Regulation of Biofilm Formation in Vibrio cholerae

霍乱弧菌是一种革兰氏阴性、能动的细菌病原体，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平行，而在霍乱非流行的地区，病原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。尽管霍乱弧菌在人类宿主中引起疾病，但它是一种天然的水生生物，必须在无数的环境小生境中生存，包括热带和温带沃茨。 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存活并传播给人类宿主。 像其他微生物一样，霍乱弧菌能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），由传感激酶（SK）和可溶性胞质反应调节因子（RR）组成 我们先前已经研究了霍乱弧菌RR、VpsR与小分子环状二GMP（c-di-GMP）通过表征来自PvpsL（vps-II生物膜生物合成操纵子起始处的启动子）以及生物膜形成簇内的其它启动子的VpsR激活的转录来调节绿球藻生物膜形成：PvpsU，vps-I操纵子起始处; PrbmA，在rbm簇的开始处;和PrbmF，其位于趋异的rbmF/E基因的上游。 我们发现，虽然VpsR共享序列同源性增强子结合蛋白激活sigma 54-RNA聚合酶，其激活PvspL使用sigma 70-RNA聚合酶。有趣的是，c-di-GMP不显著改变VpsR对PvpsL DNA的亲和力或VpsR在DNA上的DNA酶I足迹，并且VpsR不需要二聚化。然而，DNase I和KMnO 4足迹显示，PvpsL上的sigma 70-RNA聚合酶/ VpsR/c-di-GMP复合物采用与单独的sigma 70-RNA聚合酶、c-di-GMP或VpsR形成的构象不同的构象。我们的研究结果表明，c-di-GMP是VpsR产生激活转录所需的特定蛋白质-DNA结构所必需的，这是c-di-GMP在基因表达中以前未被认识到的作用。 与其他RR一样，VpsR具有保守残基（D59），该残基被预测为磷酸化位点。 然而，VpsR的磷酸化状态一直是未知的，一个可能的SK负责磷酸化VpsR尚未确定，磷酸化的作用，如果有的话，在VpsR转录激活尚未确定。 因此，我们询问VpsR是否直接磷酸化，以及除了c-di-GMP之外还有什么因素可以影响其活性，使用磷酸模拟变体D59 E，磷酸缺陷变体D59 A和纯化的变性/复性VpsR（VpsRren）。 我们发现，在体外VpsRren预孵育与AcP，它可以化学捐赠磷酸盐（Pi），或与磷酸钠或磷酸钾，这是不能磷酸化VpsR，激活转录从PvpsL时，c-di-GMP的浓度较低。然而，随着c-di-GMP浓度的增加，对Pi的需求减少。在体内，我们观察到当c-di-GMP水平低时，AcP的存在是PvpsL激活所需的，并且我们观察到在c-di-GMP浓度不变的情况下，vpsL表达从低磷酸盐增加到高磷酸盐。 我们的研究结果认为，VpsR的活性受到AcP或Pi的存在的影响，而不是通过蛋白质磷酸化，并且这些分子中的任何一个与c-di-GMP一起直接调节VpsR激活生物膜形成的能力。 我们得出结论，磷酸盐和c-di-GMP水平之间的错综复杂的连接调节霍乱弧菌生物膜的形成。

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.