Isolation and characterization of novel virulence factors of enteropathogenic Escherichia coli

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The knew findings obtained from this project are follows ;1. The bfpTVW operon of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. One of promoters, thus selected, was positively regulated by bfpTVW and is not present in non-virulent Ecoli strains. This promoter was found to be located in a 4.9 kb EPEC specific sequence and upstream of orfl, which was revealed to encode a chaperone-like protein involving expression of BFP. Hence, orfl was designated trcA (bfpT-regulated chaperone-like protein gene) and the EPEC-specific region was LIM (Locus for improving microcolony formation).2. The complete nucleotide sequence and organization of the EAF plasmid of EPEC, strain B171 (Olll:NM), were determined. Besides the bfp and bfpTVW operons, the 69 kb EAF plasmid, henceforth designated pB171, contains potential virulence-associated genes. Of the newly identified ORFs, two ORFs which comprise a single operon were found that would encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E coli. Another ORF showed high similarity with trcA of EPEC. Two sites were found to be putative replication regions, one of that is similar to RepFIIA of p307 or F and the other to RepFIB of NR1. In addition to these, a third region was identified that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.