VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae.

biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the polysaccharides () operons I and II as well as the cluster of ribomatrix () 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 σ (σ-RNAP) at the promoter (P ), which precedes the -II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ-RNAP transcription from other promoters within the biofilm formation cluster, including P , at the beginning of the -I operon, P , at the start of the cluster, and P , which lies upstream of the divergent and 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 and P ), and as a class I activator, which functions farther upstream (P and P ). 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., 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 σ from the biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ-RNA polymerase at the first genes of the 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.