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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 directly affects motility by repressing flhDC expression. (A) Motility of strain W3110 (wt) and its derivatives carrying ΔfliZ, or the low copy plasmids pCAB18 (vector control) or pFliZ was tested at 28°C. (B) Expression of a single-copy transcriptional lacZ fusion to the flhDC promoter (flhDC1::lacZ) was determined in fliZ+ and fliZ cells growing in LB medium at 28°C. (C) Expression in wild-type (wt) and ΔfliZ cells of the same fusion (flhDC1::lacZ) and of a fusion (flhDC3::lacZ) that does not include the full vegetative flhDC promoter but carries the ‘−10 σS-promoter-like element’ (F); cells were grown on LB/agar plates without salt at 28°C for 7 days. (D) Binding of FliZ to DNA fragments with (flhDC-long) or without (flhDC-short) the ‘−10 σS-promoter-like element’ downstream of the flhDC transcriptional start site was compared by EMSA (80, 160, 320 nM FliZ). (E) The FliZ-binding site in the flhDC upstream regulatory region was determined by non-radioactive DNaseI footprint analysis and the binding site was mapped to the promoter sequence (F). A core binding site and potential upstream and downstream extensions are indicated by smaller and larger bars (E) and boxes (F). The transcriptional start site (62) is printed as a bold, underlined letter and the ‘−10 σS-promoter-like element’ downstream of the flhDC promoter is printed in bold, larger letters. The start of the region present in flhDC3::lacZ and the end of the flhDC-short DNA fragment used in (D) are indicated.
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gks055-F6: FliZ directly affects motility by repressing flhDC expression. (A) Motility of strain W3110 (wt) and its derivatives carrying ΔfliZ, or the low copy plasmids pCAB18 (vector control) or pFliZ was tested at 28°C. (B) Expression of a single-copy transcriptional lacZ fusion to the flhDC promoter (flhDC1::lacZ) was determined in fliZ+ and fliZ cells growing in LB medium at 28°C. (C) Expression in wild-type (wt) and ΔfliZ cells of the same fusion (flhDC1::lacZ) and of a fusion (flhDC3::lacZ) that does not include the full vegetative flhDC promoter but carries the ‘−10 σS-promoter-like element’ (F); cells were grown on LB/agar plates without salt at 28°C for 7 days. (D) Binding of FliZ to DNA fragments with (flhDC-long) or without (flhDC-short) the ‘−10 σS-promoter-like element’ downstream of the flhDC transcriptional start site was compared by EMSA (80, 160, 320 nM FliZ). (E) The FliZ-binding site in the flhDC upstream regulatory region was determined by non-radioactive DNaseI footprint analysis and the binding site was mapped to the promoter sequence (F). A core binding site and potential upstream and downstream extensions are indicated by smaller and larger bars (E) and boxes (F). The transcriptional start site (62) is printed as a bold, underlined letter and the ‘−10 σS-promoter-like element’ downstream of the flhDC promoter is printed in bold, larger letters. The start of the region present in flhDC3::lacZ and the end of the flhDC-short DNA fragment used in (D) are indicated.

Mentions: A ΔfliZ mutant showed a subtle but reproducible increase in swimming motility as compared to the wild-type, while FliZ expression from the low-copy plasmid reduced motility (Figure 6A), indicating that in E. coli, FliZ acts as a weak negative regulator of motility. Flagellar assembly is based on the hierarchical expression of three classes of flagellar genes (32) (Supplementary Figure S1). A single-copy chromosomal lacZ fusion to the promoter of the class I flhDC operon, which encodes the flagellar master regulator complex FlhD4C2, showed increased expression in the ΔfliZ mutant (Figure 6B). Similar effects were also observed for lacZ fusions to the class II flgAMN promoter and to the class III gene yhjH (Supplementary Figure S8A). However, FliZ did not bind to classes II and III promoter fragments tested in vitro (Supplementary Figure S8B), indicating that the FliZ effect on the expression of the master regulator FlhDC (class I) is relayed to the classes II and III genes.Figure 6.


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 directly affects motility by repressing flhDC expression. (A) Motility of strain W3110 (wt) and its derivatives carrying ΔfliZ, or the low copy plasmids pCAB18 (vector control) or pFliZ was tested at 28°C. (B) Expression of a single-copy transcriptional lacZ fusion to the flhDC promoter (flhDC1::lacZ) was determined in fliZ+ and fliZ cells growing in LB medium at 28°C. (C) Expression in wild-type (wt) and ΔfliZ cells of the same fusion (flhDC1::lacZ) and of a fusion (flhDC3::lacZ) that does not include the full vegetative flhDC promoter but carries the ‘−10 σS-promoter-like element’ (F); cells were grown on LB/agar plates without salt at 28°C for 7 days. (D) Binding of FliZ to DNA fragments with (flhDC-long) or without (flhDC-short) the ‘−10 σS-promoter-like element’ downstream of the flhDC transcriptional start site was compared by EMSA (80, 160, 320 nM FliZ). (E) The FliZ-binding site in the flhDC upstream regulatory region was determined by non-radioactive DNaseI footprint analysis and the binding site was mapped to the promoter sequence (F). A core binding site and potential upstream and downstream extensions are indicated by smaller and larger bars (E) and boxes (F). The transcriptional start site (62) is printed as a bold, underlined letter and the ‘−10 σS-promoter-like element’ downstream of the flhDC promoter is printed in bold, larger letters. The start of the region present in flhDC3::lacZ and the end of the flhDC-short DNA fragment used in (D) are indicated.
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Related In: Results  -  Collection

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gks055-F6: FliZ directly affects motility by repressing flhDC expression. (A) Motility of strain W3110 (wt) and its derivatives carrying ΔfliZ, or the low copy plasmids pCAB18 (vector control) or pFliZ was tested at 28°C. (B) Expression of a single-copy transcriptional lacZ fusion to the flhDC promoter (flhDC1::lacZ) was determined in fliZ+ and fliZ cells growing in LB medium at 28°C. (C) Expression in wild-type (wt) and ΔfliZ cells of the same fusion (flhDC1::lacZ) and of a fusion (flhDC3::lacZ) that does not include the full vegetative flhDC promoter but carries the ‘−10 σS-promoter-like element’ (F); cells were grown on LB/agar plates without salt at 28°C for 7 days. (D) Binding of FliZ to DNA fragments with (flhDC-long) or without (flhDC-short) the ‘−10 σS-promoter-like element’ downstream of the flhDC transcriptional start site was compared by EMSA (80, 160, 320 nM FliZ). (E) The FliZ-binding site in the flhDC upstream regulatory region was determined by non-radioactive DNaseI footprint analysis and the binding site was mapped to the promoter sequence (F). A core binding site and potential upstream and downstream extensions are indicated by smaller and larger bars (E) and boxes (F). The transcriptional start site (62) is printed as a bold, underlined letter and the ‘−10 σS-promoter-like element’ downstream of the flhDC promoter is printed in bold, larger letters. The start of the region present in flhDC3::lacZ and the end of the flhDC-short DNA fragment used in (D) are indicated.
Mentions: A ΔfliZ mutant showed a subtle but reproducible increase in swimming motility as compared to the wild-type, while FliZ expression from the low-copy plasmid reduced motility (Figure 6A), indicating that in E. coli, FliZ acts as a weak negative regulator of motility. Flagellar assembly is based on the hierarchical expression of three classes of flagellar genes (32) (Supplementary Figure S1). A single-copy chromosomal lacZ fusion to the promoter of the class I flhDC operon, which encodes the flagellar master regulator complex FlhD4C2, showed increased expression in the ΔfliZ mutant (Figure 6B). Similar effects were also observed for lacZ fusions to the class II flgAMN promoter and to the class III gene yhjH (Supplementary Figure S8A). However, FliZ did not bind to classes II and III promoter fragments tested in vitro (Supplementary Figure S8B), indicating that the FliZ effect on the expression of the master regulator FlhDC (class I) is relayed to the classes II and III genes.Figure 6.

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