Limits...
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.

Show MeSH

Related in: MedlinePlus

Genome-wide distributions of NdgR binding regions. (A) An overview of NdgR binding profiles across the S. coelicolor genome when grown on solid minimal media supplemented with N-acetylglucosamine and L-asparagine. Black and white dots indicate previously known and newly found NdgR binding regions, respectively. (B) Examples of binding profiles of previously known targets of NdgR. Red lines indicate the locations of putative binding motifs derived from FIMO and the values are the scores for the match of a position. Black arrows indicate the target genes within the transcription units that are directly regulated by NdgR. (C) MEME logo representation of the NdgR-DNA binding profile. This motif is present in 18 out of 19 enriched regions identified by ChIP-seq.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Genome-wide distributions of NdgR binding regions. (A) An overview of NdgR binding profiles across the S. coelicolor genome when grown on solid minimal media supplemented with N-acetylglucosamine and L-asparagine. Black and white dots indicate previously known and newly found NdgR binding regions, respectively. (B) Examples of binding profiles of previously known targets of NdgR. Red lines indicate the locations of putative binding motifs derived from FIMO and the values are the scores for the match of a position. Black arrows indicate the target genes within the transcription units that are directly regulated by NdgR. (C) MEME logo representation of the NdgR-DNA binding profile. This motif is present in 18 out of 19 enriched regions identified by ChIP-seq.

Mentions: NdgR and its orthologs have been characterized in vitro by DNA-binding experiments [15-18]. However, in vivo analysis of direct interaction between NdgR and its cognate DNA has not been described. To study the binding characteristics of NdgR to the S. coelicolor chromosome, we exploited a ChIP-seq method that has been used for genome-wide determination of in vivo binding locations of regulatory proteins. We constructed a 6× myc-tagged NdgR strain using homologous recombination for the immunoprecipitation (IP) using a specific anti-c-myc antibody [21]. For cell growth, specific nutrient conditions have often been used to elucidate the function of unknown regulators if the phenotypic differences between wild-type (WT) and deletion mutants were undetectable when the cells were grown in complex media [22]. Thus, we used solid minimal media supplemented with N-acetylglucosamine and L-asparagine for the perturbation of phenotypes [21]. To determine the NdgR-binding regions at the genome scale, we constructed a sequencing library using IP-DNA and performed next-generation sequencing. Sequencing of the library yielded short sequence reads of 36 nucleotides that were uniquely mapped onto the S. coelicolor genome (NC_003888). Using the MACS program, 19 NdgR-binding loci were detected with stringent cut-off conditions (p-value <1.0e-10, fold enrichment > 3) (Table 1). The peaks were distributed across the entire S. coelicolor genome (Figure 1A).Table 1


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)

Genome-wide distributions of NdgR binding regions. (A) An overview of NdgR binding profiles across the S. coelicolor genome when grown on solid minimal media supplemented with N-acetylglucosamine and L-asparagine. Black and white dots indicate previously known and newly found NdgR binding regions, respectively. (B) Examples of binding profiles of previously known targets of NdgR. Red lines indicate the locations of putative binding motifs derived from FIMO and the values are the scores for the match of a position. Black arrows indicate the target genes within the transcription units that are directly regulated by NdgR. (C) MEME logo representation of the NdgR-DNA binding profile. This motif is present in 18 out of 19 enriched regions identified by ChIP-seq.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Genome-wide distributions of NdgR binding regions. (A) An overview of NdgR binding profiles across the S. coelicolor genome when grown on solid minimal media supplemented with N-acetylglucosamine and L-asparagine. Black and white dots indicate previously known and newly found NdgR binding regions, respectively. (B) Examples of binding profiles of previously known targets of NdgR. Red lines indicate the locations of putative binding motifs derived from FIMO and the values are the scores for the match of a position. Black arrows indicate the target genes within the transcription units that are directly regulated by NdgR. (C) MEME logo representation of the NdgR-DNA binding profile. This motif is present in 18 out of 19 enriched regions identified by ChIP-seq.
Mentions: NdgR and its orthologs have been characterized in vitro by DNA-binding experiments [15-18]. However, in vivo analysis of direct interaction between NdgR and its cognate DNA has not been described. To study the binding characteristics of NdgR to the S. coelicolor chromosome, we exploited a ChIP-seq method that has been used for genome-wide determination of in vivo binding locations of regulatory proteins. We constructed a 6× myc-tagged NdgR strain using homologous recombination for the immunoprecipitation (IP) using a specific anti-c-myc antibody [21]. For cell growth, specific nutrient conditions have often been used to elucidate the function of unknown regulators if the phenotypic differences between wild-type (WT) and deletion mutants were undetectable when the cells were grown in complex media [22]. Thus, we used solid minimal media supplemented with N-acetylglucosamine and L-asparagine for the perturbation of phenotypes [21]. To determine the NdgR-binding regions at the genome scale, we constructed a sequencing library using IP-DNA and performed next-generation sequencing. Sequencing of the library yielded short sequence reads of 36 nucleotides that were uniquely mapped onto the S. coelicolor genome (NC_003888). Using the MACS program, 19 NdgR-binding loci were detected with stringent cut-off conditions (p-value <1.0e-10, fold enrichment > 3) (Table 1). The peaks were distributed across the entire S. coelicolor genome (Figure 1A).Table 1

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