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<UL> <LI>Identify appropriate primers and PCR amplify the promoters and upstream regions for eaeA, stx2, hns, and rpoS. <LI> Ligate the amplified promoters into a promoter-less fluorescent protein vector. <LI> Transform into E. coli O157:H7 and compare the survival with the plasmid-free parent strain. <LI>Monitor the promoter-EFP expression during growth and following exposure to various physiochemical parameters. <LI> Conduct studies on ground beef inoculated with the engineered strains to determine the utility of these strains in identifying food-related parameters that influence the expression of these virulence and regulatory genes.
Approach: The proposed research will involve cooperative research between the MFS and UW to produce a set of E. coli O157:H7 strains with reporter genes linked to primary virulence markers. The resulting strains will permit us to examine linkages between the expression of virulence genes to the kinetics of bacteria growth and inactiviation, thus identifying food conditions that may control the virulence of E. coli O157:H7. The approach also addresses inherent limitations of microarray technology where higher cell numbers are required to measure nucleic acid signals. In contrast, these reporter strains can be studied in situ at very low levels that are relevant to food contaminaiton and human infection. The data will enhance the value of our models to ARS customers, especially risk assessors.