In species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of and the master quorum-sensing (QS) regulator DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the promoter region overlaps that of LuxR-binding site I, which mediates the positive control of promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in , a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the gene itself was identified to be positively autoregulated, and AphB also positively controlled and expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.

Fish pathogen Vibrio anguillarum, a mesophile bacterium, is usually found in estuarine and marine coastal ecosystems worldwide that pose a constant stress to local organism by its fluctuation in salinity as well as notable temperature change. Though V. anguillarum is able to proliferate while maintain its pathogenicity under low temperature (5-18°C), so far, coldadaption molecular mechanism of the bacteria is unknown. In this study, V. anguillarum was found possessing a putative glycine betaine synthesis system, which is encoded by betABI and synthesizes glycine betaine from its precursor choline. Furthermore, significant up-regulation of the bet gene at the transcriptional level was noted in log phase in response to cold-stress. Moreover, the accumulation of betaine glycine was only found appearing at low growth temperatures, suggesting that response regulation of both synthesis system and transporter system are cold-dependent. Furthermore, in-frame deletion mutation in the two putative ABC transporters and three putative BCCT family transporters associated with glycine betaine uptake could not block cellular accumulation of betaine glycine in V. anguillarum under coldstress, suggesting the redundant feature in V. anguillarum betaine transporter system. These findings confirmed that glycine betaine serves as an effective cold stress protectant and highlighted an underappreciated facet of the acclimatization of V. anguillarum to cold environments.

The capability of cold-adaptation is a prerequisite of microorganisms that survive in an environment with frequent fluctuations in temperature. As a global causative agent of vibriosis in marine fish farming, Vibrio anguillarum can efficiently grow and proliferate under cold-stress conditions, which is 15°C lower than the optimal growth temperatures (25-30°C). Our data showed that V. anguillarum was able to synthesize ectoine de novo and that ectoine was essential for its growth under cold stress. Using H nuclear magnetic resonance spectroscopy and mutants lacking ectABC and proVWX (ectoine synthesis and transporter system genes, respectively), we confirmed that accumulation of this compatible solute occurs strictly at low temperatures and that the expression of ectA and proV is highly activated in the stationary growth phase. However, the synthesis of ectoine was repressed by exogenous choline (precursor of glycine betaine), suggesting that ectoine is an alternative compatible solute as a cold-stress protectant in V. anguillarum. Based on these results, we present possible scenarios of the synthesis and uptake of ectoine, which will facilitate the understanding of the molecular mechanism of V. anguillarum adaptation to cold environments and help improve freezing-dry processes for the V. anguillarum live vaccine.

The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of and from a library of 285 TFs encoded by the fish pathogen A total of 7 TFs were identified to bind to the promoters of both and Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of and as well as the binding sites of LuxR, AphA, and VqsA in the gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and virulence of Collectively, our data showed that VqsA is an important QS regulator in Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.

Pages 19 and 20: The first paragraph of the Acknowledgments should read as follows: This work was supported by grants from National Natural Science Foundation of China (grants 31772891 to Q.W., 31772893 to Y.M., and 41376128 to Y.Z.), the Ministry of Agriculture of China (grant CARS-47-G17), the Shanghai Pujiang Program (grant 16PJD018), and the Science and Technology Commission of Shandong and Shanghai Municipality (grants 2017CXGC0103 and 17391902000).