H-NS Is a Negative Regulator of the Two Hemolysin/Cytotoxin Gene Clusters in Vibrio anguillarum

Hemolysins produced by Vibrio anguillarum have been implicated in the development of hemorrhagic septicemia during vibriosis, a fatal fish disease. Previously, two hemolysin gene clusters responsible for the hemolysis and cytotoxicity of V. anguillarum were identified: the vah1-plp gene cluster and the rtxACHBDE gene cluster. In this study, we identified the hns gene, which encodes the H-NS protein and acts as a negative regulator of both gene clusters. The V. anguillarum H-NS protein shares strong homology with other bacterial H-NS proteins. An hns mutant exhibited increased hemolytic activity and cytotoxicity compared to the wild-type strain. Complementation of the hns mutation restored hemolytic activity and cytotoxicity levels to nearly wild-type levels. Furthermore, expression of rtxA, rtxH, rtxB, vah1, and plp increased in the hns mutant and decreased in the hns-complemented mutant strain compared to expression in the wild-type strain. Additionally, experiments using DNase I showed that purified recombinant H-NS protected multiple sites in the promoter regions of both gene clusters. The hns mutant also exhibited significantly attenuated virulence against rainbow trout. Complementation of the hns mutation restored virulence to wild-type levels, suggesting that H-NS regulates many genes that affect fitness and virulence. Previously, we showed that HlyU is a positive regulator of expression for both gene clusters. In this study, we demonstrate that upregulation by hlyU is hns dependent, suggesting that H-NS acts to repress or silence both gene clusters and HlyU acts to relieve that repression or silencing.