Role of hha and ler in transcriptional regulation of the esp operon of enterohemorrhagic Escherichia coli O157:H7.
The locus of enterocyte effacement (LEE), which includes five major operons (LEE1 through LEE4 and tir), enables enterohemorrhagic Escherichia coli (EHEC) O157:H7 to produce attaching and effacing lesions on host cells. Expression of LEE2, LEE3, and tir is positively regulated by ler, a gene located in LEE1. Transcriptional regulation of the esp operon (LEE4), however, is not well defined. Transposon mutagenesis was used to identify transcriptional regulators of the esp operon by screening for mutants with increased beta-galactosidase activity in an EHEC O157:H7 strain harboring an esp::lac transcriptional fusion. All mutants with significant increases in beta-galactosidase activity had transposon insertions in hha (hha::Tn). Specific complementation of the hha::Tn mutation with a plasmid-encoded copy of hha reduced beta-galactosidase activity to the level expressed in the parental esp::lac strain. Purified Hha, however, bound poorly to the esp promoter, suggesting that Hha might repress the transcription of a positive regulator of esp. Transposon mutagenesis of a Deltahha esp::lac strain expressing elevated levels of beta-galactosidase resulted in ler mutants with reduced beta-galactosidase activity. Purified Hha bound to the ler promoter with a higher affinity, and complementation of a Deltahha mutation in a Deltahha ler::lac strain repressed beta-galactosidase activity to the level expressed in a ler::lac strain. A positive regulatory role of ler in esp expression was demonstrated by specific binding of Ler to the esp promoter, reduced expression of beta-galactosidase in Deltaler esp::lac strains with and without hha, and severalfold-increased transcription of ler and espA in strains lacking hha. These results indicate that hha-mediated repression of ler causes reduced expression of the esp operon.
Sharma
Vijay K
VK
National Animal Disease Center, USDA/ARS, P.O. Box 70, Ames, IA 50010, USA. vsharma@nadc.ars.usda.gov
Zuerner
Richard L
RL
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D016254:Mutagenesis, Insertional; D009876:Operon; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C457340:LEE protein, E coli; C465556:Ler protein, E coli; D010750:Phosphoproteins; D015534:Trans-Activators; C079209:hha protein, E coli