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

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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.

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Oxidised RsrR binding to full site (class 1) and half site (class 2) RsrR targets.EMSAs showing DNA probes unbound (U) and bound (B) by [2Fe-2S]2+. Ratios of [2Fe-2S] RsrR and [RsrR] to DNA are indicated. DNA concentration was 4 nM for each probe. EMSA’s using class 2 DNA probes sven0247 and sven0519 (a), class 1 probes from the RsrR rsrR binding region (b) and the four possible half sites from the rsrR class 1 sites (c) were used. For (a) the reaction mixtures were separated at 30 mA for 1 h and the polyacrylamide gel was pre-run at 30 mA for 2 min prior to use. For (b,c) the reaction mixtures were separated at 30 mA for 30 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. A representation of the rsrR promoter breakdown is also available in Supplementary Figure S3b.
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f5: Oxidised RsrR binding to full site (class 1) and half site (class 2) RsrR targets.EMSAs showing DNA probes unbound (U) and bound (B) by [2Fe-2S]2+. Ratios of [2Fe-2S] RsrR and [RsrR] to DNA are indicated. DNA concentration was 4 nM for each probe. EMSA’s using class 2 DNA probes sven0247 and sven0519 (a), class 1 probes from the RsrR rsrR binding region (b) and the four possible half sites from the rsrR class 1 sites (c) were used. For (a) the reaction mixtures were separated at 30 mA for 1 h and the polyacrylamide gel was pre-run at 30 mA for 2 min prior to use. For (b,c) the reaction mixtures were separated at 30 mA for 30 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. A representation of the rsrR promoter breakdown is also available in Supplementary Figure S3b.

Mentions: To further investigate the DNA binding activities of [2Fe-2S]2+ RsrR, EMSAs were performed on DNA probes containing the two class 2 RsrR binding sites at sven0247 and sven519 (Fig. 5a). Both probes were shifted by oxidized [2Fe-2S] RsrR showing that RsrR binds to both class 1 and 2 probes in vitro. To further test the idea of RsrR recognizing full and half sites, we constructed a series of probes based on the divergent nmrA-rsrR promoters carrying both or each individual natural class 1 sites (Fig. 5b) and artificial half sites (Fig. 5c). The combinations of artificial half sites are illustrated in Supplemental Figure S3 in regards to the original promoter region. The results show that RsrR binds strongly to both full class 1 binding sites at the nmrA-rsrR promoters (Fig. 5b) but only weakly to artificial half sites (Fig. 5c). This suggests that although MEME only calls half sites in most of the RsrR target genes identified by ChIP-seq these class 2 targets must contain sufficient sequence information in the other half to enable strong binding by RsrR.


Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily
Oxidised RsrR binding to full site (class 1) and half site (class 2) RsrR targets.EMSAs showing DNA probes unbound (U) and bound (B) by [2Fe-2S]2+. Ratios of [2Fe-2S] RsrR and [RsrR] to DNA are indicated. DNA concentration was 4 nM for each probe. EMSA’s using class 2 DNA probes sven0247 and sven0519 (a), class 1 probes from the RsrR rsrR binding region (b) and the four possible half sites from the rsrR class 1 sites (c) were used. For (a) the reaction mixtures were separated at 30 mA for 1 h and the polyacrylamide gel was pre-run at 30 mA for 2 min prior to use. For (b,c) the reaction mixtures were separated at 30 mA for 30 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. A representation of the rsrR promoter breakdown is also available in Supplementary Figure S3b.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5015018&req=5

f5: Oxidised RsrR binding to full site (class 1) and half site (class 2) RsrR targets.EMSAs showing DNA probes unbound (U) and bound (B) by [2Fe-2S]2+. Ratios of [2Fe-2S] RsrR and [RsrR] to DNA are indicated. DNA concentration was 4 nM for each probe. EMSA’s using class 2 DNA probes sven0247 and sven0519 (a), class 1 probes from the RsrR rsrR binding region (b) and the four possible half sites from the rsrR class 1 sites (c) were used. For (a) the reaction mixtures were separated at 30 mA for 1 h and the polyacrylamide gel was pre-run at 30 mA for 2 min prior to use. For (b,c) the reaction mixtures were separated at 30 mA for 30 min and the polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. A representation of the rsrR promoter breakdown is also available in Supplementary Figure S3b.
Mentions: To further investigate the DNA binding activities of [2Fe-2S]2+ RsrR, EMSAs were performed on DNA probes containing the two class 2 RsrR binding sites at sven0247 and sven519 (Fig. 5a). Both probes were shifted by oxidized [2Fe-2S] RsrR showing that RsrR binds to both class 1 and 2 probes in vitro. To further test the idea of RsrR recognizing full and half sites, we constructed a series of probes based on the divergent nmrA-rsrR promoters carrying both or each individual natural class 1 sites (Fig. 5b) and artificial half sites (Fig. 5c). The combinations of artificial half sites are illustrated in Supplemental Figure S3 in regards to the original promoter region. The results show that RsrR binds strongly to both full class 1 binding sites at the nmrA-rsrR promoters (Fig. 5b) but only weakly to artificial half sites (Fig. 5c). This suggests that although MEME only calls half sites in most of the RsrR target genes identified by ChIP-seq these class 2 targets must contain sufficient sequence information in the other half to enable strong binding by RsrR.

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.


Related in: MedlinePlus