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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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The regulatory modes of NdgR. (A) Measurement of expression levels of NdgR target genes in the sulfate assimilation pathway in various combinations of input signals. D, N and T denote diamide treatment, ndgR gene and target genes of NdgR, respectively. D = 0 or 1 indicates nontreatment or treatment of diamide, respectively. The absence or presence of the ndgR gene is denoted as 0 or 1, respectively. Expression of target genes above threshold are denoted as 0 (OFF) or 1 (ON) of output signals. Expression levels were normalized relative to the expression levels of controls (D = 0, N = 0). All the values are represented with standard deviations. (B) The logic gates of NdgR regulatory networks. NdgR regulates the sulfate assimilation pathway using coherent and incoherent FFL.
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Fig5: The regulatory modes of NdgR. (A) Measurement of expression levels of NdgR target genes in the sulfate assimilation pathway in various combinations of input signals. D, N and T denote diamide treatment, ndgR gene and target genes of NdgR, respectively. D = 0 or 1 indicates nontreatment or treatment of diamide, respectively. The absence or presence of the ndgR gene is denoted as 0 or 1, respectively. Expression of target genes above threshold are denoted as 0 (OFF) or 1 (ON) of output signals. Expression levels were normalized relative to the expression levels of controls (D = 0, N = 0). All the values are represented with standard deviations. (B) The logic gates of NdgR regulatory networks. NdgR regulates the sulfate assimilation pathway using coherent and incoherent FFL.

Mentions: The members of the IclR family of regulators have been demonstrated to be activators, repressors, and dual-role proteins in many cases [4]. However, to ascertain the physiological role of a regulator, a comprehensive understanding of its regulatory modes, including higher and lower levels of regulation, would be helpful. In order to elucidate how NdgR regulates target gene expression in response to oxidative stress, we quantified mRNA levels of the relevant genes using qRT-PCR (Figure 5A). We selected sulfur assimilation into the cysteine biosynthesis pathway as a target due to its significance in the stress response [28]. The selected genes were cysI in cysIHCDN operon, cysA and cysM in cysteine biosynthesis, and ndgR. First, we confirmed that the transcriptional level of ndgR is induced by diamide. All of the target genes were also induced by diamide regardless of the presence of ndgR. This result indicates that other transcription factors related to oxidative stress could also exist for the regulation of them. Next, we observed two regulatory modes based on the measurement of expression levels affected by NdgR under diamide treatment. cysA and cysI were induced by NdgR regardless of diamide. Meanwhile, cysM seems to be hardly expressed without diamide treatment regardless of the presence of ndgR. Although NdgR may bind to the promoter of cysM in absence of diamide treatment, the expression level may not be decreased because cysM is not expressed originally. However, NdgR repressed the expression of cysM in the presence of diamide; thus, the role of NdgR as a dual regulator was confirmed.Figure 5


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)

The regulatory modes of NdgR. (A) Measurement of expression levels of NdgR target genes in the sulfate assimilation pathway in various combinations of input signals. D, N and T denote diamide treatment, ndgR gene and target genes of NdgR, respectively. D = 0 or 1 indicates nontreatment or treatment of diamide, respectively. The absence or presence of the ndgR gene is denoted as 0 or 1, respectively. Expression of target genes above threshold are denoted as 0 (OFF) or 1 (ON) of output signals. Expression levels were normalized relative to the expression levels of controls (D = 0, N = 0). All the values are represented with standard deviations. (B) The logic gates of NdgR regulatory networks. NdgR regulates the sulfate assimilation pathway using coherent and incoherent FFL.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: The regulatory modes of NdgR. (A) Measurement of expression levels of NdgR target genes in the sulfate assimilation pathway in various combinations of input signals. D, N and T denote diamide treatment, ndgR gene and target genes of NdgR, respectively. D = 0 or 1 indicates nontreatment or treatment of diamide, respectively. The absence or presence of the ndgR gene is denoted as 0 or 1, respectively. Expression of target genes above threshold are denoted as 0 (OFF) or 1 (ON) of output signals. Expression levels were normalized relative to the expression levels of controls (D = 0, N = 0). All the values are represented with standard deviations. (B) The logic gates of NdgR regulatory networks. NdgR regulates the sulfate assimilation pathway using coherent and incoherent FFL.
Mentions: The members of the IclR family of regulators have been demonstrated to be activators, repressors, and dual-role proteins in many cases [4]. However, to ascertain the physiological role of a regulator, a comprehensive understanding of its regulatory modes, including higher and lower levels of regulation, would be helpful. In order to elucidate how NdgR regulates target gene expression in response to oxidative stress, we quantified mRNA levels of the relevant genes using qRT-PCR (Figure 5A). We selected sulfur assimilation into the cysteine biosynthesis pathway as a target due to its significance in the stress response [28]. The selected genes were cysI in cysIHCDN operon, cysA and cysM in cysteine biosynthesis, and ndgR. First, we confirmed that the transcriptional level of ndgR is induced by diamide. All of the target genes were also induced by diamide regardless of the presence of ndgR. This result indicates that other transcription factors related to oxidative stress could also exist for the regulation of them. Next, we observed two regulatory modes based on the measurement of expression levels affected by NdgR under diamide treatment. cysA and cysI were induced by NdgR regardless of diamide. Meanwhile, cysM seems to be hardly expressed without diamide treatment regardless of the presence of ndgR. Although NdgR may bind to the promoter of cysM in absence of diamide treatment, the expression level may not be decreased because cysM is not expressed originally. However, NdgR repressed the expression of cysM in the presence of diamide; thus, the role of NdgR as a dual regulator was confirmed.Figure 5

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