S/C18S) protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.