Induction of oxidative stress by high hydrostatic pressure in Escherichia coli

Abstract

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.

FIG. 1.

Level of endogenous oxidative stress (bars), measured as relative AP activity in E. coli MG1655 and LMM1010 stationary-phase cells expressing a Δ2-22 leaderless alkaline phosphatase, after treatment at different pressures (20°C, 15 min). Bacterial survival as determined by plate counts (log₁₀ N) is presented for MG1655 (♦) and LMM1010 (▴). Results are means ± standard deviations and are from three independent experiments.

FIG. 2.

Level of endogenous oxidative stress (bars), measured as relative AP activity in E. coli MG1655 exponential-phase cells expressing a Δ2-22 leaderless alkaline phosphatase, after treatment at different pressures (20°C, 15 min). Bacterial survival as determined by plate counts (log₁₀ N) is indicated by diamonds; open symbols represent counts below the detection limit. Results are means ± standard deviations and are from three independent experiments.

FIG. 3.

Inactivation of stationary-phase cells of MG1655 by pressure treatment at 300 MPa and 400 MPa (15 min, 20°C), expressed as log₁₀ (N₀/N), with N₀ and N, respectively, being the plate count before and after pressure treatment. Cells were either plated on TSA and incubated aerobically or plated on TSA plus 10 mM cysteine and incubated anaerobically. Results are means ± standard deviations and are from three independent experiments.

FIG. 4.

Inactivation of stationary-phase cells of wild-type MG1655 and oxidative stress-sensitive mutants by pressure treatment at 350 MPa (15 min, 20°C), expressed as log₁₀ (N₀/N), with N₀ and N, respectively, being the plate count before and after pressure treatment. The dashed line represents the detection limit. Results are means ± standard deviations and are from three independent experiments.

FIG. 5.

Inactivation of stationary-phase cells of MG1655 by oxidants (0.11 M t-BHP, 1 h; 1 mM plumbagin, 1 h), by high pressure (250 MPa, 20°C, 15 min), and by high pressure followed by oxidant treatment. Inactivation is expressed as log₁₀ (N₀/N), with N₀ and N, respectively, being the plate count before and after treatment. The dashed line represents the detection limit. Results are means ± standard deviations and are from three independent experiments.

FIG. 6.

Survival of stationary-phase cultures of MG1655 (▪) and its pressure-resistant mutants LMM1010 (▴), LMM1020 (♦), and LMM1030 (•) upon exposure to 0.8 mM plumbagin (A) or 0.22 M t-butylhydroperoxide (B), expressed as (N/N₀) × 100%, with N₀ and N, respectively, being the plate count before and after treatment. Open symbols represent counts below the detection limit. Representative results from two independent experiments are shown.