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. 2007 May;75(5):2432-40.
doi: 10.1128/IAI.02003-06. Epub 2007 Mar 5.

V体育平台登录 - QseA directly activates transcription of LEE1 in enterohemorrhagic Escherichia coli

Faith C Sharp  1 , Vanessa Sperandio
Affiliations

QseA directly activates transcription of LEE1 in enterohemorrhagic Escherichia coli

Faith C Sharp (V体育官网入口) et al. Infect Immun. 2007 May.

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 VSports手机版. 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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Figures

FIG. 1.
FIG. 1.
Alignment of the sequences of the LEE1 regulatory region of EHEC and EPEC. Lightly shaded areas correspond to the distal promoter (P1); darkly shaded areas correspond to the EHEC-specific proximal promoter (P2). Unshaded box 1 corresponds to the assigned ATG based on a longer reading frame; unshaded box 2 corresponds to the ATG that is associated with a putative ribosome binding sequence.
FIG. 2.
FIG. 2.
Transcription of LEE1::lacZ reporter fusion constructs within an EHEC and an E. coli K-12 background. β-Galactosidase activities are depicted in Miller units, and the numbering of base pairs is in relation to the P2 (proximal) LEE1 promoter transcriptional start site.
FIG. 3.
FIG. 3.
(A) Schematic of nested-deletion analysis. The numbering of the bases (−393 to +323) is based on the P2 transcriptional start site. (B) Deletion analysis of the LEE1 promoter in EHEC. Fragments of the LEE1 promoter were electroporated into 86-24 (WT), VS145 (qseA mutant), and VS151 (qseA complemented) and assessed for β-galactosidase activity. Error bars indicate standard deviations. (C) Deletion analysis of the LEE1 promoter in E. coli K-12. Fragments of the LEE1 promoter were transformed into MC4100 (WT), FS02 (qseA mutant), and FS76 (qseA complemented) and assessed for β-galactosidase activity. Error bars indicate standard deviations.
FIG. 4.
FIG. 4.
(A) EMSA of the LEE1 promoter fragment containing bp −393 to +42 with increasing amounts of QseA. (B) EMSA of the bla promoter fragment with QseA (negative control).
FIG. 5.
FIG. 5.
EMSA of the LEE1 promoter fragments containing bp −393 to −300 and −173 to −42 with increasing amounts of QseA.
FIG. 6.
FIG. 6.
Schematic depiction of LEE regulation. Factors shown in gray are present in both E. coli K-12 and EHEC (and also in EPEC), while regulators shown in black are specific to EHEC (some are shared with EPEC, e.g., GrlR, GrlA, and Ler). Solid lines represent regulators whose direct interactions with the target promoter have been biochemically defined, and dashed lines represent interactions which occur indirectly or have not yet been shown to bind to the target gene. IHF, integration host factor.
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References

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