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

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