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The global repressor FliZ antagonizes gene expression by σS-containing RNA polymerase due to overlapping DNA binding specificity.

Pesavento C, Hengge R - Nucleic Acids Res. (2012)

Bottom Line: R108 as well as C(-13) are also crucial for DNA binding by FliZ.However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function.Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie-Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany.

ABSTRACT
FliZ, a global regulatory protein under the control of the flagellar master regulator FlhDC, was shown to antagonize σ(S)-dependent gene expression in Escherichia coli. Thereby it plays a pivotal role in the decision between alternative life-styles, i.e. FlhDC-controlled flagellum-based motility or σ(S)-dependent curli fimbriae-mediated adhesion and biofilm formation. Here, we show that FliZ is an abundant DNA-binding protein that inhibits gene expression mediated by σ(S) by recognizing operator sequences that resemble the -10 region of σ(S)-dependent promoters. FliZ does so with a structural element that is similar to region 3.0 of σ(S). Within this element, R108 in FliZ corresponds to K173 in σ(S), which contacts a conserved cytosine at the -13 promoter position that is specific for σ(S)-dependent promoters. R108 as well as C(-13) are also crucial for DNA binding by FliZ. However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function. Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

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FliZ binding to promoter DNA. Electrophoretic mobility shift assays with FliZ (20, 40, 80 nM) are shown for (A) DNA-fragments (6 nM) comprising the promoter regions of the σS-dependent genes mlrA and yciR and control fragments containing part of the translated region of the mlrA gene (mlrA-TR) and the σ70-dependent rpoS promoter, and (B) DNA-fragments containing promoters from other FliZ-controlled genes previously identified (10).
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gks055-F1: FliZ binding to promoter DNA. Electrophoretic mobility shift assays with FliZ (20, 40, 80 nM) are shown for (A) DNA-fragments (6 nM) comprising the promoter regions of the σS-dependent genes mlrA and yciR and control fragments containing part of the translated region of the mlrA gene (mlrA-TR) and the σ70-dependent rpoS promoter, and (B) DNA-fragments containing promoters from other FliZ-controlled genes previously identified (10).

Mentions: In order to test whether FliZ is able to bind to DNA in vitro, the E. coli protein was purified and subject to EMSA with DNA fragments containing the promoters of mlrA and yciR. The mlrA gene encodes a MerR-like regulator essential for transcriptional activation of the central curli regulator CsgD (Supplementary Figure S1) which shows premature induction in post-exponential phase in a fliZ mutant (10). Similarly, the yciR gene, which specifies a cyclic di-GMP-specific PDE involved in the regulation of csgD expression (18), was previously shown to be repressed by FliZ (10). FliZ bound to both promoter regions (Figure 1A). By contrast, FliZ did not bind to a control fragment comprising part of the translated region of the mlrA gene, nor to a region including the σ70-dependent promoter of the rpoS gene, which encodes σS (Figure 1A).Figure 1.


The global repressor FliZ antagonizes gene expression by σS-containing RNA polymerase due to overlapping DNA binding specificity.

Pesavento C, Hengge R - Nucleic Acids Res. (2012)

FliZ binding to promoter DNA. Electrophoretic mobility shift assays with FliZ (20, 40, 80 nM) are shown for (A) DNA-fragments (6 nM) comprising the promoter regions of the σS-dependent genes mlrA and yciR and control fragments containing part of the translated region of the mlrA gene (mlrA-TR) and the σ70-dependent rpoS promoter, and (B) DNA-fragments containing promoters from other FliZ-controlled genes previously identified (10).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367168&req=5

gks055-F1: FliZ binding to promoter DNA. Electrophoretic mobility shift assays with FliZ (20, 40, 80 nM) are shown for (A) DNA-fragments (6 nM) comprising the promoter regions of the σS-dependent genes mlrA and yciR and control fragments containing part of the translated region of the mlrA gene (mlrA-TR) and the σ70-dependent rpoS promoter, and (B) DNA-fragments containing promoters from other FliZ-controlled genes previously identified (10).
Mentions: In order to test whether FliZ is able to bind to DNA in vitro, the E. coli protein was purified and subject to EMSA with DNA fragments containing the promoters of mlrA and yciR. The mlrA gene encodes a MerR-like regulator essential for transcriptional activation of the central curli regulator CsgD (Supplementary Figure S1) which shows premature induction in post-exponential phase in a fliZ mutant (10). Similarly, the yciR gene, which specifies a cyclic di-GMP-specific PDE involved in the regulation of csgD expression (18), was previously shown to be repressed by FliZ (10). FliZ bound to both promoter regions (Figure 1A). By contrast, FliZ did not bind to a control fragment comprising part of the translated region of the mlrA gene, nor to a region including the σ70-dependent promoter of the rpoS gene, which encodes σS (Figure 1A).Figure 1.

Bottom Line: R108 as well as C(-13) are also crucial for DNA binding by FliZ.However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function.Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie-Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany.

ABSTRACT
FliZ, a global regulatory protein under the control of the flagellar master regulator FlhDC, was shown to antagonize σ(S)-dependent gene expression in Escherichia coli. Thereby it plays a pivotal role in the decision between alternative life-styles, i.e. FlhDC-controlled flagellum-based motility or σ(S)-dependent curli fimbriae-mediated adhesion and biofilm formation. Here, we show that FliZ is an abundant DNA-binding protein that inhibits gene expression mediated by σ(S) by recognizing operator sequences that resemble the -10 region of σ(S)-dependent promoters. FliZ does so with a structural element that is similar to region 3.0 of σ(S). Within this element, R108 in FliZ corresponds to K173 in σ(S), which contacts a conserved cytosine at the -13 promoter position that is specific for σ(S)-dependent promoters. R108 as well as C(-13) are also crucial for DNA binding by FliZ. However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function. Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

Show MeSH
Related in: MedlinePlus