Issued also as thesis, Paris.

No description available.

No description available.

Graduation date: 1995

Polycyclic aromatic hydrocarbons (PAHs) are widespread hazardous, hydrophobic pollutants made of fused benzene rings. Besides of being present in large quantities in petroleum and related products they are formed generally during the incomplete combustion of organic matter. During evolution, bacteria have become capable of using such recalcitrant compounds as growth substrates. Hundreds of species of bacteria have been identified that can metabolise a wide range of contaminants, being of potential use in bioremediation strategies. The types of molecules that can be degraded depend on the metabolic pathway in each species. The genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The a and b subunits of a dioxygenase complex (FlnA1A2), showing 63% and 51% sequence identity respectively, with the subunits of an angular dioxygenase from the Gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in E. coli, FlnA1A2 was responsible for the angular oxidation of fluorene, fluorenol, fluorenone, dibenzofuran and dibenzo-p-dioxin. Moreover, FlnA1A2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. Quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1A2. Sphingomonas sp. strain LH128 harbors a naphthalene dioxygenase (PhnA1A2f) responsible for the oxidation of PAHs made of up to four rings, monochlorinated biphenyls and dibenzo-p-dioxin. PhnA1A2f shows exceptionally broad substrate specificity towards various pollutants in contrast to other naphthalene dioxygenases whose activity is limited to two- and three-ring PAHs. Moreover a conserved catabolic gene cluster could be isolated harboring 13 genes involved in PAH degradation. Sequence comparison with the initial dioxygenase of Sphingomonas sp. strain CHY-1 showed that the amino acids lining the catalytic pocket are conserved. The variation in substrate specificity of the two dioxygenases demonstrates that substitutions outside of the catalytic pocket can have marked effects on the substrate range of dioxygenase enzymes.

No description available.

No description available.

No description available.

No description available.

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