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Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

View Article: PubMed Central - PubMed

ABSTRACT

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

No MeSH data available.


Cluster- and oxidation state-dependent DNA binding by [2Fe-2S] RsrR.EMSAs showing DNA probes unbound (U), bound (B), and non-specifically bound (NS) by (a) [2Fe-2S]2+ and apo-RsrR (b) [2Fe-2S]2+ RsrR and (c) [2Fe-2S]1+ RsrR. Ratios of [2Fe-2S] containing RsrR (Holo) and [RsrR] (apo) to DNA are indicated for (a) while the concentration of [2Fe-2S] RsrR only is reported in (b,c). DNA concentration was 3.5 nM for the [2Fe-2S]2+/1+ and apo-RsrR experiments. For (a,b) the reaction mixtures were separated at 30 mA for 50 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. For (c) the reaction mixtures were separated at 30 mA for 1 h 45 min and the polyacrylamide gel was pre-run at 30 mA for 50 min prior to use using the de-gassed running buffer containing 5 mM sodium dithionite. For (a) both holo and apo protein concentrations are represented as the sample contained both forms due to incomplete cluster loading. The concentrations reported are of the [2Fe-2S] concentration.
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f4: Cluster- and oxidation state-dependent DNA binding by [2Fe-2S] RsrR.EMSAs showing DNA probes unbound (U), bound (B), and non-specifically bound (NS) by (a) [2Fe-2S]2+ and apo-RsrR (b) [2Fe-2S]2+ RsrR and (c) [2Fe-2S]1+ RsrR. Ratios of [2Fe-2S] containing RsrR (Holo) and [RsrR] (apo) to DNA are indicated for (a) while the concentration of [2Fe-2S] RsrR only is reported in (b,c). DNA concentration was 3.5 nM for the [2Fe-2S]2+/1+ and apo-RsrR experiments. For (a,b) the reaction mixtures were separated at 30 mA for 50 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. For (c) the reaction mixtures were separated at 30 mA for 1 h 45 min and the polyacrylamide gel was pre-run at 30 mA for 50 min prior to use using the de-gassed running buffer containing 5 mM sodium dithionite. For (a) both holo and apo protein concentrations are represented as the sample contained both forms due to incomplete cluster loading. The concentrations reported are of the [2Fe-2S] concentration.

Mentions: To determine which forms of RsrR are able to bind DNA, we performed EMSA experiments using the intergenic region between the highly enriched ChIP target sven1847/8 as a probe. Increasing ratios of [2Fe-2S] RsrR to DNA resulted in a clear shift in the mobility of the DNA from unbound to bound, see Fig. 4a. Equivalent experiments with cluster-free (apo) RsrR did not result in a mobility shift, demonstrating that the cluster is required for DNA-binding activity. These experiments were performed aerobically and so the [2Fe-2S] cofactor was in its oxidised state. To determine if oxidation state affects DNA binding activity, EMSA experiments were performed with [2Fe-2S]2+ and [2Fe-2S]1+ forms of RsrR. The oxidised cluster was generated by exposure to air and confirmed by UV-visible absorbance. The reduced cluster was obtained by reduction with sodium dithionite, confirmed by UV-visible absorbance, and the reduced state was maintained using EMSA running buffer containing an excess of dithionite. The resulting EMSAs, Fig. 4b,c, show that DNA-binding occurred in both cases but that the oxidised form bound significantly more tightly. Tight binding could be restored to the reduced RsrR samples by allowing it to re-oxidise in air (data not shown). We cannot rule out that the apparent low affinity DNA binding observed for the reduced sample results from partial re-oxidation of the cluster during the electrophoretic experiment. Nevertheless, the conclusion is unaffected: oxidised, [2Fe-2S]2+ RsrR is the high affinity DNA-binding form and these results suggest a change in the redox state of the [2Fe-2S] cluster controls the activity of RsrR, something which has only previously been observed for SoxR, a member of the MerR superfamily27.


Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily
Cluster- and oxidation state-dependent DNA binding by [2Fe-2S] RsrR.EMSAs showing DNA probes unbound (U), bound (B), and non-specifically bound (NS) by (a) [2Fe-2S]2+ and apo-RsrR (b) [2Fe-2S]2+ RsrR and (c) [2Fe-2S]1+ RsrR. Ratios of [2Fe-2S] containing RsrR (Holo) and [RsrR] (apo) to DNA are indicated for (a) while the concentration of [2Fe-2S] RsrR only is reported in (b,c). DNA concentration was 3.5 nM for the [2Fe-2S]2+/1+ and apo-RsrR experiments. For (a,b) the reaction mixtures were separated at 30 mA for 50 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. For (c) the reaction mixtures were separated at 30 mA for 1 h 45 min and the polyacrylamide gel was pre-run at 30 mA for 50 min prior to use using the de-gassed running buffer containing 5 mM sodium dithionite. For (a) both holo and apo protein concentrations are represented as the sample contained both forms due to incomplete cluster loading. The concentrations reported are of the [2Fe-2S] concentration.
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Related In: Results  -  Collection

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f4: Cluster- and oxidation state-dependent DNA binding by [2Fe-2S] RsrR.EMSAs showing DNA probes unbound (U), bound (B), and non-specifically bound (NS) by (a) [2Fe-2S]2+ and apo-RsrR (b) [2Fe-2S]2+ RsrR and (c) [2Fe-2S]1+ RsrR. Ratios of [2Fe-2S] containing RsrR (Holo) and [RsrR] (apo) to DNA are indicated for (a) while the concentration of [2Fe-2S] RsrR only is reported in (b,c). DNA concentration was 3.5 nM for the [2Fe-2S]2+/1+ and apo-RsrR experiments. For (a,b) the reaction mixtures were separated at 30 mA for 50 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. For (c) the reaction mixtures were separated at 30 mA for 1 h 45 min and the polyacrylamide gel was pre-run at 30 mA for 50 min prior to use using the de-gassed running buffer containing 5 mM sodium dithionite. For (a) both holo and apo protein concentrations are represented as the sample contained both forms due to incomplete cluster loading. The concentrations reported are of the [2Fe-2S] concentration.
Mentions: To determine which forms of RsrR are able to bind DNA, we performed EMSA experiments using the intergenic region between the highly enriched ChIP target sven1847/8 as a probe. Increasing ratios of [2Fe-2S] RsrR to DNA resulted in a clear shift in the mobility of the DNA from unbound to bound, see Fig. 4a. Equivalent experiments with cluster-free (apo) RsrR did not result in a mobility shift, demonstrating that the cluster is required for DNA-binding activity. These experiments were performed aerobically and so the [2Fe-2S] cofactor was in its oxidised state. To determine if oxidation state affects DNA binding activity, EMSA experiments were performed with [2Fe-2S]2+ and [2Fe-2S]1+ forms of RsrR. The oxidised cluster was generated by exposure to air and confirmed by UV-visible absorbance. The reduced cluster was obtained by reduction with sodium dithionite, confirmed by UV-visible absorbance, and the reduced state was maintained using EMSA running buffer containing an excess of dithionite. The resulting EMSAs, Fig. 4b,c, show that DNA-binding occurred in both cases but that the oxidised form bound significantly more tightly. Tight binding could be restored to the reduced RsrR samples by allowing it to re-oxidise in air (data not shown). We cannot rule out that the apparent low affinity DNA binding observed for the reduced sample results from partial re-oxidation of the cluster during the electrophoretic experiment. Nevertheless, the conclusion is unaffected: oxidised, [2Fe-2S]2+ RsrR is the high affinity DNA-binding form and these results suggest a change in the redox state of the [2Fe-2S] cluster controls the activity of RsrR, something which has only previously been observed for SoxR, a member of the MerR superfamily27.

View Article: PubMed Central - PubMed

ABSTRACT

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

No MeSH data available.