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<p>The production of Stx is central to human pathogenesis of ECO157 and the development of life-threatening HUS. The genes for Stx are located in prophage that resides in the ECO157 chromosome. The production and release of Stx is tied to the induction of stx prophage; however, there is a lack of information on conditions or constituents of the intestinal tract that trigger stx prophage into their lytic cycle. This proposal will address this lack of information with the following four objectives.</p><p><ol><li> Optimize quantitative measurements for stx phage.</li><li> Evaluate intestinal tract conditions and constituents for stx phage induction.</li><li> Test intestinal tract conditions for impacts on prophage regulators; cI, cro, recA, and HflA (FtsH) protease. 3a. Screen for protein-(cI, cII, and cro) interactions by immunoprecipitation.</li><li>(4.) Confirm role of candidate effecters in stx prophage regulation using deletion mutants and plasmid-based expression.</li></ol></p>
<p>A qPCR method for monitoring stx phage induction has been reported to detect higher levels of stx phage than traditional plaque assays (Rooks et al., 2010). This may be due to multiplicity reactivation where multiple phage chromosomes, many with a damaged defective chromosome, produce function phage particles through complementation. However, the study comparing qPCR and a plaque assay for stx phage, did not screen or engineer host strains for optimal numbers of pfu. In this study, an optimal host strain(s) will be identified or engineered to optimize the plaque assay. An optimal plaque assay will ultimately be needed in future studies to generate sufficient phage particles for sequencing. We expect to find differences in stx phage production between test strains and identify a combination of conditions and intestinal tract constituents that trigger the induction of prophage. The use of Stx as a reporter will enable us to detect the induction of stx genes should they become disconnected from prophage induction or in the case of Stx1, the pStx1 promoter is preferentially used. The rationale for further characterization of the regulators of the lambda life cycle is that STEC are frequently polylysogens that may contain 18 or more prophage remnants that could impact stx prophage regulation. There are many unanswered questions related to polylysogens and prophage-like elements. Prophage-like elements have been shown to contribute to the formation of phage mosaics in which defective phage are complemented by genes present in prophage-like elements (Asadulghani et al., 2009). It is possible that new effecter proteins of regulators for the lambda lysogenic/lytic cycles will be identified but this result is not anticipated. Rather it is expected that new information on the timing of known lambda regulators with effecter proteins and the persistence of these protein-protein interactions will be obtained in conditions simulating the intestinal tract. There is a paucity of information on prophage regulation in polylysogens and hosts with multiple prophage-like elements. Deletion mutants and/or plasmid constructs with an inducible promoter will be used to establish the role of an effecter protein in the lysogenic/lytic cycles of stx prophage. The information obtained from this and previous objectives will be applied to future studies to control STEC in their bovine reservoir by prophage-mediated lysis.</p>