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Journal of Bacteriology, November 2004, p. 7290-7301, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7290-7301.2004

Role of hha and ler in Transcriptional Regulation of the esp Operon of Enterohemorrhagic Escherichia coli O157:H7

Vijay K. Sharma1* and Richard L. Zuerner2

Pre-Harvest Food Safety and Enteric Diseases,1 Bacterial Diseases of Livestock Research Units, National Animal Disease Center, USDA Agricultural Research Service, Ames, Iowa2

Received 10 May 2004/ Accepted 30 July 2004

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 ß-galactosidase activity in an EHEC O157:H7 strain harboring an esp::lac transcriptional fusion. All mutants with significant increases in ß-galactosidase activity had transposon insertions in hha (hha::Tn). Specific complementation of the hha::Tn mutation with a plasmid-encoded copy of hha reduced ß-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 {Delta}hha esp::lac strain expressing elevated levels of ß-galactosidase resulted in ler mutants with reduced ß-galactosidase activity. Purified Hha bound to the ler promoter with a higher affinity, and complementation of a {Delta}hha mutation in a {Delta}hha ler::lac strain repressed ß-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 ß-galactosidase in {Delta}ler 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.

* Corresponding author. Mailing address: USDA, ARS, National Animal Disease Center, P.O. Box 70, Ames, IA 50010. Phone: (515) 663-7406. Fax: (515) 663-7458. E-mail: vsharma{at}nadc.ars.usda.gov.

Journal of Bacteriology, November 2004, p. 7290-7301, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7290-7301.2004




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