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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Anderson, James J
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Loyola University Chicago
Schools of Medicine
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