Identification And Characterization Of Vibrio Alginolyticus Alternative Sigma Factor X

Vibrio alginolyticus is a marine-born pathogenic bacterium, which is widely distributed worldwide and causes huge economic losses to aquaculture industries. Quorum-sensing (QS) system plays an important role in virulence regulation of V. alginolyticus. The alternative sigma factor E can regulate expression of luxR, the gene encoding QS central regulator LuxR. Previous investigation showed that rpoE expression and function are temperature dependent. RNA-Seq results show that in the case of rpoE deletion, the expression of a gene encoding homolog to an uncharacterized alternative sigma factor drastically decreased. We name this sigma factor as ax and the encoding gene as rpoX. V. alginolyticus rpoX gene shows 43% identity with Vibrio fischeri ES114 rpoS like sigma factor rpoQ. This work further characterized the roles and regulation function of rpoX in V. alginolyticus.This work verified that σE regulates rpoX at the transcription and translation level by binding directly to rpoX promoter. As aE is a kind of sigma factor controled by temperature, we also investigated the temperiature-dependent expression of rpoX and confirmed that rpoX was upregluted at high temperatures. Moreover, rpoX expression was triggered under the condition of low salt (0.5%NaCl). σx could activate motility ability and formation of biofilm with no effect on the infection ability of V. alginolyticus to zebrafish as compared the rpoX deletion mutant with wild type strain. Further analysis indicated that rpoX has no regulation function on Asp expression and QS regulator LuxR, and LuxO has no regulation function on rpoX expression.The genes regulated by σx were further analyzed through the methods of RNA-Seq and ChIP-Seq. RNA-Seq results showed that in V. alginolyticus 1043 genes were regulated by σx, among which 149 genes were activated and the expressions of 894 genes inhibited by σx. These genes were classified into groups of stress response, flagellar biosynthesis and regulation, membrane proteins, virulence, and transcription factors. With ChIP-Seq and EMSA, it was demonstrated that σx regulates the expression of itself. To sum up, we characterized a novel alternative sigma factor which will improve the understanding of pathgoensis and gene regulation in V. alginolyticus and other vibrios.