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(QS)是种群密度依赖的基因调控的一种保守机制。在革兰氏阴性细菌中，这通常涉及自身产生的酰基高丝氨酸内酯(AHL)信号的释放和感知。已获得一项拨款，用于研究斯图尔特枯萎病病原菌(Pantoea Stewartii)群体感应控制荚膜多糖(CPS)合成的机制。亚种。斯图瓦蒂。P.stewartii群体感应的关键调控成分是Esai酰基高丝氨酸内酯合成酶和同源ESAR反应调节因子。ESAI基因的破坏会导致CPS缺乏，而ESAR基因的失活会导致CPS的过度生产或粘液过多。这两种情况都扰乱了玉米斯图尔特枯萎病的正常发展。ESAR是一种LuxR同源物，它通过基因抑制和AHL依赖的去抑制来控制其自身的表达。推测，通过抑制的群体感应在CPS合成的控制中也起着作用。目前的模型预测，ESAR通过直接抑制由CPS调节子编码的基因或其他可能与高粘液表型有关的生物合成功能来抑制CPS的合成。或者，ESAR可能通过中介转录因子间接调控CPS的合成。该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。将建立一个共识的ESAR结合序列，以帮助在缺乏明确的Lux盒样DNA靶标的情况下定位这些位点。其次，对野生型和高粘液型菌株的脂肪酸和胞外聚合物组成进行比较分析，以确定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Tn5的转座子对高粘液性突变菌株进行详尽的转座子突变，将被用于分离非粘液性荧光菌株。引入功能性ESAR基因后，荧光增强或减弱的菌株将被反向遗传学分析和表征，以确定对包膜合成和ESAR控制至关重要的结构和/或调控基因。最后，将使用双向凝胶电泳法对ESAR控制的蛋白质产品进行分析。预期的结果将产生对看似非传统的群体感应控制机制的全面理解，并有助于从新的角度研究表面聚合物在动植物发病中的作用。

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.