Abstract
Bioinformatic analyses reveal a gene cluster (vva0325-vva0336) on the small chromosome of Vibrio vulnificus YJ016 might regulate cyclic-di-GMP levels. The gene cluster encodes a large RTX-like protein (VVA0331), a type I protein secretion system (VVA0332-0336), and a signaling system composing of a sensor kinase (VVA0329), two response regulators with a GGDEF-(VVA0326) and an EAL-domain (VVA0328), respectively. It is hypothesized that they may play roles in pathogenesis through modulating cyclic-di-GMP levels. Previous studies have shown that VVA0326 and VVA0328 possess diguanylate cyclase and phosphodiesterase activity, respectively, in vitro, and regulate intracellular c-di-GMP in V. vulnificus. The first study was to elucidate the subcellular location and functional roles of VVA0331. Results showed VVA0331 existed primarily in a secreted form and regulated by iron. The capabilities of vva0331 mutant strains in adherence and cytotoxicity on cells and virulence in mice were examined in comparison with wild type strain, indicating no difference. The second study was to investigate biological properties of VVA0326 and VVA0328. Deletion in the GGDEF domain of VVA0326 conferred the bacterium a slightly higher swimming motility, less biofilm formation and higher cytotoxicity activity to HEp-2 cells. However, there was no obvious difference between in-frame EAL deletion mutant and the wild type strains. Comparison of the levels of rtxA, vvhA, vva0331 transcripts and RtxA protein in wild type and vva0326 mutant strains indicates that these virulence-associated factors might be regulated by the cyclic-di-GMP signaling system. Furthermore, localization analysis had demonstrated that VVA0326 and VVA0328 were localized at V. vulnificus YJ016 poles by constructing GFP fusion proteins. Taken together, the gene cluster regulated by cyclic-di-GMP may have effects on bacterial virulence in V. vulnificus YJ016. We believe that the findings would be important for understanding the V. vulnificus physiology and pathogenesis mechanism.