Physiological consequences of trichloroethylene degradation by the toluene-oxidizing bacterium Burkholderia cepacia G4

Graduation date: 2002A number of bacterial species are capable of degrading the widespread environmental\ud pollutant trichloroethylene (TCE) via aerobic cometabolism, but cytotoxic effects that\ud can debilitate the microorganism often accompany this transformation. In this\ud dissertation the effects of TCE degradation on the well-studied, toluene-oxidizing\ud bacterium Burkholderia cepacia G4 were investigated at the physiological and genetic\ud level and compared and contrasted to the effects elicited by several nonhalogenated,\ud short chain alkenes and alkynes. Linear alkynes (C₃-C₁₀) were classified as strong\ud mechanism-based inactivators of toluene 2-monooxygenase activity in B. cepacia G4,\ud with 2- and 3-alkynes providing a more potent effect than their 1-alkyne counterparts.\ud The C₂ alkyne, acetylene, was weak inactivator of toluene 2-monooxygenase activity\ud presumably because it does not bind efficiently to this oxygenase. Toluene-grown cells of B. cepacia G4 cells oxidized ethylene and propylene to their respective\ud epoxides with no observable effect on cell culturability or general respiratory activity.\ud In contrast, TCE oxidation was accompanied by a myriad of cytotoxic effects.\ud Accumulation of general cellular damage, manifested as a loss of cell culturability and\ud general respiratory activity, outpaced loss of toluene 2-monooxygenase activity during\ud TCE oxidation. Measures of the culturability of TCE-injured cells varied up to 3\ud orders of magnitude (depending on the method of assessment), and it was found that\ud TCE-injured cells were ultra sensitive to H₂O₂ on the surface of agar plates. It was\ud proposed that a toxicity threshold exists for B. cepacia G4 during TCE oxidation, and\ud once cells have degraded ≥0.5 μmol of TCE (mg of cells⁻¹) the likelihood of recovery\ud decreases significantly. Tn5 mutants of B. cepacia G4 with disruptions in genes\ud putatively encoding enzymes involved in DNA repair (including UvrB, RuvB, RecA,\ud and RecG) were ultra susceptible to killing by TCE, as well as the known DNA\ud damaging agents, UV light, mitomycin C, and H₂O₂. Physiological and genetic\ud analysis of the mutants provided suggestive evidence that nucleotide excision repair\ud and recombinational repair activities are linked to the survivability of TCE-injured B.\ud cepacia G4