An official website of the United States government.
This project will construct a set of reporter strains that harbor stx2 (most toxic of the Shiga toxins) and eaeA promoters fused to green and red fluorescent protein that emit different wavelengths of light. In addition to these two virulence factors, the promoters from two dominant global regulators, H-NS and RpoS, will be fused to variant GFP to monitor regulatory circuits involved in growth and stress tolerance.<P> This approach also addresses inherent limitations of microarray technology where high 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 contamination and human infection. The inoculation of foods with the constructed cocktail of reporter strains should provide insight into the influence of intrinsic food parameters and storage conditions on the growth, stress tolerance, and virulence of E. coli O157:H7.
Non-Technical Summary: Infections by entrohemorrhagic Escherichia coli (EHEC) can result in diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Considerable research has focused on the identification of virulence factors associated with human infections; however, comparatively little has been done on the impact of the intrinsic factors of foods or storage conditions on the expression of these virulence genes. This project will construct a set of reporter strains to monitor expression of key virulence, growth, and stress tolerance genes. The inoculation of foods with the constructed cocktail of reporter strains should provide insight into the influence of intrinsic food parameters and storage conditions on the growth, stress tolerance, and virulence of E. coli O157:H7. <P> Approach: The construction of a set of reporter strains is necessary for food-inoculation studies to monitor the impact of intrinsic and extrinsic factors of a food on the expression of genes involved in growth, stress tolerance, and virulence of E. coli O157:H7. We propose to construct a set of E coli O157:H7 strains with promoters of key growth and stress regulators as well as two virulence genes fused to variants of green and red fluorescent proteins. The promoter and at least 800-bp region upstream from the four-target genes will be fused with one of the fluorescent reporter proteins. Two of the genes to be monitored will be virulence genes, eaeA and stx2 , encoding for intimin and Shiga like toxin II, respectively. The remaining two genes that will be monitored are global regulators, hns and rpoS, associated with growth and metabolism and stress tolerance. Initially, the promoters from these four genes will be amplified by PCR and ligated into a promoterless enhanced fluorescent protein vector (pEFP-1 series and pDsRed-1, Clontech). These plasmid constructs will be transformed or electroporated into a parental E. coli O157:H7 strain associated with human illness. The growth and survival characteristics of the plasmid-containing strains will be compared with the parent strain to check for phenotypic differences. In the case of plasmid-mediated differences in growth or survival, the promoter-fluorescent protein cassette will be cloned and inserted into a BamH1 cut site of the non-functional uidA gene (encodes for B-glucuronidase) in E. coli O157:H7. The resulting plasmid (pBR322 derivative) will harbor the promoter-fluorescent protein cassette flanked by approximately 200 bp of uidA, which will be inserted into the suicide vector pCVD422 that will be conjugated into the O157:H7 recipient strain following previously defined procedures. Transconjugates containing the promoter-fluorescent protein cassette homologously recombined into the non-function gene target will be selected for further study. Following construction of the strains and comparative growth and survival analyses with the parent strain, the constructed set of strains will be used in pure culture studies to optimize detection and ascertain the level of sensitivity of each of the expressed fluorescent proteins. The ARS collaborator (Tamplin) will measure E. coli O157:H7 gene expression in ground beef and integrate the data into the existing PMP model. The inoculated ground beef will be incubated at different temperatures, samples removed, and homogenates prepared and analyzed for fluorescence microscopically and/or with a fluorometer.