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The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

Gundogdu O, da Silva DT, Mohammad B, Elmi A, Mills DC, Wren BW, Dorrell N - Front Microbiol (2015)

Bottom Line: Mutation of either rrpA or rrpB reduces catalase (KatA) expression.Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant.Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine , London, UK.

ABSTRACT
The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic mobility shift assays (EMSAs) indicate that RrpA binds to a DNA promoter sequence upstream of the rrpA (A) and katA gene (B). EMSAs were performed for RrpA protein hybridized to a PCR amplified fragment upstream of rrpA or katA labeled with IRDye® 700. 2.5 μg (≈175 pmol) recombinant native protein was hybridized with 20 ng of gel purified DNA using the Odyssey® Infrared EMSA kit (LI-COR Biosciences). Unlabelled PCR amplified fragments upstream of either rrpA, katA, or flaA were included as controls. Separation was performed under non-denaturing conditions with samples loaded onto a 6% (w/v) DNA retardation gel.
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Figure 1: Electrophoretic mobility shift assays (EMSAs) indicate that RrpA binds to a DNA promoter sequence upstream of the rrpA (A) and katA gene (B). EMSAs were performed for RrpA protein hybridized to a PCR amplified fragment upstream of rrpA or katA labeled with IRDye® 700. 2.5 μg (≈175 pmol) recombinant native protein was hybridized with 20 ng of gel purified DNA using the Odyssey® Infrared EMSA kit (LI-COR Biosciences). Unlabelled PCR amplified fragments upstream of either rrpA, katA, or flaA were included as controls. Separation was performed under non-denaturing conditions with samples loaded onto a 6% (w/v) DNA retardation gel.

Mentions: RrpB is a DNA binding protein that binds to the promoter region of the rrpB gene resulting in negative autoregulation, a feature of the MarR family of transcriptional regulators (Gundogdu et al., 2011). EMSAs were performed to investigate whether RrpA could bind to the promoter region of the rrpA gene. The full-length RrpA protein was expressed and purified from E. coli. We do not believe the 6 × His interfered with any observed phenotypes as we have constructed the rrpA 6 × His tagged in the complementation vector pRRC as a control and obtained resuscitation of the hydrogen peroxide phenotype. This recombinant RrpA protein was observed to bind to a IRDye 700 fluorescently labeled 160 bp DNA fragment upstream of the rrpA gene (Figure 1A). Unlabelled upstream regions of rrpA and flaA were used as controls. An excess of unlabelled rrpA upstream region competed with the IRDye® 700 fluorescently labeled rrpA upstream region for the RrpA recombinant protein. However, an excess of unlabelled flaA upstream region did not compete with the RrpA recombinant protein. This data indicates that RrpA acts as a DNA binding protein. MarR family transcriptional regulators are classically negative autoregulators, so whilst RrpA and RrpB have been shown to bind upstream of rrpA and rrpB respectively, it is not possible to infer whether this is negative or positive autoregulation based on this experimental data. In addition, RrpA was shown to bind upstream of katA (Figure 1B).


The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

Gundogdu O, da Silva DT, Mohammad B, Elmi A, Mills DC, Wren BW, Dorrell N - Front Microbiol (2015)

Electrophoretic mobility shift assays (EMSAs) indicate that RrpA binds to a DNA promoter sequence upstream of the rrpA (A) and katA gene (B). EMSAs were performed for RrpA protein hybridized to a PCR amplified fragment upstream of rrpA or katA labeled with IRDye® 700. 2.5 μg (≈175 pmol) recombinant native protein was hybridized with 20 ng of gel purified DNA using the Odyssey® Infrared EMSA kit (LI-COR Biosciences). Unlabelled PCR amplified fragments upstream of either rrpA, katA, or flaA were included as controls. Separation was performed under non-denaturing conditions with samples loaded onto a 6% (w/v) DNA retardation gel.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508579&req=5

Figure 1: Electrophoretic mobility shift assays (EMSAs) indicate that RrpA binds to a DNA promoter sequence upstream of the rrpA (A) and katA gene (B). EMSAs were performed for RrpA protein hybridized to a PCR amplified fragment upstream of rrpA or katA labeled with IRDye® 700. 2.5 μg (≈175 pmol) recombinant native protein was hybridized with 20 ng of gel purified DNA using the Odyssey® Infrared EMSA kit (LI-COR Biosciences). Unlabelled PCR amplified fragments upstream of either rrpA, katA, or flaA were included as controls. Separation was performed under non-denaturing conditions with samples loaded onto a 6% (w/v) DNA retardation gel.
Mentions: RrpB is a DNA binding protein that binds to the promoter region of the rrpB gene resulting in negative autoregulation, a feature of the MarR family of transcriptional regulators (Gundogdu et al., 2011). EMSAs were performed to investigate whether RrpA could bind to the promoter region of the rrpA gene. The full-length RrpA protein was expressed and purified from E. coli. We do not believe the 6 × His interfered with any observed phenotypes as we have constructed the rrpA 6 × His tagged in the complementation vector pRRC as a control and obtained resuscitation of the hydrogen peroxide phenotype. This recombinant RrpA protein was observed to bind to a IRDye 700 fluorescently labeled 160 bp DNA fragment upstream of the rrpA gene (Figure 1A). Unlabelled upstream regions of rrpA and flaA were used as controls. An excess of unlabelled rrpA upstream region competed with the IRDye® 700 fluorescently labeled rrpA upstream region for the RrpA recombinant protein. However, an excess of unlabelled flaA upstream region did not compete with the RrpA recombinant protein. This data indicates that RrpA acts as a DNA binding protein. MarR family transcriptional regulators are classically negative autoregulators, so whilst RrpA and RrpB have been shown to bind upstream of rrpA and rrpB respectively, it is not possible to infer whether this is negative or positive autoregulation based on this experimental data. In addition, RrpA was shown to bind upstream of katA (Figure 1B).

Bottom Line: Mutation of either rrpA or rrpB reduces catalase (KatA) expression.Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant.Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine , London, UK.

ABSTRACT
The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

No MeSH data available.


Related in: MedlinePlus