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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.


Related in: MedlinePlus

Spectroscopic characterization of RsrR.UV-visible absorption (a), CD (b) and EPR spectra (c) of 309 μM [2Fe-2S] RsrR (~75% cluster-loaded). Black lines – as isolated, red lines – oxidised, grey lines reduced proteins. In (a,b), initial exposure to ambient O2 for 30 min was followed by 309 μM sodium dithionite treatment; in (c) – as isolated protein was first anaerobically reduced by 309 μM sodium dithionite and then exposed to ambient O2 for 50 min. A 1 mm pathlength cuvette was used for optical measurements. Inset in (a) shows details of the iron-sulfur cluster absorbance in the 300–700 nm region.
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f2: Spectroscopic characterization of RsrR.UV-visible absorption (a), CD (b) and EPR spectra (c) of 309 μM [2Fe-2S] RsrR (~75% cluster-loaded). Black lines – as isolated, red lines – oxidised, grey lines reduced proteins. In (a,b), initial exposure to ambient O2 for 30 min was followed by 309 μM sodium dithionite treatment; in (c) – as isolated protein was first anaerobically reduced by 309 μM sodium dithionite and then exposed to ambient O2 for 50 min. A 1 mm pathlength cuvette was used for optical measurements. Inset in (a) shows details of the iron-sulfur cluster absorbance in the 300–700 nm region.

Mentions: The genes bound by RsrR do not include any NO detoxification genes and this suggested it is not an NsrR homologue but instead has an alternative function. To learn more about the protein we purified it from E. coli under strictly anaerobic conditions. The anaerobic RsrR solution is pink in colour but rapidly turns brown when exposed to O2, suggesting the presence of a redox-active cofactor. Consistent with this, the UV-visible absorbance spectrum of the as-isolated protein revealed broad weak bands in the 300–640 nm region but following exposure to O2, the spectrum changed significantly, with a more intense absorbance band at 460 nm and a pronounced shoulder feature at 330 nm (Fig. 2a). The form of the reduced and oxidized spectra are similar to those previously reported for [2Fe-2S] clusters that are coordinated by three Cys residues and one His2122. The anaerobic addition of dithionite to the previously air-exposed sample (at a 1:1 ratio with [2Fe-2S] cluster as determined by iron content) resulted in a spectrum very similar to that of the as-isolated protein (Fig. 2a), demonstrating that the cofactor undergoes redox cycling.


Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily
Spectroscopic characterization of RsrR.UV-visible absorption (a), CD (b) and EPR spectra (c) of 309 μM [2Fe-2S] RsrR (~75% cluster-loaded). Black lines – as isolated, red lines – oxidised, grey lines reduced proteins. In (a,b), initial exposure to ambient O2 for 30 min was followed by 309 μM sodium dithionite treatment; in (c) – as isolated protein was first anaerobically reduced by 309 μM sodium dithionite and then exposed to ambient O2 for 50 min. A 1 mm pathlength cuvette was used for optical measurements. Inset in (a) shows details of the iron-sulfur cluster absorbance in the 300–700 nm region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Spectroscopic characterization of RsrR.UV-visible absorption (a), CD (b) and EPR spectra (c) of 309 μM [2Fe-2S] RsrR (~75% cluster-loaded). Black lines – as isolated, red lines – oxidised, grey lines reduced proteins. In (a,b), initial exposure to ambient O2 for 30 min was followed by 309 μM sodium dithionite treatment; in (c) – as isolated protein was first anaerobically reduced by 309 μM sodium dithionite and then exposed to ambient O2 for 50 min. A 1 mm pathlength cuvette was used for optical measurements. Inset in (a) shows details of the iron-sulfur cluster absorbance in the 300–700 nm region.
Mentions: The genes bound by RsrR do not include any NO detoxification genes and this suggested it is not an NsrR homologue but instead has an alternative function. To learn more about the protein we purified it from E. coli under strictly anaerobic conditions. The anaerobic RsrR solution is pink in colour but rapidly turns brown when exposed to O2, suggesting the presence of a redox-active cofactor. Consistent with this, the UV-visible absorbance spectrum of the as-isolated protein revealed broad weak bands in the 300–640 nm region but following exposure to O2, the spectrum changed significantly, with a more intense absorbance band at 460 nm and a pronounced shoulder feature at 330 nm (Fig. 2a). The form of the reduced and oxidized spectra are similar to those previously reported for [2Fe-2S] clusters that are coordinated by three Cys residues and one His2122. The anaerobic addition of dithionite to the previously air-exposed sample (at a 1:1 ratio with [2Fe-2S] cluster as determined by iron content) resulted in a spectrum very similar to that of the as-isolated protein (Fig. 2a), demonstrating that the cofactor undergoes redox cycling.

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