The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.