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BosR (BB0647) controls the RpoN-RpoS regulatory pathway and virulence expression in Borrelia burgdorferi by a novel DNA-binding mechanism.

Ouyang Z, Deka RK, Norgard MV - PLoS Pathog. (2011)

Bottom Line: However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog.We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes.Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ⁵⁴ (RpoN) directly activates transcription of another alternative σ factor, σ(S) (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ⁵⁴-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ⁵⁴-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ⁵⁴-dependent gene regulation in bacteria.

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BosR binds to the rpoS promoter.(A) In a typical EMSA, 30 fmol of digoxigenin-labeled rpoS promoter (PrpoS) was incubated with the indicated concentrations of BosR at 37°C for 30 min. Probe name is indicated at the position of unbound DNA. Bound DNA is denoted by arrows. (B) DNase I footprinting analysis of the PrpoS probe with BosR. Lanes A, T, G and C represent sequencing ladders. Lane 1–5 contains 0, 200, 500, 1000, 1500 nM of BosR, respectively. The protected regions are marked on the right. (C) A summary of the DNase I footprinting assay results. The -24/-12 σ54 promoter sequence and the ATG start codon are indicated in boldface. The rpoS transcription start site is marked by the asterisk. The BosR protected regions (BS1-3) are indicated with the dotted-line box. The predicted direct repeat (DR) sequence is underlined.
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ppat-1001272-g005: BosR binds to the rpoS promoter.(A) In a typical EMSA, 30 fmol of digoxigenin-labeled rpoS promoter (PrpoS) was incubated with the indicated concentrations of BosR at 37°C for 30 min. Probe name is indicated at the position of unbound DNA. Bound DNA is denoted by arrows. (B) DNase I footprinting analysis of the PrpoS probe with BosR. Lanes A, T, G and C represent sequencing ladders. Lane 1–5 contains 0, 200, 500, 1000, 1500 nM of BosR, respectively. The protected regions are marked on the right. (C) A summary of the DNase I footprinting assay results. The -24/-12 σ54 promoter sequence and the ATG start codon are indicated in boldface. The rpoS transcription start site is marked by the asterisk. The BosR protected regions (BS1-3) are indicated with the dotted-line box. The predicted direct repeat (DR) sequence is underlined.

Mentions: In silico analysis predicted that Bb BosR contains an N-terminal winged helix-turn-helix DNA binding domain and a C-terminal dimerization domain. Three-dimensional (3D) protein modeling using the Swiss-model program (http://swissmodel.expasy.org/) indicated that the structure of the DNA-binding domain of BosR is quite similar to the Vibrio cholerae Fur protein [52] and the B. subtilis PerR protein [51] (Fig. S3), suggesting that, consistent with previous reports [28]–[29], BosR may be a DNA-binding protein. Moreover, our aforementioned data revealed that BosR impacted rpoS expression at the transcription level. Thus, EMSAs were performed to examine potential interactions between BosR and the rpoS promoter. Consistent with previous studies [28]–[29], BosR bound to the promoter of Bb napA (from −336 to +48, relative to the ATG start codon) (Fig. 4A). However, BosR did not bind to the ospC or dbpBA promoters under our tested conditions (Fig. 4B and C), providing support that BosR likely does not impact ospC and dbpA directly. Although BosR did not bind to the probe ZM126 that encompasses the rpoS promoter from −67 to −8 (Fig. 4D), BosR, in a dose-dependent manner, bound to the rpoS promoter (PrpoS) encompassing 277 bp of the rpoS upstream region and 245 bp of the rpoS encoding region (Fig. 5A). Of note, binding of rBosR generated multiple shifted bands, suggesting the possible existence of multiple BosR binding sites (BSs) in the probe. As an initial approach to identify the BosR binding sequence, DNase I footprinting assays were performed. As shown in Fig. 5B, three BosR BSs were recognized in the PrpoS DNA. Specifically, BosR BS1, BS2, or BS3 spanned regions of −193 to −137, −120 to −46, or −29 to +43 (relative to the ATG start codon, where A is +1), respectively (Fig. 5C).


BosR (BB0647) controls the RpoN-RpoS regulatory pathway and virulence expression in Borrelia burgdorferi by a novel DNA-binding mechanism.

Ouyang Z, Deka RK, Norgard MV - PLoS Pathog. (2011)

BosR binds to the rpoS promoter.(A) In a typical EMSA, 30 fmol of digoxigenin-labeled rpoS promoter (PrpoS) was incubated with the indicated concentrations of BosR at 37°C for 30 min. Probe name is indicated at the position of unbound DNA. Bound DNA is denoted by arrows. (B) DNase I footprinting analysis of the PrpoS probe with BosR. Lanes A, T, G and C represent sequencing ladders. Lane 1–5 contains 0, 200, 500, 1000, 1500 nM of BosR, respectively. The protected regions are marked on the right. (C) A summary of the DNase I footprinting assay results. The -24/-12 σ54 promoter sequence and the ATG start codon are indicated in boldface. The rpoS transcription start site is marked by the asterisk. The BosR protected regions (BS1-3) are indicated with the dotted-line box. The predicted direct repeat (DR) sequence is underlined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037356&req=5

ppat-1001272-g005: BosR binds to the rpoS promoter.(A) In a typical EMSA, 30 fmol of digoxigenin-labeled rpoS promoter (PrpoS) was incubated with the indicated concentrations of BosR at 37°C for 30 min. Probe name is indicated at the position of unbound DNA. Bound DNA is denoted by arrows. (B) DNase I footprinting analysis of the PrpoS probe with BosR. Lanes A, T, G and C represent sequencing ladders. Lane 1–5 contains 0, 200, 500, 1000, 1500 nM of BosR, respectively. The protected regions are marked on the right. (C) A summary of the DNase I footprinting assay results. The -24/-12 σ54 promoter sequence and the ATG start codon are indicated in boldface. The rpoS transcription start site is marked by the asterisk. The BosR protected regions (BS1-3) are indicated with the dotted-line box. The predicted direct repeat (DR) sequence is underlined.
Mentions: In silico analysis predicted that Bb BosR contains an N-terminal winged helix-turn-helix DNA binding domain and a C-terminal dimerization domain. Three-dimensional (3D) protein modeling using the Swiss-model program (http://swissmodel.expasy.org/) indicated that the structure of the DNA-binding domain of BosR is quite similar to the Vibrio cholerae Fur protein [52] and the B. subtilis PerR protein [51] (Fig. S3), suggesting that, consistent with previous reports [28]–[29], BosR may be a DNA-binding protein. Moreover, our aforementioned data revealed that BosR impacted rpoS expression at the transcription level. Thus, EMSAs were performed to examine potential interactions between BosR and the rpoS promoter. Consistent with previous studies [28]–[29], BosR bound to the promoter of Bb napA (from −336 to +48, relative to the ATG start codon) (Fig. 4A). However, BosR did not bind to the ospC or dbpBA promoters under our tested conditions (Fig. 4B and C), providing support that BosR likely does not impact ospC and dbpA directly. Although BosR did not bind to the probe ZM126 that encompasses the rpoS promoter from −67 to −8 (Fig. 4D), BosR, in a dose-dependent manner, bound to the rpoS promoter (PrpoS) encompassing 277 bp of the rpoS upstream region and 245 bp of the rpoS encoding region (Fig. 5A). Of note, binding of rBosR generated multiple shifted bands, suggesting the possible existence of multiple BosR binding sites (BSs) in the probe. As an initial approach to identify the BosR binding sequence, DNase I footprinting assays were performed. As shown in Fig. 5B, three BosR BSs were recognized in the PrpoS DNA. Specifically, BosR BS1, BS2, or BS3 spanned regions of −193 to −137, −120 to −46, or −29 to +43 (relative to the ATG start codon, where A is +1), respectively (Fig. 5C).

Bottom Line: However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog.We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes.Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ⁵⁴ (RpoN) directly activates transcription of another alternative σ factor, σ(S) (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ⁵⁴-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ⁵⁴-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ⁵⁴-dependent gene regulation in bacteria.

Show MeSH
Related in: MedlinePlus