The role of catalase isozymes in the culturability of the root colonizer Pseudomonas putida after exposure to hydrogen peroxide and antibiotics

Abstract

The culturability of Pseudomonas putida cells after exposure to hydrogen peroxide and antibiotics was correlated with growth-dependent expression of catalase isozymes. Exponential phase wild-type cells, which contained catalase isozyme A, survived a 15-min treatment with less than 4 mM hydrogen peroxide, but were killed by higher concentrations. The culturability of P. putida mutant JIM, which lacked any functional catalase in exponential phase, was reduced by more than 75% after a 15-min exposure to ≥ 0.25 mM hydrogen peroxide. Because submillimolar concentrations of hydrogen peroxide are physiologically relevant in the bacterial cell, our results demonstrate that catalase isozyme A has essential housekeeping functions for growing cultures of P. putida. The accumulation of catalase isozymes B and C during growth into stationary phase coincided with a decrease in the sensitivity of wild-type and JIM cells of P. putida to hydrogen peroxide. Late stationary phase wild-type cells survived a 15-min exposure to even 50 mM hydrogen peroxide and mutant J1M cells survived exposure to 20 mM but not 50 mM hydrogen peroxide. The antibiotics tetracycline and kanamycin, which inhibit protein synthesis, were used to study the role of catalase induction in resistance to hydrogen peroxide. More than 40 and 80% of exponential phase cells of P. putida wild-type and J1M strains, respectively, were rendered nonculturable after a 20-min exposure to 45 μM tetracycline. Surprisingly, stationary phase cells of both P. putida strains were culturable after a 20-min exposure to tetracycline but remained sensitive to kanamycin. Exposure to tetracycline of stationary phase cells did not reduce the resistance of these cells to hydrogen peroxide. Tetracycline but not kanamycin increased the activity of catalase in lysates prepared from P. putida wild-type and mutant cells in early stationary growth phase. At this growth phase, only catalase isozyme B is operational in both strains, which suggests that tetracycline affects the activity of this enzyme.Key words: Pseudomonas putida, antibiotics, catalase, culturability, growth phase.