The LuxR-Type Regulator VpsT Negatively Controls the Transcription of rpoS, Encoding the General Stress Response Regulator, in Vibrio cholerae Biofilms

Abstract

Cholera is a waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. Expression of the general stress response regulator RpoS and formation of biofilm communities enhance the capacity of V. cholerae to persist in aquatic environments. The transition of V. cholerae between free-swimming (planktonic) and biofilm life-styles is regulated by the second messenger cyclic di-GMP (c-di-GMP). We previously reported that increasing the c-di-GMP pool by overexpression of a diguanylate cyclase diminished RpoS expression. Here we show that c-di-GMP repression of RpoS expression is eliminated by deletion of the genes vpsR and vpsT, encoding positive regulators of biofilm development. To determine the mechanism of this regulation, we constructed a strain expressing a vpsT-FLAG allele from native transcription and translation signals. Increasing the c-di-GMP pool induced vpsT-FLAG expression. The interaction between VpsT-FLAG and the rpoS promoter was demonstrated by chromatin immunoprecipitation. Furthermore, purified VpsT interacted with the rpoS promoter in a c-di-GMP-dependent manner. Primer extension analysis identified two rpoS transcription initiation sites located 43 bp (P1) and 63 bp (P2) upstream of the rpoS start codon. DNase I footprinting showed that the VpsT binding site at the rpoS promoter overlaps the primary P1 transcriptional start site. Deletion of vpsT significantly enhanced rpoS expression in V. cholerae biofilms that do not make HapR. This result suggests that VpsT and c-di-GMP contribute to the transcriptional silencing of rpoS in biofilms prior to cells entering the quorum-sensing mode.

Identification and Characterization of VpsR and VpsT Binding Sites in Vibrio cholerae

“…The VpsT recognition sequence identified in this study is different from the one identified in the regulatory region of rpoS (AAAGGTTGTAAATC) (30). Thus, it appears that the VpsT recognition sequence can vary substantially.…”

“…Recent studies showed that VpsT can bind to the regulatory region of vpsL and rpoS in a c-di-GMP-dependent manner (25,30). Two inverted repeats were found in the VpsT recognition site at the rpoS promoter, yet this motif was not found in the regulatory region of vps genes (30).…”

“…Notably, VpsR was shown to bind to a region containing the predicted VpsR binding motif located upstream of aphA; the VpsR recognition site overlaps the recognition site of HapR (29). Recent studies showed that VpsT can bind to the regulatory region of vpsL and rpoS in a c-di-GMP-dependent manner (25,30). Two inverted repeats were found in the VpsT recognition site at the rpoS promoter, yet this motif was not found in the regulatory region of vps genes (30).…”

Role of Non-coding Regulatory RNA in the Virulence of Human Pathogenic Vibrios

“…It also suppresses luxM by coupled degradation and activates aphA by revealing the ribosome binding site, while at the same time the sRNA is degraded (Feng et al, 2015). The activation of the translation of AphA (Rutherford et al, 2011; Van Kessel et al, 2013a) indirectly activates the transcription of virulence and biofilm genes, which promotes bacterial colonization and infection (Wang et al, 2014), so an important function of sRNAs in Vibrios is the link between QS and virulence (Carignan et al, 2016). Also, HapR/LuxR/SmcR/OpaR regulates the expression of genes involved in the synthesis and degradation of c-di-GMP reducing the levels of this second messenger that controls the switch between biofilm formation and motility and other fundamental bacterial behavior including fimbrial synthesis, T3SS and RNA modulation (Srivastava and Waters, 2012; Wang et al, 2014; Sengupta et al, 2016).…”

“…HapR negatively regulates the expression of aphA and directly suppresses biofilm formation by binding to the promoter of the gene vpsT , which codes a regulator of the exopolysaccharide production cluster vps , and indirectly through aphA , since AphA is a positive regulator of VpsT (Teschler et al, 2015; Carignan et al, 2016) ( Figure 1 ). HapR also suppresses the transcription of vpsR , which codes another transcription factor that also activates genes involved in biofilm formation and integrates signals from at least three different QS systems, which converge to the regulators of the LuxO response (Wang et al, 2014; Vakulskas et al, 2015) ( Figure 1 ).…”