SummaryEnteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram-negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, de®ning transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid-encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single-copy lacZ fusions in K-12-derived strains, we determined that Per only directly activated the LEE1::lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single-copy LEE2::lacZ and LEE3::lacZ fusions in trans and modestly increased the expression of LEE4::lacZ in K-12 strains. Orf1 was therefore designated Ler, for LEE-encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

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Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. ⌬ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important enteric pathogens for humans. EPEC is the most common bacterial cause of diarrhea in infants (35), while EHEC, especially those of serotype O157: H7, are important emerging pathogens causing diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (35). Central to the pathogenesis of both EPEC and EHEC infections is the formation of attaching and effacing (AE) lesions on infected host intestinal epithelial cells. The AE lesion is characterized by the loss of microvilli (effacement) and the induction of a pedestal of polymerized actin and other cytoskeletal elements that forms underneath and around the infecting bacterium (13,24,35). In EPEC strain E2348/69, the AE phenotype is encoded by a 35.6-kb pathogenicity island, the locus of enterocyte effacement (LEE) (31, 32). The LEE contains genes encoding an outer membrane protein (intimin), a type III secretion system (Esc, Sep, and Ces proteins), secreted proteins (Esp), and the translocated intimin receptor (Tir), as well as a number of open reading frames of undetermined function (8). These genes are also found in the same organization on the LEE of EHEC (36) and are necessary but not sufficient for AE lesion formation by EHEC in vitro (11).In addition to the LEE pathogenicity island and the AE phenotype, other parts of the genome in both EPEC and EHEC encode additional virulence factors and pathogenic mechanisms. The EPEC virulence plasmid encodes the regulator Per (18) and the type IV bundle-forming pili (BFP) (16), which are necessary both for in vitro EPEC adherence to HEp-2 cells in the characteristic localized-adherence pattern and for full virulence in humans (2). The EHEC virulence plasmid encodes a large...

We report the complete 43,359-bp sequence of the locus of enterocyte effacement (LEE) from EDL933, an enterohemorrhagicEscherichia coli O157:H7 serovar originally isolated from contaminated hamburger implicated in an outbreak of hemorrhagic colitis. The locus was isolated from the EDL933 chromosome with a homologous-recombination-driven targeting vector. Recent completion of the LEE sequence from enteropathogenic E. coli (EPEC) E2348/69 afforded the opportunity for a comparative analysis of the entire pathogenicity island. We have identified a total of 54 open reading frames in the EDL933 LEE. Of these, 13 fall within a putative P4 family prophage designated 933L. The prophage is not present in E2348/69 but is found in a closely related EPEC O55:H7 serovar and other O157:H7 isolates. The remaining 41 genes are shared by the two complete LEEs, and we describe the nature and extent of variation among the two strains for each gene. The rate of divergence is heterogeneous along the locus. Most genes show greater than 95% identity between the two strains, but other genes vary more than expected for clonal divergence among E. coli strains. Several of these highly divergent genes encode proteins that are known to be involved in interactions with the host cell. This pattern suggests recombinational divergence coupled with natural selection and has implications for our understanding of the interaction of both pathogens with their host, for the emergence of O157:H7, and for the evolutionary history of pathogens in general.