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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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Analyses of purified recombinant BosR.(A) 12.5% (w/v) SDS–PAGE of purified recombinant BosR (right lane). Molecular masses are indicated in the left lane in kDa. (B) Native BosR in Bb cultivated in BSK-H at 37°C was probed with α-BosR. Right lane: molecular mass markers; left lane: Bb lysates. (C) Size exclusion chromatogram of purified BosR on a Superdex 200 column. Protein molecular mass standards used to calibrate the gel-filtration column are indicated by arrows (aldolase  = 158 kDa; ovalbumin  = 43 kDa; ribonuclease A  = 13.7 kDa). (D) Metal content analysis of recombinant BosR. Metal content was determined by ICP-AES.
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ppat-1001272-g003: Analyses of purified recombinant BosR.(A) 12.5% (w/v) SDS–PAGE of purified recombinant BosR (right lane). Molecular masses are indicated in the left lane in kDa. (B) Native BosR in Bb cultivated in BSK-H at 37°C was probed with α-BosR. Right lane: molecular mass markers; left lane: Bb lysates. (C) Size exclusion chromatogram of purified BosR on a Superdex 200 column. Protein molecular mass standards used to calibrate the gel-filtration column are indicated by arrows (aldolase  = 158 kDa; ovalbumin  = 43 kDa; ribonuclease A  = 13.7 kDa). (D) Metal content analysis of recombinant BosR. Metal content was determined by ICP-AES.

Mentions: Recombinant BosR (rBosR) was hyper-expressed in E. coli and purified to apparent homogeneity. SDS-PAGE analysis indicated that BosR has an apparent molecular mass of ∼18.7 kDa (Fig. 3A), which is in agreement with the apparent mass of native BosR in Bb (Fig. 3B). Furthermore, when analyzed by size-exclusion chromatography, purified BosR eluted predominantly as a dimer with a molecular mass of ∼38 kDa (Fig. 3C). Although recombinant BosR has been obtained previously and Zn2+ was found to affect BosR's in vitro binding to DNA [28]–[29], it remained unclear whether BosR contains bound metal. Therefore, metal content analysis was carried out by inductively coupled plasma atomic emission spectrometry (ICP-AES) [27], [49]. rBosR did not contain detectable levels (<0.001 ppm) of metal ions such as Fe or Mn (Fig. 3D). Rather, it contained 1.4 mol of zinc per mol of protein. Moreover, in order to remove bound metal(s) from BosR, we also dialyzed the protein in the presence of 10 mM EDTA. However, 0.3 mol of zinc/mol of proteins remained in the demetallated BosR (Fig. 3D), suggesting that the recombinant protein bound zinc avidly. Of note, these properties are typical of the dimeric bacterial Fur protein [50] or the Bacillus subtilis H2O2 stress response regulator PerR [51].


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)

Analyses of purified recombinant BosR.(A) 12.5% (w/v) SDS–PAGE of purified recombinant BosR (right lane). Molecular masses are indicated in the left lane in kDa. (B) Native BosR in Bb cultivated in BSK-H at 37°C was probed with α-BosR. Right lane: molecular mass markers; left lane: Bb lysates. (C) Size exclusion chromatogram of purified BosR on a Superdex 200 column. Protein molecular mass standards used to calibrate the gel-filtration column are indicated by arrows (aldolase  = 158 kDa; ovalbumin  = 43 kDa; ribonuclease A  = 13.7 kDa). (D) Metal content analysis of recombinant BosR. Metal content was determined by ICP-AES.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037356&req=5

ppat-1001272-g003: Analyses of purified recombinant BosR.(A) 12.5% (w/v) SDS–PAGE of purified recombinant BosR (right lane). Molecular masses are indicated in the left lane in kDa. (B) Native BosR in Bb cultivated in BSK-H at 37°C was probed with α-BosR. Right lane: molecular mass markers; left lane: Bb lysates. (C) Size exclusion chromatogram of purified BosR on a Superdex 200 column. Protein molecular mass standards used to calibrate the gel-filtration column are indicated by arrows (aldolase  = 158 kDa; ovalbumin  = 43 kDa; ribonuclease A  = 13.7 kDa). (D) Metal content analysis of recombinant BosR. Metal content was determined by ICP-AES.
Mentions: Recombinant BosR (rBosR) was hyper-expressed in E. coli and purified to apparent homogeneity. SDS-PAGE analysis indicated that BosR has an apparent molecular mass of ∼18.7 kDa (Fig. 3A), which is in agreement with the apparent mass of native BosR in Bb (Fig. 3B). Furthermore, when analyzed by size-exclusion chromatography, purified BosR eluted predominantly as a dimer with a molecular mass of ∼38 kDa (Fig. 3C). Although recombinant BosR has been obtained previously and Zn2+ was found to affect BosR's in vitro binding to DNA [28]–[29], it remained unclear whether BosR contains bound metal. Therefore, metal content analysis was carried out by inductively coupled plasma atomic emission spectrometry (ICP-AES) [27], [49]. rBosR did not contain detectable levels (<0.001 ppm) of metal ions such as Fe or Mn (Fig. 3D). Rather, it contained 1.4 mol of zinc per mol of protein. Moreover, in order to remove bound metal(s) from BosR, we also dialyzed the protein in the presence of 10 mM EDTA. However, 0.3 mol of zinc/mol of proteins remained in the demetallated BosR (Fig. 3D), suggesting that the recombinant protein bound zinc avidly. Of note, these properties are typical of the dimeric bacterial Fur protein [50] or the Bacillus subtilis H2O2 stress response regulator PerR [51].

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