Regulation of a gene associated with c-di-GMP production and biofilm formation in Vibrio cholerae

项目摘要/摘要 霍乱是一种腹泻疾病，其特征是危及生命的脱水。它是由血吸虫的血清型引起的 产生霍乱毒素的革兰氏阴性菌霍乱弧菌。一种与地方性疾病有关的菌株 霍乱暴发是O1血清型El Tor。霍乱弧菌El Tor形成生物膜，具有保护性 细菌不受环境压力影响的群落。第二信使二-(3‘-5’)- 环-二鸟苷一磷酸(c-di-GMP)是运动生物膜向固着生物膜过渡的关键。 生活方式，高水平的c-di-GMP与生物膜的形成有关。C-di-GMP的合成需要 二鸟苷环化酶。由VCA0956编码的一种这样的二鸟苷环化酶，命名为 与生物膜的形成和高感染性细菌聚集体的形成有关 在晚期感染期间形成，并从肠道释放，为细菌重新进入肠道做准备 水环境。调控cdgF表达的机制尚不清楚。在这个项目中，我们 我将研究cdgF表达受不同编码的Marr家族控制的假设 转录因子，我们将其命名为DgcR，DgcR与配体c-di-GMP反应，从而产生一个 支持c-di-GMP合成的正反馈环。我们还提出了cdgF的初始表达式 当DgcR被氧化时发生，这一事件削弱了其结合cdgF启动子和抑制的能力 抄写。DgcR的DNA和配体结合将在体外通过DNase I足迹和通过 DgcR在没有和存在配体的情况下的生物物理分析。体内调控cdgF启动子活性的研究 在大肠杆菌中使用cdgF启动子-报告结构也表达可诱导的dgcR，并且 C-di-GMP或氧化剂抑制基因表达的能力将被确定。体外转录检测 将被实施以补充体内检测。拟议中的实验有望完成。 描绘了一种c-di-GMP维持自身合成的新型反馈系统，并有望 确定氧化应激和c-di-GMP积累之间的直接联系。知道通过什么机制 产生临床上相关的cdgF酶对于更好地理解El Tor霍乱弧菌是重要的 致病性，它将为干扰这一调控开辟前景，从而防止 常规抗菌剂对其无效的生物膜群落。

PROJECT SUMMARY/ABSTRACT Cholera is a diarrheal disease characterized by life-threatening dehydration. It is caused by serotypes of the Gram-negative bacterium Vibrio cholerae, which produce cholera toxin. One of the strains linked to endemic cholera outbreaks is the O1 serotype El Tor. V. cholerae El Tor forms biofilms, which are protective communities in which the bacteria are shielded from environmental stress. The second messenger bis-(3'-5')- cyclic-diguanylate monophosphate (c-di-GMP) is key to the transition between motile and sessile (biofilm) lifestyles, with high levels of c-di-GMP associated with biofilm formation. Synthesis of c-di-GMP requires diguanylate cyclase enzymes. One such diguanylate cyclase, which is encoded by VCA0956 and named CdgF, has been linked to biofilm formation and to formation of the hyper-infective bacterial aggregates that form during late infection and are released from the intestinal tract in preparation for bacterial re-entry into the aqueous environment. Mechanisms by which cdgF expression is regulated are unknown. In this project, we will investigate the hypotheses that cdgF expression is controlled by the divergently encoded MarR family transcription factor that we named DgcR, and that DgcR responds to the ligand c-di-GMP, thus creating a positive feed-back loop that sustains c-di-GMP synthesis. We also propose that initial expression of cdgF occurs when DgcR is oxidized, an event that attenuates its ability to bind the cdgF promoter and repress transcription. DNA and ligand binding by DgcR will be determined in vitro by DNase I footprinting and by biophysical analyses of DgcR in absence and presence of ligand. Control of cdgF promoter activity in vivo will be addressed using cdgF promoter-reporter constructs in E. coli also expressing inducible dgcR, and the ability of c-di-GMP or oxidant to de-repress gene expression will be determined. In vitro transcription assays will be implemented to complement the in vivo assays. Completion of the proposed experiments is expected to delineate a novel feed-back system in which c-di-GMP sustains its own synthesis, and it is expected to define a direct link between oxidative stress and c-di-GMP accumulation. Knowing the mechanisms by which the clinically relevant CdgF enzyme is produced is important for a better understanding of V. cholerae El Tor pathogenicity, and it will open prospects for interfering with this regulation and hence prevent formation of the biofilm communities against which conventional antimicrobial agents are ineffective.