Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus

Abstract

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.

VqsA, a Novel LysR-Type Transcriptional Regulator, Coordinates Quorum Sensing (QS) and Is Controlled by QS To Regulate Virulence in the Pathogen Vibrio alginolyticus

“…The LTTR protein MetR has been shown to be involved in QS regulation in response to methionine metabolism in V. harveyi (31). AphB, a low-pH-and oxygen-responsive LTTR protein (32,33), regulates QS by directly binding to the luxR promoter and controlling LuxR expression at the transcriptional level in V. alginolyticus (25). Moreover, identification of the oxidativestress-responsive SoxR (34) and the feast/famine regulatory protein AsnC (35) as being involved in QS regulation also suggested that various physiological signals might affect QS regulation.…”

“…The genes of 285 probable transcription factors were amplified from V. alginolyticus EPGS by using gene-specific primers (a subset of the primers are shown in Table 3) and cloned into the pTRG plasmid. The promoters of the V. alginolyticus aphA and luxR genes were amplified using primers specific to these promoters (Table 3) and inserted into the reporter vector pBXcmT, as previously described (25,26). The E. coli XL1-Blue MRF9 kanamycin-resistant (Km r ) strain was used for propagating all pBXcmT and pTRG recombinant plasmids.…”

“…Autorepression of LuxR occurs via the binding of LuxR to the promoter at two sites, known as LuxR-binding site I (RBSI) and RBSII (6). In addition to LuxR and AphA, the promoter of luxR is also indirectly regulated by LuxT and directly regulated by AphB and the alternative sigma factor RpoE, which works in conjunction with core RNA polymerase in response to changes in temperature (6,23,25). However, whether there are other factors that can regulate QS in the bacterium remains largely unknown.…”

Vibrio vulnificusin aquariums is a novel threat to marine mammals and public health

“…In Asia, Vibrio parahaemolyticus is a common cause of foodborne disease (Sakata, Yonekita, & Kawatsu, ), while Vibrio alginolyticus is implicated in wound infections and otitis. Vibrio alginolyticus was recently recognized as a human pathogen after excessive exposure to seawater (Gao et al., ). Vibrio fluvialis has been considered an emerging pathogen that induces foodborne diarrhea (Ramamurthy, Chowdhury, Pazhani, & Shinoda, ).…”

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501

“…Samples were first extracted with phenol-chloroform and then precipitated with ethanol, and the pellets were dissolved in 30 μl of Milli-Q water. The preparation of the DNA ladder, electrophoresis, and DNA sequencing were performed as described previously (45, 46).…”