ABSTRACT
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 TraVC18S mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraVC10S or TraVC10S/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 TraVC10S and TraVC18S proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraVC10S/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.
The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope.