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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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Related in: MedlinePlus

Analysis of the DR in the upstream region of bosR.(A) Sequences of the probes used in EMSAs. The DR is underlined. Mutated nucleotides are indicated in boldface. (B) EMSAs. The concentration of BosR (nM) is designated above each lane. Probe name is indicated below the image. Bound DNA is denoted by arrows. The numbers in parenthesis indicate numbers of omitted nucleotides (dash lines) between the probe and the ATG start codon.
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ppat-1001272-g012: Analysis of the DR in the upstream region of bosR.(A) Sequences of the probes used in EMSAs. The DR is underlined. Mutated nucleotides are indicated in boldface. (B) EMSAs. The concentration of BosR (nM) is designated above each lane. Probe name is indicated below the image. Bound DNA is denoted by arrows. The numbers in parenthesis indicate numbers of omitted nucleotides (dash lines) between the probe and the ATG start codon.

Mentions: Previously, BosR was reported to bind to the Bb napA promoter (PnapA). Using footprinting assays, Boylan et al. [29] found that BosR protected a 50-bp region located at -222 to -173 (relative to the ATG start codon) in PnapA. Katona et al. [28], using EMSAs, reported that BosR also bound to a DNA fragment encompassing PnapA from −152 to +3. In addition, the latter researchers also reported that BosR bound to upstream regions of bosR. Interestingly, these two genes were not identified in our search (Table 2). However, when scrutinizing the upstream regions of bosR and napA, multiple imperfect DR sequences were detected (Fig. 11A, 12A). Therefore, EMSAs using synthesized dsDNA were employed to examine the roles of these imperfect DR sequences in BosR binding. Specifically, two dsDNA fragments, ZM215 and ZM217, were used to represent the BosR binding region in PnapA identified in previous studies [28]–[29]. As shown in Fig. 11B, BosR bound to both DNA fragments. When a mutation was introduced into the DR, binding of BosR to each probe was abolished. Similar data were also obtained for the probe ZM219 representing the bosR upstream region; BosR binding to the probe was abolished when the predicted DR was mutated (ZM220) (Fig. 12B). These data further substantiate the critical role of the DR in BosR binding.


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)

Analysis of the DR in the upstream region of bosR.(A) Sequences of the probes used in EMSAs. The DR is underlined. Mutated nucleotides are indicated in boldface. (B) EMSAs. The concentration of BosR (nM) is designated above each lane. Probe name is indicated below the image. Bound DNA is denoted by arrows. The numbers in parenthesis indicate numbers of omitted nucleotides (dash lines) between the probe and the ATG start codon.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001272-g012: Analysis of the DR in the upstream region of bosR.(A) Sequences of the probes used in EMSAs. The DR is underlined. Mutated nucleotides are indicated in boldface. (B) EMSAs. The concentration of BosR (nM) is designated above each lane. Probe name is indicated below the image. Bound DNA is denoted by arrows. The numbers in parenthesis indicate numbers of omitted nucleotides (dash lines) between the probe and the ATG start codon.
Mentions: Previously, BosR was reported to bind to the Bb napA promoter (PnapA). Using footprinting assays, Boylan et al. [29] found that BosR protected a 50-bp region located at -222 to -173 (relative to the ATG start codon) in PnapA. Katona et al. [28], using EMSAs, reported that BosR also bound to a DNA fragment encompassing PnapA from −152 to +3. In addition, the latter researchers also reported that BosR bound to upstream regions of bosR. Interestingly, these two genes were not identified in our search (Table 2). However, when scrutinizing the upstream regions of bosR and napA, multiple imperfect DR sequences were detected (Fig. 11A, 12A). Therefore, EMSAs using synthesized dsDNA were employed to examine the roles of these imperfect DR sequences in BosR binding. Specifically, two dsDNA fragments, ZM215 and ZM217, were used to represent the BosR binding region in PnapA identified in previous studies [28]–[29]. As shown in Fig. 11B, BosR bound to both DNA fragments. When a mutation was introduced into the DR, binding of BosR to each probe was abolished. Similar data were also obtained for the probe ZM219 representing the bosR upstream region; BosR binding to the probe was abolished when the predicted DR was mutated (ZM220) (Fig. 12B). These data further substantiate the critical role of the DR in BosR binding.

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