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Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni.

Hwang S, Kim M, Ryu S, Jeon B - PLoS ONE (2011)

Bottom Line: Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide.Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type.Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea.

ABSTRACT
CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

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DNase I footprinting of the sodB and ahpC promoter regions.The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6], [9], the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC. Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.
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pone-0022300-g006: DNase I footprinting of the sodB and ahpC promoter regions.The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6], [9], the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC. Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.

Mentions: Among the oxidative stress proteins identified by 2DGE, CosR negatively and positively regulated SodB and AhpC, respectively (Figure 5A), and bound to the promoter region of the encoding genes (Figure 5B). DNase I footprinting assays were performed to determine the CosR binding sites in the promoter region of sodB and ahpC genes. DNase I footprinting results were interpreted based on the previous studies of the sodB and ahpC promoter sequences in C. jejuni [6], [9]. The results showed a region immediately downstream of the −10 region of the sodB promoter, which spans the leader sequence and the start codon of sodB, was protected from DNase I treatment (Figure 6A). Two CosR binding sites were found in the ahpC promoter, including the −37 to −17 region overlapping the −35 region of the ahpC promoter and the +1 to +21 region that is between the −10 region and the ahpC start codon (Figure 6B). The nucleotide sequences of the three CosR binding sites obtained from the DNase I footprinting assays were aligned to predict the consensus CosR binding sequence (Figure 6C). The alignment revealed a 21-bp CosR-binding sequence (tttaAanAaAAaTtAtgaTTt, ‘n’ represents any nucleotide, lowercase letters indicate less conserved residues, and capital letters are highly conserved residues; Figure 6C), suggesting that CosR binds to a specific target site in the CosR-regulated genes. Putative CosR binding sites were also identified in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence (Figure 6D).


Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni.

Hwang S, Kim M, Ryu S, Jeon B - PLoS ONE (2011)

DNase I footprinting of the sodB and ahpC promoter regions.The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6], [9], the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC. Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.
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Related In: Results  -  Collection

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pone-0022300-g006: DNase I footprinting of the sodB and ahpC promoter regions.The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6], [9], the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC. Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.
Mentions: Among the oxidative stress proteins identified by 2DGE, CosR negatively and positively regulated SodB and AhpC, respectively (Figure 5A), and bound to the promoter region of the encoding genes (Figure 5B). DNase I footprinting assays were performed to determine the CosR binding sites in the promoter region of sodB and ahpC genes. DNase I footprinting results were interpreted based on the previous studies of the sodB and ahpC promoter sequences in C. jejuni [6], [9]. The results showed a region immediately downstream of the −10 region of the sodB promoter, which spans the leader sequence and the start codon of sodB, was protected from DNase I treatment (Figure 6A). Two CosR binding sites were found in the ahpC promoter, including the −37 to −17 region overlapping the −35 region of the ahpC promoter and the +1 to +21 region that is between the −10 region and the ahpC start codon (Figure 6B). The nucleotide sequences of the three CosR binding sites obtained from the DNase I footprinting assays were aligned to predict the consensus CosR binding sequence (Figure 6C). The alignment revealed a 21-bp CosR-binding sequence (tttaAanAaAAaTtAtgaTTt, ‘n’ represents any nucleotide, lowercase letters indicate less conserved residues, and capital letters are highly conserved residues; Figure 6C), suggesting that CosR binds to a specific target site in the CosR-regulated genes. Putative CosR binding sites were also identified in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence (Figure 6D).

Bottom Line: Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide.Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type.Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea.

ABSTRACT
CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.

Show MeSH
Related in: MedlinePlus