Regulation of Biofilm Formation in Vibrio cholerae

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Biofilms are comprised of matrix proteins, extracellular DNA, and polysaccharides. Over 50% of the biofilm matrix mass of the pathogen Vibrio cholerae is comprised of Vibrio polysaccharides(VPS). Essential for the three-dimensional structure observed in biofilms, V. cholerae secretes VPS after initial attachment to either biotic or abiotic surfaces. Both the biofilm structure modulator A protein, RbmA, as well as the biofilm matrix protein, Bap1, are also secreted from the cell surface, promoting cell-cell adhesion and cell-VPS adherence. As the biofilm grows and develops, other matrix proteins, outer-membrane vesicles, and extracellular DNA help encase the biofilm. Genes involved in VPS synthesis are located on the V. cholerae's larger chromosome and organized into two operons, vps-I and vps-II. Vps-I contains 12 genes, including vpsU and vpsA-K while vps-II contains six genes, vpsL-vpsQ. These two operons are separated by 8.3 kilobase pairs that contain six ribomatrix genes, rbmA, rbmC, and rbmBDEF, which also contribute to biofilm formation. We have previously investigated how the response regulator VpsR, together with the small molecule cyclic di-GMP (c-di-GMP) regulates V. chlolerae biofilm formation by characterizing VpsR-activated transcription from PvpsL, the promoter at the beginning of the vps-II biofilm biosynthesis operon. We found that while VpsR shares homology with enhancer binding proteins that activate sigma54-RNA polymerase, its activation of PvspL uses sigma70-RNA polymerase. Interestingly, c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO4 footprints reveal that the sigma70-RNA polymerase / VpsR/c-di-GMP complex on PvpsL adopts a different conformation from that formed by sigma70-RNA polymerase alone, with c-di-GMP, or with VpsR. Our results suggested that c-di-GMP is required for VpsR to generate the specific protein-DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression. We have also identified the transcription start sites for other promoters within the biofilm formation cluster: PvpsU, at the beginning of the vps-I operon; PrbmA, at the start of the rbm cluster; and PrbmF, which lies upstream of the divergent rbmF/E genes. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we have shown that VpsR together with c-di-GMP directly activates sigma70-RNAP transcription from these promoters also. In this capacity, we have found that VpsR is able to behave both as a Class II activator, which functions immediately adjacent/overlapping the core promoter sequence (PvpsL and PvpsU), and as a Class I activator, which functions farther upstream (PrbmA and PrbmF). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we have suggested 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. Our findings demonstrate that VpsR is an adaptable, direct activator, functioning as both a Class I and Class II transcription activator, to induce multiple promoters of genes essential for biofilm formation in V. cholerae. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and provide a greater understanding of the regulation of biofilm formation.