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Polychlorinated biphenyl (PCB) metabolism in psychrotolerant and mesophilic bacteria : from substrate uptake to catalysis Master, Emma R.

Abstract

Bioremediation of soil contaminated with polychlorinated biphenyls (PCBs) is potentially a cost-effective cleanup strategy as it can be performed on-site. My general 5 aims were to isolate PCB-degrading bacteria from PCB-contaminated Arctic soil and to determine if these bacteria are adapted for PCB degradation at low temperature. An Arctic soil bacterium and a mesophilic bacterium were compared on the basis of PCB degradation by whole cells, activities of purified biphenyl dioxygenases, regulation of genes encoding these enzymes and the mechanism of biphenyl uptake by these bacteria. 10 In addition to these studies, I demonstrated bioremediation of weathered PCBcontaminated Arctic soil in a microcosm. PCB-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil. 16S rDNA sequences indicate that these isolates are members of the genus Pseudomonas. At 7°C, PCB congeners that were transformed by the Arctic bacteria and by the 15 mesophilic PCB-degrader, Burkholderia sp. strain LB400, were transformed at higher initial rates by the Arctic soil isolates. Furthermore, in contrast to PCB transformation by LB400, PCB transformation by the Arctic bacteria was diminished at high temperature (50°C). These observations suggest that the Arctic soil bacteria are adapted to degrade PCBs at low temperature. The effect of temperature on the kinetics of biphenyl 20 dioxygenase and the regulation of bph genes from LB400 and the Arctic soil bacterium Pseudomonas sp. strain Cam-1 were determined. The biphenyl dioxygenase from Cam-1 (BPDO[sub Cam1]) and LB400 (BPDO[sub LB400]) were overproduced in E.coli and purified anaerobically. Consistent with observations using whole cells, BPDO[sub Cam1] displayed high catalytic activity with biphenyl ([sup app][sub cat]) at low temperature and low thermal stability compared to BPDOLB4OO- However, the respective catalytic efficiencies (k[sup app][sub cat]/k[sup app][sub in]) of BPDO[sub Cam1] and BPDO[sub LB400] were not significantly different at 15°C and 25°C. The constitutive expression of bphA in LB400 was diminished at low temperature. In contrast, the expression of bphA in Cam-1 was 5 inducible, and was expressed at 7°C and 30°C. Thus, lower expression of BPDO in LB400 than in Cam-1 might contribute to the difference in PCB transformation activities by whole cells at 7°C. The induction of bphA in Cam-1 was highest with biphenyl although naphthalene, salicylate, 2-chlorobiphenyl and 4-chlorobiphenyl induced bphA in Cam-1 to levels above background. 10 To begin to elucidate how PCB-uptake affects PCB metabolism, biphenyl-uptake by Cam-1 and LB400 was investigated. In both microorganisms, biphenyl was actively transported and uptake did not depend on biphenyl catalysis. Moreover, biphenyl-uptake showed saturable kinetics with respect to biphenyl concentration. These data suggest that biphenyl-uptake occurs via an active biphenyl transport system. 15 To date, the enzymology of PCB degradation has been the main target for generating recombinant strains with improved ability to degrade PCBs. This thesis demonstrates that the regulation of bph genes and PCB uptake by bacteria are also important targets for improving the potential of PCB bioremediation. Thus, future investigations should include identifying potential biphenyl uptake systems and 20 characterizing regulatory proteins involved in bph gene expression. As a result, it might be possible to design biphenyl uptake systems with improved ability to transport PCBs, and regulatory proteins that respond to different inducers, thereby optimizing bph gene expression in different environments.

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