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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 BosR binding to BS2.(A) Detailed sequences of probes representing BS2. The DRs are underlined. Scrambled sequences are italicized. 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.
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ppat-1001272-g010: Analysis of BosR binding to BS2.(A) Detailed sequences of probes representing BS2. The DRs are underlined. Scrambled sequences are italicized. 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.

Mentions: Using this same strategy, we also examined the two DRs in BS2. As shown in Fig. 10, although BosR still bound to probe ZM149 having sequences downstream of the -24/-12 site scrambled, BosR binding to BS2 was abolished when sequences upstream of the -24/-12 site were scrambled (ZM147), suggesting that the functional BosR binding sites are located in the sequence upstream of the -24/-12 site. Because sequences flanking the binding motif often play important roles in protein-DNA interactions, we synthesized another dsDNA (ZM212) to represent the 5′ of BS2, allowing added flanking sequences to the predicted DR sequences. EMSAs indicated that BosR still bound to probe (ZM213) with the DR1 mutated (Fig. 10B). When a mutation was introduced into DR2 (ZM214), BosR binding was dramatically reduced (Fig. 10B). Moreover, when both DR sequences were mutated, protein binding was completely abolished (Fig. 10B). These data suggest that DR1 and DR2 in BS2 are required for 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 BosR binding to BS2.(A) Detailed sequences of probes representing BS2. The DRs are underlined. Scrambled sequences are italicized. 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.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001272-g010: Analysis of BosR binding to BS2.(A) Detailed sequences of probes representing BS2. The DRs are underlined. Scrambled sequences are italicized. 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.
Mentions: Using this same strategy, we also examined the two DRs in BS2. As shown in Fig. 10, although BosR still bound to probe ZM149 having sequences downstream of the -24/-12 site scrambled, BosR binding to BS2 was abolished when sequences upstream of the -24/-12 site were scrambled (ZM147), suggesting that the functional BosR binding sites are located in the sequence upstream of the -24/-12 site. Because sequences flanking the binding motif often play important roles in protein-DNA interactions, we synthesized another dsDNA (ZM212) to represent the 5′ of BS2, allowing added flanking sequences to the predicted DR sequences. EMSAs indicated that BosR still bound to probe (ZM213) with the DR1 mutated (Fig. 10B). When a mutation was introduced into DR2 (ZM214), BosR binding was dramatically reduced (Fig. 10B). Moreover, when both DR sequences were mutated, protein binding was completely abolished (Fig. 10B). These data suggest that DR1 and DR2 in BS2 are required for 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