Acetyl Phosphate as a Global Stress Signal

乙酰磷酸作为整体压力信号

• 批准号: 7057754
• 负责人: ALAN J WOLFE
• 金额: $ 28.9万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057754
• 关键词: Escherichia coli; acetates; bacterial genetics; bioinformatics; biological signal transduction; environmental stressor; gene induction /repression; genetic enhancer element; genetic transcription; microarray technology; microorganism metabolism; phosphoric ester; phosphorylation; polymerase chain reaction; posttranslational modifications; transcription factor

DESCRIPTION (provided by applicant): The network of two-component signaling pathways controls a multitude of genes in response to diverse environmental signals. For example, during their colonization of host tissue, bacterial pathogens use multiple two-component pathways to ensure they express the proper subset of virulence factors. Less well understood is the impact made on this signaling network by the small molecule acetyl phosphate. We recently reported that acetyl phosphate affects expression of about 100 genes involved in the synthesis of flagella, type 1 pili, capsule and stress effectors - structures implicated in biofilm development. In the 1st aim, we will combine microarray technology, bioinformatics and transcriptional analyses to obtain a comprehensive list of uropathogenic and non-pathogenic E. coli genes that respond to acetyl phosphate and the transcription factors that mediate that response. If we find that most of these transcription factors are response regulators, then we will have obtained evidence that acetyl phosphate can influence gene expression through response regulators. An increasing number of published reports rely either explicitly or implicitly on the "fact" that acetyl phosphate acts as a phospho-donor for response regulators in vivo. Although alternative explanations exist, in our opinion, the "direct phospho-donor" model best explains the existing data. Most, but not all, of the connections predicted by this hypothesis have been documented and no new players or mechanisms need to be found or envisioned. In the 2nd aim, we propose to make the final, critical connection - to determine whether in vivo that a specific response occurs because acetyl phosphate donates its phosphate to a specific response regulator. We also will use biochemical means to test the hypothesis that molecular crowding can increase the efficiency of the phosphorylation reaction, thereby increasing the capacity of acetyl phosphate to act as a phospho-donor in vivo. We possess evidence indicating that OmpR plays a previously unknown role required by cells that can synthesize acetyl phosphate. In the 3rd aim, we will elucidate the linkage between acetyl phosphate and OmpR. We will dissect an OmpR-dependent phenotype that requires the synthesis of acetyl phosphate - the propensity of ompR ackA mutants to lyse during late exponential phase. We will test the three specific hypotheses outlined in this aim, and we will identify the suppressor mutations that permit a small subset of cells to escape lysis. This approach should either verify that acetyl phosphate acts directly through response regulators or lead us to alternative explanations.

描述（由申请人提供）：二元信号通路网络控制大量基因以响应不同的环境信号。例如，在宿主组织定植期间，细菌病原体使用多个二元途径来确保它们表达正确的毒力因子子集。人们不太了解小分子乙酰磷酸对这一信号网络的影响。我们最近报道，乙酰磷酸会影响大约 100 个基因的表达，这些基因涉及鞭毛、1 型菌毛、荚膜和应激效应子的合成，这些结构与生物膜的发育有关。第一个目标是，我们将结合微阵列技术、生物信息学和转录分析，获得对乙酰磷酸做出反应的尿路致病性和非致病性大肠杆菌基因以及介导该反应的转录因子的完整列表。如果我们发现这些转录因子大部分都是反应调节因子，那么我们就获得了乙酰磷酸可以通过反应调节因子影响基因表达的证据。越来越多的已发表报告明确或隐含地依赖于乙酰磷酸作为体内反应调节剂的磷酸供体的“事实”。尽管存在其他解释，但我们认为“直接磷酸供体”模型最好地解释了现有数据。该假设预测的大多数（但不是全部）联系已被记录下来，不需要发现或设想新的参与者或机制。在第二个目标中，我们建议建立最后的关键联系 - 确定体内是否会因为乙酰磷酸将其磷酸盐捐赠给特定的反应调节剂而发生特定的反应。我们还将使用生化手段来检验分子拥挤可以提高磷酸化反应的效率，从而提高乙酰磷酸在体内充当磷酸供体的能力的假设。我们拥有的证据表明，OmpR 发挥着细胞合成乙酰磷酸所需的先前未知的作用。在第三个目标中，我们将阐明乙酰磷酸和 OmpR 之间的联系。我们将剖析需要合成乙酰磷酸的 OmpR 依赖性表型 - ompR ackA 突变体在指数后期裂解的倾向。我们将测试该目标中概述的三个具体假设，并且我们将识别允许一小部分细胞逃脱裂解的抑制突变。这种方法应该要么验证乙酰磷酸直接通过反应调节剂起作用，要么引导我们找到替代的解释。