VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

Abstract

<jats:p><jats:italic>Vibrio cholerae</jats:italic> biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the <jats:italic><jats:underline>V</jats:underline>ibrio</jats:italic> <jats:underline>p</jats:underline>oly<jats:underline>s</jats:underline>accharides (<jats:italic>vps</jats:italic>) operons I and II as well as the cluster of <jats:underline>r</jats:underline>i<jats:underline>b</jats:underline>o<jats:underline>m</jats:underline>atrix (<jats:italic>rbm</jats:italic>) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the <jats:italic>vpsL</jats:italic> promoter (P<jats:italic><jats:sub>vpsL</jats:sub></jats:italic>), which precedes the <jats:italic>vps</jats:italic>-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using <jats:italic>in vitro</jats:italic> transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster: P<jats:italic><jats:sub>vpsU</jats:sub></jats:italic>, at the beginning of the <jats:italic>vps</jats:italic>-I operon; P<jats:italic><jats:sub>rbmA</jats:sub></jats:italic>, at the start of the <jats:italic>rbm</jats:italic> cluster; and P<jats:italic><jats:sub>rbmF</jats:sub></jats:italic>, which lies upstream of the divergent <jats:italic>rbmF/E</jats:italic> genes. In this capacity, we find that VpsR is able to behave both as a Class II activator, which functions immediately adjacent/overlapping the core promoter sequence (P<jats:italic><jats:sub>vpsL</jats:sub></jats:italic> and P<jats:italic><jats:sub>vpsU</jats:sub></jats:italic>), and as a Class I activator, which functions farther upstream (P<jats:italic><jats:sub>rbmA</jats:sub></jats:italic> and P<jats:italic><jats:sub>rbmF</jats:sub></jats:italic>). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR's mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation.</jats:p> <jats:p><jats:bold>Importance</jats:bold> <jats:italic>Vibrio cholerae</jats:italic>, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the <jats:italic>vpsL</jats:italic> biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the <jats:italic>vps</jats:italic> and ribomatrix operons. In this regard, it functions as either a Class I or Class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.</jats:p>

Diguanylate Cyclases in Vibrio cholerae: Essential Regulators of Lifestyle Switching

“…Both VpsR and VpsT bind directly to the vps promoter regions and have recognition sites in vps-1, vps-2 and vps-L operons which act as regulatory sequences in the expression of extracellular polysaccharide and matrix protein synthesis (Fong et al, 2010). A recent report relates the activation of the vps operons to the concentration of VpsR as well as c-di-GMP (Hsieh et al, 2020) directly affecting the σ70 RNAP. Additionally, VpsT can act as a regulatory protein with recognition sequences for RbmA, whereas RbmC and Bap1 promoters also contain recognition sites for VpsR (Boyd and O'Toole, 2012;Zhao-Xun, 2015).…”

Modulation ofVibrio choleraegene expression through conjugative delivery of engineered regulatory small RNAs

“…Exopolysaccharide (EPS) is one of the main components of biofilm in V. cholerae and plays a major role in transmission and intestinal colonization as well as survival in aquatic environments (32). EPS is synthesized by several proteins encoded in two regions: vps-I (coding for vpsU and vpsA-K ) and vps-II (coding for vpsL-Q ) (32, 33). We designed sRNAs targeting three different genes previously shown to be essential for EPS production: vpsU (TarVpsU) , vpsA (TarVpsA), and vpsL (TarVpsL) (Table S3, File S1).…”

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

“…4), which suggested that the regulation of mRNA and protein levels of MshA by VC1371 and VcRfaH might not be through MshH, but rather by directly activating the mshH promoter and enhancing the production of MSHA pili. We also tested the mRNA levels of some genes in the operon of Vibrio polysaccharide (VPS, vc0917-vc0927) and rbmA (vc0928), which are essential for biofilm formation in V. cholerae [46,47], by qRT-PCR in Δvc1371 and ΔVcrfaH; however, we did not find VC1371 or VcRfaH affecting the expression of these genes (Fig. S6).…”

“…We also tested the mRNA levels of some genes in the operon of Vibrio polysaccharide (VPS, vc0917-vc0927 ) and rbmA ( vc0928 ), which are essential for biofilm formation in V. cholerae [46, 47], by qRT-PCR in Δ vc1371 and Δ VcrfaH ; however, we did not find VC1371 or VcRfaH affecting the expression of these genes (Fig. S6).…”