Acetyl Phosphate as a Global Stress Signal

乙酰磷酸作为整体压力信号

• 批准号: 7229512
• 负责人: ALAN J WOLFE
• 金额: $ 28.07万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229512
• 关键词: Affect; Anabolism; Bacteria; Binding; Biochemical; Biogenesis; Bioinformatics; Biological Assay; Cells; Condition; Crowding; Cytolysis; DNA; Data; Development; Ensure; Environment; Escherichia coli; Feedback; Flagella; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Harvest; In Vitro; Knowledge; Lead; Learning; Mediating; Metabolism; Microarray Analysis; Microbial Biofilms; Modeling; Molecular; Numbers; Pathogenesis; Pathway interactions; Phase; Phenotype; Phosphorylation; Pilum; Play; Proteins; Publishing; Reaction; Reporter; Reporting; Repression; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stress; Structure; Suppressor Mutations; Testing; Time; Tissues; Virulence Factors; acetyl phosphate; base; capsule; in vivo; inorganic phosphate; mutant; pathogen; pathogenic Escherichia coli; pill; protein-histidine kinase; research study; response; small molecule; 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(申请人提供)：双组分信号通路网络控制多种基因，以响应不同的环境信号。例如，在寄主组织的定植过程中，细菌病原体使用多个双组分途径来确保它们表达适当的毒力因子子集。小分子乙酰磷酸对这一信号网络的影响还不是很清楚。我们最近报道了乙酰磷酸影响大约100个基因的表达，这些基因参与了鞭毛、1型菌毛、被膜和应激效应的合成-这些结构与生物膜的发育有关。在第一个目标中，我们将结合微阵列技术、生物信息学和转录分析来获得对乙酰磷酸反应的泌尿系致病和非致病大肠杆菌基因以及介导这一反应的转录因子的全面清单。如果我们发现这些转录因子中的大多数是反应调节因子，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。越来越多的已发表的报告或明确或隐含地依赖于这样一个事实，即乙酰磷酸在体内作为反应调节器的磷酸供体。尽管存在其他解释，但在我们看来，“直接磷供体”模型最好地解释了现有数据。这一假说预测的大部分(但不是全部)联系已经被记录在案，不需要找到或设想新的参与者或机制。在第二个目标中，我们建议进行最后的、关键的连接--确定在体内是否因为乙酰磷酸将其磷酸盐贡献给特定的反应调节器而发生特定的反应。我们还将使用生化手段来验证分子拥挤可以提高磷酸化反应的效率，从而增加乙酰磷酸在体内作为磷酸供体的能力的假说。我们有证据表明，OmpR在合成乙酰磷酸的细胞中扮演着一个以前未知的角色。在第三个目标中，我们将阐明乙酰磷酸和OmpR之间的联系。我们将剖析一个依赖OmpR的表型，它需要合成乙酰磷酸--ompR ackA突变体在指数后期裂解的倾向。我们将测试这一目标中概述的三个具体假设，并确定允许一小部分细胞逃脱裂解的抑制突变。这种方法应该要么证实乙酰磷酸直接通过反应调节器起作用，要么引导我们找到另一种解释。