| Summary: | A number of bacterial species are capable of degrading the widespread environmental
pollutant trichloroethylene (TCE) via aerobic cometabolism, but cytotoxic effects that
can debilitate the microorganism often accompany this transformation. In this
dissertation the effects of TCE degradation on the well-studied, toluene-oxidizing
bacterium Burkholderia cepacia G4 were investigated at the physiological and genetic
level and compared and contrasted to the effects elicited by several nonhalogenated,
short chain alkenes and alkynes. Linear alkynes (C���-C������) were classified as strong
mechanism-based inactivators of toluene 2-monooxygenase activity in B. cepacia G4,
with 2- and 3-alkynes providing a more potent effect than their 1-alkyne counterparts.
The C��� alkyne, acetylene, was weak inactivator of toluene 2-monooxygenase activity
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
epoxides with no observable effect on cell culturability or general respiratory activity.
In contrast, TCE oxidation was accompanied by a myriad of cytotoxic effects.
Accumulation of general cellular damage, manifested as a loss of cell culturability and
general respiratory activity, outpaced loss of toluene 2-monooxygenase activity during
TCE oxidation. Measures of the culturability of TCE-injured cells varied up to 3
orders of magnitude (depending on the method of assessment), and it was found that
TCE-injured cells were ultra sensitive to H���O��� on the surface of agar plates. It was
proposed that a toxicity threshold exists for B. cepacia G4 during TCE oxidation, and
once cells have degraded ���0.5 ��mol of TCE (mg of cells�����) the likelihood of recovery
decreases significantly. Tn5 mutants of B. cepacia G4 with disruptions in genes
putatively encoding enzymes involved in DNA repair (including UvrB, RuvB, RecA,
and RecG) were ultra susceptible to killing by TCE, as well as the known DNA
damaging agents, UV light, mitomycin C, and H���O���. Physiological and genetic
analysis of the mutants provided suggestive evidence that nucleotide excision repair
and recombinational repair activities are linked to the survivability of TCE-injured B.
cepacia G4. === Graduation date: 2002
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