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对不同环境信号的响应。例如，在它们在宿主组织的定殖期间，细菌病原体使用多个双组分途径来确保它们表达毒力因子的适当子集。不太清楚的是小分子乙酰磷酸对这个信号网络的影响。我们最近报道，乙酰磷酸盐影响约100个基因的表达，这些基因参与鞭毛、1型皮利、荚膜和应激效应子的合成，这些结构与生物膜的发育有关。第一个目标是将联合收割机技术、生物信息学和转录分析相结合，获得一个完整的泌尿系致病性和非致病性大肠杆菌的列表。大肠杆菌中对乙酰磷酸起反应的基因以及调节这种反应的转录因子。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”。虽然存在其他解释，但我们认为，“直接磷酸供体”模型最能解释现有数据。这一假说所预测的大部分（但不是全部）联系都已被记录在案，不需要找到或设想新的参与者或机制。在第二个目标中，我们提出了最后的，关键的连接-以确定是否在体内发生的特定反应，因为乙酰磷酸捐赠其磷酸盐的特定反应调节。我们还将使用生物化学手段来测试分子拥挤可以增加磷酸化反应的效率，从而增加乙酰磷酸作为体内磷酸供体的能力的假设。我们拥有的证据表明，OmpR发挥了以前未知的作用所需的细胞，可以合成乙酰磷酸。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，需要乙酰磷酸的合成-ompR ackA突变体的倾向，在后期指数期裂解。我们将测试在这个目标中概述的三个特定的假设，我们将确定抑制突变，允许一小部分细胞逃避溶解。这种方法应该验证乙酰磷酸直接通过反应调节剂起作用，或者引导我们找到替代解释。

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.