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Genome-scale analysis reveals a role for NdgR in the thiol oxidative stress response in Streptomyces coelicolor.

Kim JN, Jeong Y, Yoo JS, Roe JH, Cho BK, Kim BG - BMC Genomics (2015)

Bottom Line: Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions.Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation.The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, Korea. realkjw1@snu.ac.kr.

ABSTRACT

Background: NdgR is an IclR-type transcription factor that regulates leucine biosynthesis and other metabolic pathways in Streptomyces coelicolor. Recent study revealed that NdgR is one of the regulatory targets of SigR, an oxidative stress response sigma factor, suggesting that the NdgR plays an important physiological role in response to environmental stresses. Although the regulatory functions of NdgR were partly characterized, determination of its regulon is required for better understanding of the transcriptional regulatory network related with the oxidative stress response.

Results: We determined genome-wide binding loci of NdgR by using chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and explored its physiological roles. The ChIP-seq profiles revealed 19 direct binding loci with a 15-bp imperfect palindromic motif, including 34 genes in their transcription units. Most genes in branched-chain amino acid and cysteine biosynthesis pathways were involved in the NdgR regulon. We proved that ndgR is induced by SigR under the thiol oxidation, and that an ndgR mutant strain is sensitive to the thiol oxidizing agent, diamide. Through the expression test of NdgR and the target genes for NdgR under diamide treatment, regulatory motifs were suggested. Interestingly, NdgR constitutes two regulatory motifs, coherent and incoherent feed-forward loops (FFL), in order to control its regulon under the diamide treatment. Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions.

Conclusions: Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation. The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

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SigR-dependent transcription activation of thendgRgene inS. coelicolor.(A) Quantitation of ndgR transcripts using quantitative realtime-PCR analysis (qRT-PCR) from diamide-treated cells reveals that the ndgR gene is induced using the SigR promoter. mRNA level of ndgR in WT (black bars) was induced under diamide treatment compared to the level of ndgR in sigRrsrA deletion mutant (gray bars). All levels are normalized by the levels of each sample at 0 min. (B) Sensitivity test of WT, ndgR deletion mutant (BG11) and complemented mutant (BG13) under thiol oxidative stress. Serially diluted spores of WT, BG11 and BG13 were spotted on R5 agar plates with or without added diamide (0.6 mM). Plates were incubated at 30°C for 5 days.
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Fig4: SigR-dependent transcription activation of thendgRgene inS. coelicolor.(A) Quantitation of ndgR transcripts using quantitative realtime-PCR analysis (qRT-PCR) from diamide-treated cells reveals that the ndgR gene is induced using the SigR promoter. mRNA level of ndgR in WT (black bars) was induced under diamide treatment compared to the level of ndgR in sigRrsrA deletion mutant (gray bars). All levels are normalized by the levels of each sample at 0 min. (B) Sensitivity test of WT, ndgR deletion mutant (BG11) and complemented mutant (BG13) under thiol oxidative stress. Serially diluted spores of WT, BG11 and BG13 were spotted on R5 agar plates with or without added diamide (0.6 mM). Plates were incubated at 30°C for 5 days.

Mentions: To ascertain the physiological role of the IclR family of regulators in the cell, identification of the interaction between the ligand and the substrate-recognition domain of the regulator would provide valuable insight. The effector molecule of NdgR has not been identified despite several attempts [15,16]. Instead, we explored a higher level of the NdgR regulatory network. Previous ChIP-chip experiments revealed ndgR as one of the targets of the oxidative stress response sigma factor SigR [19]. We measured mRNA expression level to validate the transcription of ndgR by SigR in response to thiol oxidative stress. NdgR was transiently induced by diamide treatment in the presence of SigR (Figure 4A); hence, NdgR is expected to regulate its target genes in response to thiol oxidative stress. However, NdgR was expressed constitutively in the absence of SigR, suggesting that there are additional transcriptional regulators in addition to the SigR.Figure 4


Genome-scale analysis reveals a role for NdgR in the thiol oxidative stress response in Streptomyces coelicolor.

Kim JN, Jeong Y, Yoo JS, Roe JH, Cho BK, Kim BG - BMC Genomics (2015)

SigR-dependent transcription activation of thendgRgene inS. coelicolor.(A) Quantitation of ndgR transcripts using quantitative realtime-PCR analysis (qRT-PCR) from diamide-treated cells reveals that the ndgR gene is induced using the SigR promoter. mRNA level of ndgR in WT (black bars) was induced under diamide treatment compared to the level of ndgR in sigRrsrA deletion mutant (gray bars). All levels are normalized by the levels of each sample at 0 min. (B) Sensitivity test of WT, ndgR deletion mutant (BG11) and complemented mutant (BG13) under thiol oxidative stress. Serially diluted spores of WT, BG11 and BG13 were spotted on R5 agar plates with or without added diamide (0.6 mM). Plates were incubated at 30°C for 5 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340878&req=5

Fig4: SigR-dependent transcription activation of thendgRgene inS. coelicolor.(A) Quantitation of ndgR transcripts using quantitative realtime-PCR analysis (qRT-PCR) from diamide-treated cells reveals that the ndgR gene is induced using the SigR promoter. mRNA level of ndgR in WT (black bars) was induced under diamide treatment compared to the level of ndgR in sigRrsrA deletion mutant (gray bars). All levels are normalized by the levels of each sample at 0 min. (B) Sensitivity test of WT, ndgR deletion mutant (BG11) and complemented mutant (BG13) under thiol oxidative stress. Serially diluted spores of WT, BG11 and BG13 were spotted on R5 agar plates with or without added diamide (0.6 mM). Plates were incubated at 30°C for 5 days.
Mentions: To ascertain the physiological role of the IclR family of regulators in the cell, identification of the interaction between the ligand and the substrate-recognition domain of the regulator would provide valuable insight. The effector molecule of NdgR has not been identified despite several attempts [15,16]. Instead, we explored a higher level of the NdgR regulatory network. Previous ChIP-chip experiments revealed ndgR as one of the targets of the oxidative stress response sigma factor SigR [19]. We measured mRNA expression level to validate the transcription of ndgR by SigR in response to thiol oxidative stress. NdgR was transiently induced by diamide treatment in the presence of SigR (Figure 4A); hence, NdgR is expected to regulate its target genes in response to thiol oxidative stress. However, NdgR was expressed constitutively in the absence of SigR, suggesting that there are additional transcriptional regulators in addition to the SigR.Figure 4

Bottom Line: Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions.Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation.The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, Korea. realkjw1@snu.ac.kr.

ABSTRACT

Background: NdgR is an IclR-type transcription factor that regulates leucine biosynthesis and other metabolic pathways in Streptomyces coelicolor. Recent study revealed that NdgR is one of the regulatory targets of SigR, an oxidative stress response sigma factor, suggesting that the NdgR plays an important physiological role in response to environmental stresses. Although the regulatory functions of NdgR were partly characterized, determination of its regulon is required for better understanding of the transcriptional regulatory network related with the oxidative stress response.

Results: We determined genome-wide binding loci of NdgR by using chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and explored its physiological roles. The ChIP-seq profiles revealed 19 direct binding loci with a 15-bp imperfect palindromic motif, including 34 genes in their transcription units. Most genes in branched-chain amino acid and cysteine biosynthesis pathways were involved in the NdgR regulon. We proved that ndgR is induced by SigR under the thiol oxidation, and that an ndgR mutant strain is sensitive to the thiol oxidizing agent, diamide. Through the expression test of NdgR and the target genes for NdgR under diamide treatment, regulatory motifs were suggested. Interestingly, NdgR constitutes two regulatory motifs, coherent and incoherent feed-forward loops (FFL), in order to control its regulon under the diamide treatment. Using the regulatory motifs, NdgR regulates cysteine biosynthesis in response to thiol oxidative stress, enabling cells to maintain sulfur assimilation with homeostasis under stress conditions.

Conclusions: Our analysis revealed that NdgR is a global transcriptional regulator involved in the regulation of branched-chain amino acids biosynthesis and sulphur assimilation. The identification of the NdgR regulon broadens our knowledge regarding complex regulatory networks governing amino acid biosynthesis in the context of stress responses in S. coelicolor.

Show MeSH
Related in: MedlinePlus