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Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress.

Loui C, Chang AC, Lu S - BMC Microbiol. (2009)

Bottom Line: Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge.An abundant protein flagellin was elevated at both the protein and mRNA levels in the DeltaarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the DeltaarcA mutant to H2O2.The resistance of the DeltaarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, CA 94720, USA. csloui@berkeley.edu

ABSTRACT

Background: The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.

Results: To investigate the mechanism of ROS resistance mediated by ArcAB, we generated deletion mutants of ArcA and ArcB in E. coli. Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge. Mutagenesis analysis of ArcA indicated that ROS resistance was mediated through a distinct signaling pathway from that used in anaerobic conditions. An abundant protein flagellin was elevated at both the protein and mRNA levels in the DeltaarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the DeltaarcA mutant to H2O2. The resistance of the DeltaarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance.

Conclusion: Our results suggest that in addition to its role as a global regulator for anaerobic growth of bacteria, ArcAB system is also important for bacterial resistance to ROS in aerobic conditions, possibly through its influence on bacterial metabolism, especially amino acid and/or protein assimilation and synthesis.

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Resistance of the ΔarcA and ΔarcB mutant of E. coli to H2O2. (A and B) Growth and survival of wild type E. coli (diamond), ΔarcA mutant E. coli (square), ΔarcA mutant E. coli transformed with plasmid pRB3-273C (triangle) and ΔarcA mutant E. coli transformed with plasmid pRB3-arcA (cross) in LB broth with 1.5 mM H2O2 (A) or LB broth alone (B). (C and D) Growth and survival of wild type E. coli (diamond), ΔarcB mutant E. coli (square) and ΔarcB revertant mutant E. coli (cross) in LB broth with 1.5 mM H2O2 (C) or LB broth alone (D). Bacterial concentration was determined by plating and plotted against the indicated incubation time period. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation and sometimes fall within the data label.
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Figure 1: Resistance of the ΔarcA and ΔarcB mutant of E. coli to H2O2. (A and B) Growth and survival of wild type E. coli (diamond), ΔarcA mutant E. coli (square), ΔarcA mutant E. coli transformed with plasmid pRB3-273C (triangle) and ΔarcA mutant E. coli transformed with plasmid pRB3-arcA (cross) in LB broth with 1.5 mM H2O2 (A) or LB broth alone (B). (C and D) Growth and survival of wild type E. coli (diamond), ΔarcB mutant E. coli (square) and ΔarcB revertant mutant E. coli (cross) in LB broth with 1.5 mM H2O2 (C) or LB broth alone (D). Bacterial concentration was determined by plating and plotted against the indicated incubation time period. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation and sometimes fall within the data label.

Mentions: To determine if the ArcAB global regulatory system plays a role in the survival of E. coli under stress by reactive oxygen species (ROS), we generated deletion mutants of ArcA (the global regulator) and ArcB (the cognate sensor-kinase of ArcA) in E. coli (Table 1). Both ΔarcA and ΔarcB mutant E. coli formed smaller colonies than their parental E. coli, but otherwise showed similar colony morphology. The ΔarcA and ΔarcB mutant E. coli were tested for their growth properties in complete (Luria Bertani broth) or minimal (M9) medium with glucose as carbon source. Overnight culture of each bacterial strain was diluted 1:100 in LB or M9 medium, and the growth of bacteria was measured by the optical density of the culture at 550 nm (OD550 nm) every 2 hours for 8 hours and then at 24 hours. This incubation period includes both log phase of growth and stationary phase of bacteria. We found that OD550 nm of both ΔarcA and ΔarcB mutants appeared to be lower than that of the wild type E. coli during the log phase of growth. However, both mutants had similar bacterial concentrations and growth curves to those of the wild type E. coli when their growth was quantified by plating (Figure 1B and 1D). Therefore, no gross defect was observed in ΔarcA and ΔarcB mutants in spite of lower OD550 nm of their cultures. The anaerobic growth of the ΔarcA and ΔarcB mutant E. coli was also tested and compared to that of the wild type E. coli. No defect was detected (data not shown). Similar results were obtained with LB broth and M9 minimal medium, results obtained with LB broth are shown (Figure 1).


Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress.

Loui C, Chang AC, Lu S - BMC Microbiol. (2009)

Resistance of the ΔarcA and ΔarcB mutant of E. coli to H2O2. (A and B) Growth and survival of wild type E. coli (diamond), ΔarcA mutant E. coli (square), ΔarcA mutant E. coli transformed with plasmid pRB3-273C (triangle) and ΔarcA mutant E. coli transformed with plasmid pRB3-arcA (cross) in LB broth with 1.5 mM H2O2 (A) or LB broth alone (B). (C and D) Growth and survival of wild type E. coli (diamond), ΔarcB mutant E. coli (square) and ΔarcB revertant mutant E. coli (cross) in LB broth with 1.5 mM H2O2 (C) or LB broth alone (D). Bacterial concentration was determined by plating and plotted against the indicated incubation time period. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation and sometimes fall within the data label.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748088&req=5

Figure 1: Resistance of the ΔarcA and ΔarcB mutant of E. coli to H2O2. (A and B) Growth and survival of wild type E. coli (diamond), ΔarcA mutant E. coli (square), ΔarcA mutant E. coli transformed with plasmid pRB3-273C (triangle) and ΔarcA mutant E. coli transformed with plasmid pRB3-arcA (cross) in LB broth with 1.5 mM H2O2 (A) or LB broth alone (B). (C and D) Growth and survival of wild type E. coli (diamond), ΔarcB mutant E. coli (square) and ΔarcB revertant mutant E. coli (cross) in LB broth with 1.5 mM H2O2 (C) or LB broth alone (D). Bacterial concentration was determined by plating and plotted against the indicated incubation time period. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation and sometimes fall within the data label.
Mentions: To determine if the ArcAB global regulatory system plays a role in the survival of E. coli under stress by reactive oxygen species (ROS), we generated deletion mutants of ArcA (the global regulator) and ArcB (the cognate sensor-kinase of ArcA) in E. coli (Table 1). Both ΔarcA and ΔarcB mutant E. coli formed smaller colonies than their parental E. coli, but otherwise showed similar colony morphology. The ΔarcA and ΔarcB mutant E. coli were tested for their growth properties in complete (Luria Bertani broth) or minimal (M9) medium with glucose as carbon source. Overnight culture of each bacterial strain was diluted 1:100 in LB or M9 medium, and the growth of bacteria was measured by the optical density of the culture at 550 nm (OD550 nm) every 2 hours for 8 hours and then at 24 hours. This incubation period includes both log phase of growth and stationary phase of bacteria. We found that OD550 nm of both ΔarcA and ΔarcB mutants appeared to be lower than that of the wild type E. coli during the log phase of growth. However, both mutants had similar bacterial concentrations and growth curves to those of the wild type E. coli when their growth was quantified by plating (Figure 1B and 1D). Therefore, no gross defect was observed in ΔarcA and ΔarcB mutants in spite of lower OD550 nm of their cultures. The anaerobic growth of the ΔarcA and ΔarcB mutant E. coli was also tested and compared to that of the wild type E. coli. No defect was detected (data not shown). Similar results were obtained with LB broth and M9 minimal medium, results obtained with LB broth are shown (Figure 1).

Bottom Line: Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge.An abundant protein flagellin was elevated at both the protein and mRNA levels in the DeltaarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the DeltaarcA mutant to H2O2.The resistance of the DeltaarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, CA 94720, USA. csloui@berkeley.edu

ABSTRACT

Background: The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.

Results: To investigate the mechanism of ROS resistance mediated by ArcAB, we generated deletion mutants of ArcA and ArcB in E. coli. Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge. Mutagenesis analysis of ArcA indicated that ROS resistance was mediated through a distinct signaling pathway from that used in anaerobic conditions. An abundant protein flagellin was elevated at both the protein and mRNA levels in the DeltaarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the DeltaarcA mutant to H2O2. The resistance of the DeltaarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance.

Conclusion: Our results suggest that in addition to its role as a global regulator for anaerobic growth of bacteria, ArcAB system is also important for bacterial resistance to ROS in aerobic conditions, possibly through its influence on bacterial metabolism, especially amino acid and/or protein assimilation and synthesis.

Show MeSH
Related in: MedlinePlus