QseA Directly Activates Transcription of LEE1 in Enterohemorrhagic Escherichia coli

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Abstract
Quorum sensing (QS) in enterohemorrhagic Escherichia coli (EHEC) regulates the expression of the locus of enterocyte effacement (LEE). The LEE contains five major operons named LEE1 through LEE5 . QseA was previously shown to be activated through QS and to activate the transcription of LEE1 . The LEE1 operon encodes Ler, the transcription activator of all other LEE genes, and has two promoters: a distal promoter (P1) and a proximal promoter (P2). We have previously reported that QseA acts on P1 and not P2. To identify the minimal region of LEE1 that is necessary for QseA-mediated activation, a series of nested-deletion constructs of the LEE1 promoter fused to a lacZ reporter were constructed in both the EHEC and E. coli K-12 backgrounds. In an EHEC background, QseA-dependent activation of LEE1 can be observed for the entire regulatory region (beginning at nucleotide −393 and ending at nucleotide −123). In contrast to what occurred in EHEC, in K-12 there was no QseA-dependent activation of LEE1 transcription between base pairs −393 and −343. These data indicate that a QseA-dependent EHEC-specific regulator is required for the activation of transcription in this region. We also observed QseA-dependent LEE1 activation from nucleotides −218 to −123 in K-12, similar to results of the nested-deletion analysis performed with EHEC. Electrophoretic mobility shift assays established that QseA directly binds to the region of LEE1 from bp −173 to −42 and not to the region from bp −393 to −343. These studies suggest that QseA activates the transcription of LEE1 by directly binding upstream of its P1 promoter region.

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