Involvement of catalase and superoxide dismutase in hydrophobic organic solvent tolerance of Escherichia coli

Abstract

Escherichia coli strains are generally sensitive to hydrophobic organic solvents such as n-hexane and cyclohexane. Oxidative stress in E. coli by exposure to these hydrophobic organic solvents has been poorly understood. In the present study, we examined organic solvent tolerance and oxygen radical generation in E. coli mutants deficient in reactive oxygen species (ROS)-scavenging enzymes. The organic solvent tolerances in single gene mutants lacking genes encoding superoxide dismutase (sodA, sodB, and sodC), catalase (katE and katG), and alkyl hydroperoxide reductase (ahpCF) were similar to that of parent strain BW25113. We constructed a BW25113-based katE katG double mutant (BW25113∆katE∆katG) and sodA sodB double mutant (BW25113sodA∆sodB). These double-gene mutants were more sensitive to hydrophobic organic solvents than BW25113. In addition, the intracellular ROS levels in E. coli strains increased by the addition of n-hexane or cyclohexane. The ROS levels in BW25113∆katE∆katG and BW25113∆sodA∆sodB induced by exposure to the solvents were higher than that in BW25113. These results suggested that ROS-scavenging enzymes contribute to the maintenance of organic solvent tolerance in E. coli. In addition, the promoter activities of sodA and sodB were significantly increased by exposure to n-hexane.

Keywords: Catalase; Escherichia coli; Organic solvent tolerance; Reactive oxygen species; Superoxide dismutase.

Fig. 1

Colony-forming efficiency of E. coli BW25113 and its mutants deficient in ROS-scavenging enzymes on LBGMg agar medium. Each strain was grown in the absence of an organic solvent (A) and in the presence of n-hexane (B). Each strain was spotted at a tenfold dilution and incubated at 25 °C for 48 h

Fig. 2

Colony-forming efficiency of BW25113-based recombinant E. coli strains. Each strain was grown on LBGMg agar medium containing ampicillin (50 μg/ml) and isopropyl-β-d-thiogalactopyranoside (IPTG; 0.5 mM) in the absence of an organic solvent (A) and in the presence of n-hexane (B). Each strain was spotted at a tenfold dilution and incubated at 25 °C for 48 h

Fig. 3

Growth of E. coli BW25113, BW25113∆katE∆katG and BW25113∆sodA∆sodB in LBGMg liquid medium at 37 °C in the absence of an organic solvent (A) and in the presence of 10% (vol/vol) cyclooctane (B), 10% (vol/vol) n-hexane (C), or a 10% (vol/vol) n-hexane and cyclohexane mixture (9:1 vol/vol) (D). A 100-μl culture of an overnight-grown E. coli strain was inoculated into 10 ml of fresh LBGMg liquid medium containing an organic solvent. This two-phase culture was incubated at 37 °C. Growth was monitored by measuring turbidity (OD₆₆₀). Symbols: filled circle, BW25113; open square, BW25113∆katE∆katG; open triangle, BW25113∆sodA∆sodB. Values indicate the means and standard deviations of the results from three independent experiments

Fig. 4

Levels of intracellular ROS in E. coli cells measured by using carboxy-H₂DCFDA. ROS levels were measured in the strains exposed to TBHP (tert-butyl hydroperoxide), n-hexane or cyclohexane as described in the Methods. The relative specific fluorescence shows the ratio of the specific fluorescence in each strain divided by that in strain BW25113 not exposed to the organic solvent. Abbreviations: BW, BW25113; ∆katE∆katG, BW25113∆katE∆katG; ∆sodA∆sodB, BW25113∆sodA∆sodB. Mean values and standard deviations for three independent experiments are shown

Fig. 5

Effect of n-hexane on the promoter activity of katE, katG, sodA, or sodB. A 100-μl culture of the overnight-grown strain BW25113(pMCkatEp) (A), BW25113(pMCkatGp) (B), BW25113(pMCsodAp) (C), or BW25113(pMCsodBp) (D) was inoculated into 10 ml of fresh LBGMg liquid medium containing 50 μg/ml ampicillin without or with 1 ml of n-hexane. The culture was incubated at 37 °C until reaching an OD₆₀₀ of approximately 0.6. Cells were treated with chloroform and assayed for β-galactosidase activity. Values indicate the means and standard deviations of the results from three independent experiments