Article Figures & Data
Figures
- Fig. 1.
Ahp scavenges H2O2 in a Kat− strain. Cultures of MG1655 (wild type, ▿), JI367 (katG katE, ◊), JI370 (ahpCF, *), JI377 (ahpCF katG katE, ○), JI374 (ahpCF katG, ■), and JI372 (ahpCF katE, ▵) were grown aerobically in LB and resuspended in PBS at an OD of 0.1. H2O2 was added at a final concentration of 1.5 μM. At various time points after addition of H2O2, the H2O2concentration was measured as described in Materials and Methods.
- Fig. 2.
Dependence of scavenging rate on H2O2 concentration. Rates of H2O2 decomposition were measured in dilute suspensions of JI370 (ahpCF, ◊) and JI367 (katG katE, ■). Rates were normalized to a value of 1.0 OD.
- Fig. 3.
Distinct efficiencies of Ahp and HPI at different H2O2 concentrations. H2O2 was added at a final concentration of 0.1 μM (right panel) and 150 μM (left panel) to cultures of JI372 (ahpCF katE), JI367 (katG katE), and JI362 (katE). Two minutes after addition of H2O2, the H2O2concentration was measured as described in Materials and Methods.
- Fig. 4.
H2O2 production by Ahp− Kat− cells. MG1655 (wild type), JI370 (ahpCF), JI367 (katG katE), and JI377 (ahpCF katG katE) were grown aerobically in LB and resuspended in 37°C minimal A salts containing 0.2% glucose. At various time points after resuspension, the H2O2 concentration of the medium was measured. (The H2O2 levels drop for the three scavenger-proficient strains because these strains degrade the 0.05 μM H2O2 that is present in the initial medium.)
- Fig. 5.
An Ahp− Kat− strain has an aerobic growth defect. (A) Growth of MG1655 (wild type, ▪), JI370 (ahpCF, ○), JI367 (katG katE, ◊), JI374 (katG ahpCF, *), and JI377 (katG katE ahpCF, ▿) in aerobic LB medium. (B) MG1655 and JI377 were grown aerobically in LB from 0.001 OD to mid-log phase, as for panel A. Cells were then subcultured into fresh LB at an OD of 0.01, and residual growth was observed. (C) Exogenous catalase protects against an aerobic growth defect in LB. MG1655 and JI377 were grown aerobically in fresh LB. Cultures were then subcultured to 0.01 OD in LB. Exogenous catalase was added to one culture of JI377 every 15 min to maintain catalase activity. At various time points, aliquots were removed from each culture and plated in selective top agar. Growth rate was determined the next day. (D) Endogenous H2O2can be toxic to cells. Exponential anaerobic MG1655 and JI377 were subcultured into fresh aerobic peroxide-free minimal A glucose (0.2%) medium, and growth was monitored.
Tables
- Table 1.
E. coli strains
Strain Genotype Source or reference UM1 katE katG14 lacY rspL thi-1 14 UM120 katE12::Tn10 hfrH thi-1 Peter Loewen UM202 katG17::Tn10 hfrH thi-1 Peter Loewen GK100 ΔcydAB::camΔ(cyoABCDE)456::kan 12 KM38 As UM1 plus ΔcydAB::cam P1(GK100) × UM1 KM39 As UMI plus Δ(cyoABCDE)456::kan P1(GK100) × UM1 SK2255 zbe-279::Tn10 thyA6 rps120 decC1 E. coli Genetic Stock Center N9716 As GC4468 plus ΔoxyR::spec Gisela Storz AS430 ΔoxyR::specΔlacU169 rpsL P1(N9716) × GC4468 AB1157 F−thr-1 leuB6 proA2 his-4 thi-1 argE2 lacY1 galK2 rspL supE44 ara-14 xyl-15 mtl-1 tsx-33 10 MG1655 F− wild-type E. coli Genetic Stock Center JI360 As MG1655 pluskatE12::Tn10 P1(UM120) × MG1655 JI361 As MG1655 pluskatG17::Tn10 P1(UM202) × MG1655 JI362 As JI360 plus Δ(katE12::Tn10)1(Tets) Tets derivative of JI360 JI364 As JI361 plus Δ(katG17::Tn10)1(Tets) Tets derivative of JI361 JI367 As JI364 pluskatE12::Tn10 P1(UM120) × JI364 JI370 As MG1655 plus ΔahpCF′ kan::′ahpF P1(MC4100ΔahpCF) × MG1655 JI372 As JI362 plus ΔahpCF′ kan::′ahpF P1(MC4100ΔahpCF) × JI362 JI374 As JI364 plus ΔahpCF′ kan::′ahpF P1(MC4100ΔahpCF) × JI364 JI377 As JI367 plus ΔahpCF′ kan::′ahpF P1(MC4100ΔahpCF) × JI367 MC4100 araD139 Δ(argF-lac)169λ−flhD5301 fruA25 relA1 rpsL150 rbsR22 deoC1 E. coli Genetic Stock Center MC4100ΔahpCF As MC4100 plus ΔahpCF′ kan::′ahpF Gisela Storz GS022 As MC4100 plus λRS45 Φ(katG::lacZ) Gisela Storz LC70 As GS022 plus ΔahpCF′ kan::′ahpF P1(MC4100ΔahpCF) × GS022 LC74 As LC70 plus ΔoxyR::spec P1(AS430) × LC70 LC80 As GS022 pluskatG17::Tn10 P1(UM202) × GS022 HDO3 As GS022 pluskatE12::Tn10 P1(UM120) × GS022 LC82 As LC80 plus Δ(katG17::Tn10)2(Tets) Tets derivative of LC80 LC84 As LC82 pluskatE12::Tn10 P1(UM120) × LC82 LC87 As MC4100ΔahpCF pluszbe-279::Tn10 P1(SK2255) × MC4100ΔahpCF LC89 As GS022 plus ΔahpCF′ kan::′ahpF zbe-279::Tn10 P1(LC87) × GS022 - Table 2.
Endogenous H2O2 activates the OxyR regulon in an ahpCF mutant
Straina Mean β-galactosidase activity (U/mg of protein) ± SD Anaerobic Aerobic Aerobic + catalaseb Ahp+ OxyR+(GSO22) 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 Ahp− OxyR+(LC70) 0.03 ± 0.01 0.35 ± 0.03 0.18 ± 0.02 Ahp− OxyR−(LC74) 0.03 ± 0.01 0.03 ± 0.01 NDc - Table 3.
HPI (katG) is the primary scavenger of supranormal H2O2concentrationsa
Strain Zone of inhibitionb(cm) Wild type 1.1 katG 1.6 ahpCF 1.1 katG katE 1.6 ahpCF katG 2.9 ahpCF katG katE 3.1 ↵a Assay was done by disk diffusion of 25 μl of 3% (880 mM) H2O2. Assay was done in triplicate, and trends were always consistent. One data set is shown. Strains used were MG1655 (wild type), JI364 (katG), JI370 (ahpCF), JI367 (katG katE), JI374 (ahpCF katG), and JI377 (ahpCF katG katE).
↵b Distance of clearing from the edge of the disk.
