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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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Comparison of the relative affinity of BosR for BS1 (ZM132) and BS2 (ZM127).(A) 30 fmol of labeled probe was incubated with various concentrations of BosR (nM). The membrane containing the bound and unbound DNA was detected using an enzyme immunoassay, and exposed to a Fujifilm LAS-3000 Imager (Fujifilm). Images were analyzed by using the MultiGauge V3.0 software (Fujifilm), and bands were quantified to determine the affinity of BosR for probes. (B) Competition of labeled BS1 (ZM132) with various amounts of unlabeled BS1, BS2, or non-specific (NS) DNA. (C) Competition of labeled BS2 (ZM127) with various amounts of unlabeled BS1, BS2, or NS DNA. In (B) and (C), 100 nM of BosR was used in EMSAs, and bound and unbound DNA was measured as described in (A). NS: non-specific competitor (ZM126).
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ppat-1001272-g008: Comparison of the relative affinity of BosR for BS1 (ZM132) and BS2 (ZM127).(A) 30 fmol of labeled probe was incubated with various concentrations of BosR (nM). The membrane containing the bound and unbound DNA was detected using an enzyme immunoassay, and exposed to a Fujifilm LAS-3000 Imager (Fujifilm). Images were analyzed by using the MultiGauge V3.0 software (Fujifilm), and bands were quantified to determine the affinity of BosR for probes. (B) Competition of labeled BS1 (ZM132) with various amounts of unlabeled BS1, BS2, or non-specific (NS) DNA. (C) Competition of labeled BS2 (ZM127) with various amounts of unlabeled BS1, BS2, or NS DNA. In (B) and (C), 100 nM of BosR was used in EMSAs, and bound and unbound DNA was measured as described in (A). NS: non-specific competitor (ZM126).

Mentions: EMSAs employing target DNA sequences (representing the three BosR binding sites) exposed to increasing concentrations of rBosR were used as means of inferring BosR binding affinities for the three binding sites. As shown in Fig. 6 and 7, concentrations of 50, 20, or 200 nM of rBosR induced shifts by ZM132 (BS1), ZM127 (BS2), or ZM161 (BS3), respectively, suggesting that BosR has an affinity for these DNA targets in the order of BS2>BS1>BS3. In addition, when 200 nM of rBosR was used, only a slight proportion (<10%) of ZM161 (BS3) was shifted (Fig. 7), and probe ZM161 could not be saturated even by 10,000 nM of BosR (data not shown). To more precisely assess the affinity of BosR for BS1 and BS2, we measured the amount of bound DNA as a function of BosR concentration in EMSA assays (Fig. 8A). The dissociation binding constants (Kd) for BS1 (ZM132) or BS2 (ZM127) were 210.2 or 36.6 nM, respectively. The relative affinities of these two DNA elements for BosR were also assessed by competition EMSA analysis (Fig. 8, B and C). Binding of labeled BS1 or BS2 was not inhibited by the non-specific competitor ZM126 (NS), but was inhibited by unlabeled (cold competitor) BS1 or BS2, respectively. Moreover, binding of labeled BS1 was inhibited approximately 90% by the addition of 200-fold unlabeled BS1, but was completely competed out by 50-fold unlabeled BS2 (Fig. 8B). The addition of 200-fold of unlabeled BS1 competed out only 15% of BS2 binding (Fig. 8C). These data indicate that BosR has a higher affinity for BS2 than for BS1.


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)

Comparison of the relative affinity of BosR for BS1 (ZM132) and BS2 (ZM127).(A) 30 fmol of labeled probe was incubated with various concentrations of BosR (nM). The membrane containing the bound and unbound DNA was detected using an enzyme immunoassay, and exposed to a Fujifilm LAS-3000 Imager (Fujifilm). Images were analyzed by using the MultiGauge V3.0 software (Fujifilm), and bands were quantified to determine the affinity of BosR for probes. (B) Competition of labeled BS1 (ZM132) with various amounts of unlabeled BS1, BS2, or non-specific (NS) DNA. (C) Competition of labeled BS2 (ZM127) with various amounts of unlabeled BS1, BS2, or NS DNA. In (B) and (C), 100 nM of BosR was used in EMSAs, and bound and unbound DNA was measured as described in (A). NS: non-specific competitor (ZM126).
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Related In: Results  -  Collection

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ppat-1001272-g008: Comparison of the relative affinity of BosR for BS1 (ZM132) and BS2 (ZM127).(A) 30 fmol of labeled probe was incubated with various concentrations of BosR (nM). The membrane containing the bound and unbound DNA was detected using an enzyme immunoassay, and exposed to a Fujifilm LAS-3000 Imager (Fujifilm). Images were analyzed by using the MultiGauge V3.0 software (Fujifilm), and bands were quantified to determine the affinity of BosR for probes. (B) Competition of labeled BS1 (ZM132) with various amounts of unlabeled BS1, BS2, or non-specific (NS) DNA. (C) Competition of labeled BS2 (ZM127) with various amounts of unlabeled BS1, BS2, or NS DNA. In (B) and (C), 100 nM of BosR was used in EMSAs, and bound and unbound DNA was measured as described in (A). NS: non-specific competitor (ZM126).
Mentions: EMSAs employing target DNA sequences (representing the three BosR binding sites) exposed to increasing concentrations of rBosR were used as means of inferring BosR binding affinities for the three binding sites. As shown in Fig. 6 and 7, concentrations of 50, 20, or 200 nM of rBosR induced shifts by ZM132 (BS1), ZM127 (BS2), or ZM161 (BS3), respectively, suggesting that BosR has an affinity for these DNA targets in the order of BS2>BS1>BS3. In addition, when 200 nM of rBosR was used, only a slight proportion (<10%) of ZM161 (BS3) was shifted (Fig. 7), and probe ZM161 could not be saturated even by 10,000 nM of BosR (data not shown). To more precisely assess the affinity of BosR for BS1 and BS2, we measured the amount of bound DNA as a function of BosR concentration in EMSA assays (Fig. 8A). The dissociation binding constants (Kd) for BS1 (ZM132) or BS2 (ZM127) were 210.2 or 36.6 nM, respectively. The relative affinities of these two DNA elements for BosR were also assessed by competition EMSA analysis (Fig. 8, B and C). Binding of labeled BS1 or BS2 was not inhibited by the non-specific competitor ZM126 (NS), but was inhibited by unlabeled (cold competitor) BS1 or BS2, respectively. Moreover, binding of labeled BS1 was inhibited approximately 90% by the addition of 200-fold unlabeled BS1, but was completely competed out by 50-fold unlabeled BS2 (Fig. 8B). The addition of 200-fold of unlabeled BS1 competed out only 15% of BS2 binding (Fig. 8C). These data indicate that BosR has a higher affinity for BS2 than for BS1.

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