Regulation of Rugosity and Biofilm Formation in Vibrio cholerae : Comparison of VpsT and VpsR Regulons and Epistasis Analysis of vpsT , vpsR , and hapR

Abstract

Vibrio cholerae undergoes phenotypic variation that generates two morphologically different variants, termed smooth and rugose. The transcriptional profiles of the two variants differ greatly, and many of the differentially regulated genes are controlled by a complex regulatory circuitry that includes the transcriptional regulators VpsR, VpsT, and HapR. In this study, we identified the VpsT regulon and compared the VpsT and VpsR regulons to elucidate the contribution of each positive regulator to the rugose variant transcriptional profile and associated phenotypes. We have found that although the VpsT and VpsR regulons are very similar, the magnitude of the gene regulation accomplished by each regulator is different. We also determined that cdgA, which encodes a GGDEF domain protein, is partially responsible for the altered vps gene expression between the vpsT and vpsR mutants. Analysis of epistatic relationships among hapR, vpsT, and vpsR with respect to a whole-genome expression profile, colony morphology, and biofilm formation revealed that vpsR is epistatic to hapR and vpsT. Expression of virulence genes was increased in a vpsR hapR double mutant relative to a hapR mutant, suggesting that VpsR negatively regulates virulence gene expression in the hapR mutant. These results show that a complex regulatory interplay among VpsT, VpsR, HapR, and GGDEF/EAL family proteins controls transcription of the genes required for Vibrio polysaccharide and virulence factor production in V. cholerae.

Quantification of high-specificity cyclic diguanylate signaling

“…To study the impact of c-di-GMP on transcriptional control, we measured induction of a VpsR target gene. However, because multiple positive-feedback loops exist between VpsR and VpsT (30), and both of these transcription factors are c-di-GMP-dependent, we reasoned a more direct assay for VpsR activation by c-di-GMP was quantification of aphA induction.…”

“…VpsR directly binds to c-di-GMP to increase transcription of vpsT (28). VpsT itself then binds to c-di-GMP (29), and both c-di-GMP- bound VpsR and VpsT are thought to induce expression of the vps genes to generate the extracellular matrix necessary for biofilm formation (30). In addition to vpsT induction, c-di-GMPbound VpsR also induces expression of the gene aphA (28), which encodes a transcription factor that activates a subset of the low-cell-density quorum sensing regulon and virulence gene cascade (31,32).…”

Biofilms in water, its role and impact in human disease transmission

“…Most are driven by a peristaltic pump and include single pass systems and recycling reactors [13]. The biofilm bait can be a silicone tubing, such as is used in catheters, or glass capillary [10,13]. Many less expensive alternatives to flow cells have been described.…”

“…Confocal scanning laser microscopy (CSLM) captures top-down images of mature biofilms that can be viewed in a qualitative manner. Additionally, those images can be sectioned along the xz axis and reconstructed to give sagittal or orthogonal profiles, for which the depth, and subsequently volume, of the biofilm can be quantified [10][11][12]. There are a number of software packages, such as COMSTAT or IMARIS, that can assist in this type of analysis [10,13].…”

“…Additionally, those images can be sectioned along the xz axis and reconstructed to give sagittal or orthogonal profiles, for which the depth, and subsequently volume, of the biofilm can be quantified [10][11][12]. There are a number of software packages, such as COMSTAT or IMARIS, that can assist in this type of analysis [10,13]. Phase contrast light microscopy and epifluorescence microscopy are used primarily to examine early development of biofilms, that is cell attachment and migration to form microcolonies, and in those cases where CSLM is not available [4 ].…”