VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

Abstract

<i>Vibrio cholerae</i> biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the <i>Vibrio</i> polysaccharides (<i>vps</i>) operons I and II as well as the cluster of ribomatrix (<i>rbm</i>) 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 <i>vpsL</i> promoter (P <i>_vpsL</i> ), which precedes the <i>vps</i>-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using <i>in vitro</i> 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, including P <i>_vpsU</i> , at the beginning of the <i>vps</i>-I operon, P <i>_rbmA</i> , at the start of the <i>rbm</i> cluster, and P <i>_rbmF</i> , which lies upstream of the divergent <i>rbmF</i> and <i>rbmE</i> 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 <i>_vpsL</i> and P <i>_vpsU</i> ), and as a class I activator, which functions farther upstream (P <i>_rbmA</i> and P <i>_rbmF</i> ). 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.<b>IMPORTANCE</b><i>Vibrio cholerae</i>, 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 <i>vpsL</i> 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 <i>vps</i> 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.