Frederick R. Blattner

Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC′DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC′DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim−) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.

Urease of enterohemorrhagic Escherichia coli: Evidence for regulation by Fur and a trans-acting factor

ABSTRACT Recent genomic analyses of Escherichia coli O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (ureDABCEFG), which are absent in nonpathogenic E. coli strain K-12. This report demonstrates that the cloned EDL933 ure gene cluster is capable of synthesizing urease in an E. coli DH5α background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified trans-acting factor in enterohemorrhagic E. coli (EHEC) is responsible for this regulation of ure expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of ureD and ureA. Enzymatic assays confirmed a difference in urease expression of cloned EHEC ure clusters in E. coli MC3100Δfur. Likewise, interruption of fur in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of Salmonella enterica serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.