Regulation of Biofilm Formation in Vibrio cholerae

霍乱弧菌是一种革兰氏阴性的活动性细菌病原体，可引起胃肠道疾病霍乱。在霍乱流行的地区，疾病的发生与季节模式和气候变化相似，而在霍乱非流行的地区，致病菌的引入加上卫生条件差，导致细菌通过粪-口途径迅速传播。尽管在人类宿主中引起疾病，但霍乱弧菌是一种天然的水生生物，必须在无数的环境中生存，包括热带和温带水域。快速检测和适应波动的环境变化对细菌的生存和增殖至关重要。特别是，霍乱弧菌必须调节生物膜的形成，因为生物膜有助于在水生环境中生存并传播给人类宿主。像其他微生物一样，霍乱弧菌可以感知大量的细胞外信号；这些信号的传递会导致基因表达的变化。在细菌中，一个主要的信号通路是双组分信号转导系统（TCS），由一个传感激酶（SK）和一个可溶性细胞质反应调节剂（RR）组成。

Vibrio cholerae is a Gram-negative, motile bacterial pathogen that causes the gastrointestinal disease cholera. In areas where cholera is endemic, disease occurrence parallels seasonal pattern and climate changes, while in regions where cholera is non-endemic, the introduction of the pathogenic bacteria together with poor sanitation leads to rapid bacterial dissemination via the fecal-oral route. Despite causing illness in human hosts, V. cholerae is a natural aquatic inhabitant and must survive in myriad environmental niches, including both tropical and temperate waters. Rapid detection and adaptation to fluctuating environmental changes are essential for bacterial survival and proliferation. In particular, V. cholerae must regulate biofilm formation since biofilm facilitates survival in the aquatic environment and transmission to the human host. Like other microorganisms, V.cholerae senses a multitude of extracellular signals; transmission of those signals then lead to changes in gene expression. In bacteria, a major signaling pathway is the two-component signal transduction system (TCS), comprised of a sensor kinase (SK) and a soluble cytoplasmic response regulator (RR) We have previously investigated how the V. cholerae RR, 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 as well as at 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. We found that while VpsR shares sequence 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 have 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. Like other RRs, VpsR has a conserved residue (D59) that has been predicted to be a site of phosphorylation. However, the phosphorylation status of VpsR has been unknown, a possible SK responsible for phosphorylating VpsR has not been identified, and the role of phosphorylation, if any, in VpsR transcriptional activation has not been determined. Consequently, we asked whether VpsR is directly phosphorylated and what factors besides c-di-GMP can affect its activity, using the phospho-mimic variant D59E, the phospho-defective variant D59A, and a purified denatured/renatured VpsR (VpsRren). We find that in vitro VpsRren preincubated either with AcP, which can chemically donate phosphate (Pi), or with sodium phosphate or potassium phosphate, which are not able to phosphorylate VpsR, activates transcription from PvpsL when concentrations of c-di-GMP are lower. However, as the concentration of c-di-GMP increases, the need for Pi diminishes. In vivo, we observe that the presence of AcP is needed for PvpsL activation when the level of c-di-GMP is low, and we observe an increase of vpsL expression from low to high phosphate at unaltered concentrations of c-di-GMP. Our results argue that the activity of VpsR is affected by the presence of either AcP or Pi, rather than by protein phosphorylation, and that either of these molecules together with c-di-GMP directly modulate the ability of VpsR to activate biofilm formation. We conclude that an intricate connection between phosphate and c-di-GMP levels modulates V. cholerae biofilm formation.