Acetyl Phosphate as a Global Stress Signal

描述（由申请人提供）：双组分信号通路网络控制多种基因响应不同的环境信号。例如，在宿主组织定植期间，细菌病原体使用多种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个信号网络的影响还不太清楚。我们最近报道了乙酰磷酸影响大约100个基因的表达，这些基因与鞭毛、1型毛、荚膜和胁迫效应物的合成有关，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尿路致病性和非致病性大肠杆菌基因的全面列表，这些基因对乙酰磷酸酯和介导这种反应的转录因子有反应。如果我们发现这些转录因子大部分都是应答调节因子，那么我们就获得了乙酰磷酸通过应答调节因子影响基因表达的证据。越来越多的已发表的报告明确或含蓄地依赖于乙酰磷酸作为体内反应调节因子的磷酸供体这一“事实”。虽然存在其他解释，但在我们看来，“直接磷供体”模型最能解释现有数据。这一假说所预测的大部分联系（但不是全部）都已被记录在案，不需要发现或设想新的参与者或机制。在第二个目标中，我们建议进行最后的关键连接-确定体内特异性反应是否因为乙酰磷酸将其磷酸盐捐赠给特定反应调节剂而发生。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效率的假设，从而增加乙酰磷酸在体内作为磷酸供体的能力。我们有证据表明，OmpR在合成乙酰磷酸的细胞中起着以前未知的作用。在第三个目标中，我们将阐明乙酰磷酸和OmpR之间的联系。我们将剖析一种需要合成乙酰磷酸的ompR依赖表型——ompR ackA突变体在指数期后期裂解的倾向。我们将测试在这个目标中概述的三个特定假设，我们将确定允许一小部分细胞逃避裂解的抑制突变。这种方法要么可以验证乙酰磷酸直接通过反应调节因子起作用，要么可以为我们提供其他解释。

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.