Quorum Sensing Mediated Regulation of Exopolymer Synthesis in Pantoea stewartii: A Critical Factor in Surface Attachment, Biofilm Formation, and Host Invasion

群体感应介导的泛菌外聚合物合成调节：表面附着、生物膜形成和宿主入侵的关键因素

• 批准号: 0211687
• 负责人: Susanne von Bodman
• 金额: $ 31.7万
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0211687&HistoricalAwards=false
• 关键词: Quorum; Sensing; Mediated; Regulation; Exopolymer

Quorum Sensing (QS) is a conserved mechanism of population density-dependent gene regulation. In Gram-negative bacteria, this generally involves the release and perception of self-produced acyl-homoserine lactone (AHL) signals. A grant has been awarded to study the mechanism of quorum sensing control of capsular polysaccharide (CPS) synthesis in the Stewart's wilt pathogen, Pantoea stewartii. subsp. stewartii. The critical regulatory components of quorum sensing in P. stewartii are the EsaI acyl-homoserine lactone synthase and the cognate EsaR response regulator. Disruption of the esaI gene leads to CPS deficiency, while the inactivation of the esaR gene leads to CPS overproduction, or hypermucoidy. Both conditions disrupt the normal development of Stewart's wilt disease in maize. EsaR, a LuxR homologue, functions by gene repression and AHL-dependent derepression in the control of its own expression. Presumably, quorum sensing by repression plays a role also in the control of CPS synthesis. The current model predicts that EsaR represses CPS synthesis by direct repression of genes encoded by the cps regulon or other biosynthetic functions that may contribute to the hypermucoid phenotype. Alternatively, EsaR may govern CPS synthesis indirectly through intermediary transcription factors. The first objective of this project is to define relevant transcript start sites upstream and within the cps gene system to identify potential EsaR contact sites. A consensus EsaR binding sequence will be established to aid the localization of such sites in absence of defined lux box-like DNA targets. Second, a comparative analysis of the fatty acid and exopolymer composition of the wild type and hypermucoid strains will be used to determine whether deregulated CPS synthesis is primarily a function of the cps gene system and/or alternate biosynthetic pathways. Third, exhaustive transposon mutagenesis of the hypermucoid mutant strain using a Tn5-based transposon with a promoterless green fluorescent protein reporter function will be used to isolate nonmucoid, fluorescent strains. Strains showing enhanced or depressed fluorescence after introduction of a functional esaR gene will be analyzed and characterized by reverse genetics to identify structural and/or regulatory genes critical for capsule synthesis and control by EsaR. Finally, two-dimensional gel electrophoresis will be employed to profile EsaR-controlled protein products. The expected results will generate a comprehensive understanding of a seemingly unconventional quorum sensing control mechanism and contribute to a novel perspective on the role of surface polymers in plant and animal pathogenesis.

群体感应（Quorum Sensing，QS）是一种保守的群体密度依赖性基因调控机制。在革兰氏阴性菌中，这通常涉及释放和感知自身产生的酰基高丝氨酸内酯（阿勒）信号。已获得一笔赠款，用于研究斯图尔特枯萎病病原体Pantoea stewartii中群体感应控制荚膜多糖（CPS）合成的机制。亚种stewartii斯氏毕赤酵母群体感应的关键调节组分是EsaI酰基高丝氨酸内酯合酶和同源EsaR反应调节剂。esaR基因的破坏导致CPS缺陷，而esaR基因的失活导致CPS过度产生或高粘液性。这两种情况都破坏了玉米中斯图尔特枯萎病的正常发展。EsaR是LuxR的同源物，通过基因阻遏和AHL依赖性去阻遏来控制其自身的表达。据推测，群体感应抑制在CPS合成的控制中也起作用。目前的模型预测EsaR通过直接抑制cps调节子编码的基因或其他可能导致高粘液表型的生物合成功能来抑制CPS合成。或者，EsaR可能通过中间转录因子间接控制CPS合成。本项目的第一个目标是确定cps基因系统上游和内部的相关转录起始位点，以确定潜在的EsaR接触位点。将建立共有EsaR结合序列，以帮助在不存在确定的勒克斯盒样DNA靶标的情况下定位这些位点。其次，将使用野生型和高类粘蛋白菌株的脂肪酸和外聚合物组成的比较分析来确定解除调节的CPS合成是否主要是cps基因系统和/或替代生物合成途径的功能。第三，使用具有无启动子绿色荧光蛋白报告功能的基于Tn 5的转座子对高粘液突变株进行穷举转座子诱变，将用于分离非粘液荧光菌株。将通过反向遗传学分析和表征在引入功能性esaR基因后显示增强或抑制的荧光的菌株，以鉴定对胶囊合成和EsaR控制至关重要的结构和/或调控基因。最后，将采用二维凝胶电泳来分析EsaR控制的蛋白质产物。预期的结果将产生一个看似非常规的群体感应控制机制的全面理解，并有助于对植物和动物的发病机理的表面聚合物的作用的新的观点。