parahaemolyticus possesses a full set of the exsACDE regulatory system, which is similar to that of P. aeruginosa and which regulates T3SS1-related gene expression. H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation in enteric bacteria (Varshavsky et al., 1977; Hulton et al., 1990). H-NS affects the expression of many unrelated genes and several virulence genes in Salmonella DZNeP molecular weight enterica serovar Typhimurium, Shigella sp. and Vibrio cholerae (Maurelli & Sansonetti, 1988; Hulton et al., 1990; Tobe et al., 1993; Harrison et al., 1994; O’Byrne & Dorman, 1994;

Nye et al., 2000). Therefore, we examined the possibility that T3SS1 genes are part of the H-NS regulon. As shown in Fig. 3a, the production of VscC1 and VepA proteins in a Δhns strain was considerably increased in both the bacterial pellet and the supernatant compared with that of the WT. A ΔhnsΔexsA mutant strain did not exhibit

increased production of these proteins (Fig. 3b), suggesting that exsA is necessary for overproduction of T3SS1-related proteins via hns gene deletion. We next examined the possibility that H-NS represses exsA expression using an exsA–lacZ transcriptional fusion reporter (Fig. 3c). Transcription of exsA–lacZ was dramatically increased in the hns deletion strain compared with that in the WT. The increase in exsA–lacZ transcription in the hns deletion strain was suppressed by Staurosporine mouse in trans complementation with the Ixazomib supplier hns gene. These results

indicate that H-NS represses T3SS1-related gene expression by suppressing exsA gene expression. In summary, we identified VP1701 and VP1702 of V. parahaemolyticus as functional orthologues of P. aeruginosa ExsC and ExsE, respectively. As VP1701 has sequence similarity with its counterpart, it was not difficult to predict its function. Indeed, the production of T3SS1-related proteins was repressed in a Δvp1701 mutant and derepressed by complementation of the vp1701 gene (Fig. 1b and c). Unlike ExsA (VP1699), ExsD (VP1698) and ExsC (VP1701), sequence annotation of the T3SS1 region on the genome of V. parahaemolyticus did not reveal any CDSs predicted to encode the homologue of P. aeruginosa ExsE. However, we found that one hypothetical CDS (VP1702) was encoded next to the vp1701 (exsC) gene. Deletion of the vp1702 gene deregulated the production of T3SS1-related proteins. Furthermore, VP1702 itself was a substrate for the T3SS1 secretion system. These properties of VP1702 of V. parahaemolyticus conform with those of its counterpart in P. aeruginosa. In P. aeruginosa, the coupling of transcription to secretion is mediated by three interacting proteins (ExsC, ExsE and ExsD) that regulate ExsA transcription activity (Yahr & Wolfgang, 2006). Although it is still unknown whether ExsC, ExsE and ExsD of V.