EXITING STATIONARY PHASE IN E COLI

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DESCRIPTION: Bacterial infections remain a leading cause of illness and death. To control bacterial pathogens it is important to understand not only host-pathogen interactions, but also how the pathogen's life cycle is affected by the environment. Bacteria can survive a variety of environmental stresses and still rapidly resume growth when conditions become favorable. This ability is an important factor in disease transmission and can also affect the initial stages of infection. The long-term goal of this project is to understand the mechanisms that regulate gene expression as the enteric bacterium E coli recovers from starvation stress. Mechanisms we identify are likely to be important in the infection cycle of pathogenic E. coli and other pathogenic bacteria. A group of nine E. coli proteins are transiently expressed during the initial period of outgrowth from stationary phase. The limited time when these proteins are made suggests that they are controlled by factors involved in regulating important aspects of the outgrowth response. The immediate goal of this proposal is to identify the genes encoding outgrowth-specific proteins. Once identified, these genes will be used as reporters to study the regulation of the outgrowth response. The first specific aim is to identify genes encoding outgrowth-specific proteins by obtaining amino acid sequence information from the outgrowth-specific proteins already described. State-of-the-art mass spectrometry methods will be used to analyze gel-purified proteins. The sequence information obtained will be used to identify the genes in the completed E. coli genome sequence. The second aim is to identify transcripts made specifically during outgrowth using PCR based differential display. The sequence of the differentially expressed cDNAs will be compared to the E. coli genome sequence to identify the genes. Once outgrowth-specific genes are identified, their function. and the mechanisms regulating their expression will be studied. Mutations will be constructed and the phenotypes of mutants characterized. Standard molecular methods, such as Northern blots and fusions to reporter genes, will be used to characterize how expression is the outgrowth-specific genes is controlled.