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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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Amino acid sequence analysis of CosR homologs and genomic organization of cosR flanking region.(A) Multiple alignment of CosR homologs. The GenBank accession number is indicated in parentheses: C. jejuni CosR (Cj0355c: YP_002343793.1), Campylobacter coli CCO0443 (ZP_00367627.1), Helicobacter pylori HP1043 (AAD05966.1), Helicobacter hepaticus HH1697 (NP_861228.1), Arcobacter butzleri Abu_0375 (YP_001489319.1) and Wolinella succinogenes WS0306 (NP_906557.1). Based on previous studies of the HP1043 and OmpR proteins [60], [74], the highly conserved residues in the receiver domain are indicated by arrowheads. The aspartate residue which differs from the highly conserved aspartate residues in the other CosR homologs is marked with a star. (B) The analysis of cosR flanking region shows the lack of its cognate sensor kinase in its vicinity.
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pone-0022300-g001: Amino acid sequence analysis of CosR homologs and genomic organization of cosR flanking region.(A) Multiple alignment of CosR homologs. The GenBank accession number is indicated in parentheses: C. jejuni CosR (Cj0355c: YP_002343793.1), Campylobacter coli CCO0443 (ZP_00367627.1), Helicobacter pylori HP1043 (AAD05966.1), Helicobacter hepaticus HH1697 (NP_861228.1), Arcobacter butzleri Abu_0375 (YP_001489319.1) and Wolinella succinogenes WS0306 (NP_906557.1). Based on previous studies of the HP1043 and OmpR proteins [60], [74], the highly conserved residues in the receiver domain are indicated by arrowheads. The aspartate residue which differs from the highly conserved aspartate residues in the other CosR homologs is marked with a star. (B) The analysis of cosR flanking region shows the lack of its cognate sensor kinase in its vicinity.

Mentions: CosR is an OmpR-type response regulator essential for the viability of C. jejuni [17], [20]. Basic Local Alignment Search Tool (BLAST) results showed that CosR is highly conserved among Campylobacter species, including C. jejuni, C. coli, C. fetus, C. hominis, C. curvus, C. lari and C. concisus (data not shown). Interestingly, CosR homologs with high amino acid sequence similarities were found primarily in ε-proteobacteria, such as Helicobacter and Wolinella (Figure 1A). Compared with C. jejuni CosR, the N-terminal receiver domain of H. pylori HP1043 exhibited lower sequence similarity (38% identity with five gaps over 112 amino acids) than the highly conserved C-terminal DNA-binding domain (83% identity over 111 amino acids; Figure 1A). The conserved aspartate residue D8 in the CosR homologs is replaced with a lysine in HP1043, and four amino acid residues in the receiver domain (51st∼54th amino acid residues in CosR) are deleted in HP1043 (Figure 1A). Although an asparagine residue (N51) in CosR is substituted for a conserved aspartate residue present in the CosR homologs of Wolinella and Arcobacter, other conserved aspartate residues (e.g. D8, D56, and D58) may serve as an alternative phosphorylation site (Figure 1A). Genes encoding the sensor kinase and response regulator of the two-component regulatory system are often positioned adjacent to each other; however, the potential cognate sensor kinase was not found near cosR (Figure 1B). In C. jejuni, the cosR gene is located downstream of ferredoxin (fdxB) and upstream of dihydroneopterin aldolase (folB; Figure 1B).


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)

Amino acid sequence analysis of CosR homologs and genomic organization of cosR flanking region.(A) Multiple alignment of CosR homologs. The GenBank accession number is indicated in parentheses: C. jejuni CosR (Cj0355c: YP_002343793.1), Campylobacter coli CCO0443 (ZP_00367627.1), Helicobacter pylori HP1043 (AAD05966.1), Helicobacter hepaticus HH1697 (NP_861228.1), Arcobacter butzleri Abu_0375 (YP_001489319.1) and Wolinella succinogenes WS0306 (NP_906557.1). Based on previous studies of the HP1043 and OmpR proteins [60], [74], the highly conserved residues in the receiver domain are indicated by arrowheads. The aspartate residue which differs from the highly conserved aspartate residues in the other CosR homologs is marked with a star. (B) The analysis of cosR flanking region shows the lack of its cognate sensor kinase in its vicinity.
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Related In: Results  -  Collection

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pone-0022300-g001: Amino acid sequence analysis of CosR homologs and genomic organization of cosR flanking region.(A) Multiple alignment of CosR homologs. The GenBank accession number is indicated in parentheses: C. jejuni CosR (Cj0355c: YP_002343793.1), Campylobacter coli CCO0443 (ZP_00367627.1), Helicobacter pylori HP1043 (AAD05966.1), Helicobacter hepaticus HH1697 (NP_861228.1), Arcobacter butzleri Abu_0375 (YP_001489319.1) and Wolinella succinogenes WS0306 (NP_906557.1). Based on previous studies of the HP1043 and OmpR proteins [60], [74], the highly conserved residues in the receiver domain are indicated by arrowheads. The aspartate residue which differs from the highly conserved aspartate residues in the other CosR homologs is marked with a star. (B) The analysis of cosR flanking region shows the lack of its cognate sensor kinase in its vicinity.
Mentions: CosR is an OmpR-type response regulator essential for the viability of C. jejuni [17], [20]. Basic Local Alignment Search Tool (BLAST) results showed that CosR is highly conserved among Campylobacter species, including C. jejuni, C. coli, C. fetus, C. hominis, C. curvus, C. lari and C. concisus (data not shown). Interestingly, CosR homologs with high amino acid sequence similarities were found primarily in ε-proteobacteria, such as Helicobacter and Wolinella (Figure 1A). Compared with C. jejuni CosR, the N-terminal receiver domain of H. pylori HP1043 exhibited lower sequence similarity (38% identity with five gaps over 112 amino acids) than the highly conserved C-terminal DNA-binding domain (83% identity over 111 amino acids; Figure 1A). The conserved aspartate residue D8 in the CosR homologs is replaced with a lysine in HP1043, and four amino acid residues in the receiver domain (51st∼54th amino acid residues in CosR) are deleted in HP1043 (Figure 1A). Although an asparagine residue (N51) in CosR is substituted for a conserved aspartate residue present in the CosR homologs of Wolinella and Arcobacter, other conserved aspartate residues (e.g. D8, D56, and D58) may serve as an alternative phosphorylation site (Figure 1A). Genes encoding the sensor kinase and response regulator of the two-component regulatory system are often positioned adjacent to each other; however, the potential cognate sensor kinase was not found near cosR (Figure 1B). In C. jejuni, the cosR gene is located downstream of ferredoxin (fdxB) and upstream of dihydroneopterin aldolase (folB; Figure 1B).

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