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The objectives of this cooperative research project are to construct gene microarrays for E. coli O157:H7 strain EDL933. These microarrays will be used to identify differences in gene expression of E. coli O157:H7 that are grown or exposed to different environmental conditions both in vivo and in vitro. E. coli O157:H7 genes that are preferentially expressed in vivo will be candidates for further evaluation to determine their specific role in colonization, survival, and persistence in cattle.
The published genomic sequence of E. coli O157:H7 will be used to design primers for PCR amplification of the 3574 open reading frame (ORFs) that are common to both O157 and K-12 strains, the 1387 O157 unique ORFs, the 528 K-12 unique ORFs, and the 100 genes that are carried on the pO157 plasmid present in O157:H7 E. coli. The PCR products will be spotted in a microarray and used in hybridization assays. RNA isolated from O157:H7 E. coli grown or exposed to different in vivo and in vitro conditions will be used to produce labeled cDNA for hybridization. This will be used to identify genes that are differentially expressed under a variety of different conditions both in vitro and in vivo.