Metabolism of dichloroethenes by the butane-oxidizing bacterium 'Pseudomonas butanovora' Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/nc580q54p

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  • Reductive dechlorination of chlorinated ethenes is dependent upon suitable substrates promoting microbial activity and creating anaerobic conditions. At the periphery of active reductive dechlorinating zones combinations of lesser chlorinated ethenes should exist along with end products of the anaerobic metabolism that is driving reductive dechlorination. Potential end-products of anaerobic metabolism were investigated for their ability to stimulate oxidative cometabolism of dichloro ethenes (DCEs) by the butane-degrading bacterium, Pseudomonas butanovora. Organic acids that supported butane-monooxygenase (BMO) activity were acetate, propionate, lactate, and butyrate. Lactate consistently supported and sustained greater rates of cooxidation than did the other organic acids. When propane replaced butane as the growth substrate, lactate remained the superior electron donor, while the ability of butyrate and acetate to support BMO activity decreased. In contrast, propionate-supported cooxidation was only observed in propane-grown cells. Lactate supported the degradation of 1,2-trans dichloroethylene (1,2-trans DCE), 1,2-cis dichloroethylene (1,2-cis DCE) and 1,1-dichloroethylene (1,1-DCE) in butane-grown P. butanovora. 78 nmoles (25 μM) of 1,2-cis DCE were completely degraded by butane-grown P. butanovora. In contrast, smaller amounts of 1,1-DCE and 1,2-trans DCE were degraded over the twenty minute time course. Decreasing rates of cooxidation over time were observed for of all three DCEs, and 50% of BMO activity was irreversibly lost after 15 min, 6 min, and 0.5 min exposures to 1,2-cis DCE, 1,2 trans-DCE, and 1,1-DCE respectively. Cell viability decreased by over 90, 95, and 99.95% during the transformation of 25 nmoles/mg protein of 1,2-cis DCE, 1,2-trans DCE and 1,1-DCE. These results indicate that cellular viability was more sensitive to cooxidation of 1,2-cis DCE and 1,2-trans DCE than was BMO. 1,2-cis DCE and 1,2-trans DCE induced BMO activity to 25 and 45% of the butane control, respectively. Induction by 1,2-trans DCE was observed at a threshold of about 20 μM and higher concentrations did not increase BMO activity. Fusion of lacZ to the BMO catabolic promoter, with consequent knock out of BMO activity, provided the opportunity to assess substrate induction without the confounding effects of enzyme inactivation and product induction. While BMO substrates, butane, 1,2-cis DCE, and ethylene, were unable to induce lacZ activity the BMO products, 1-butanol, and ethylene oxide, effectively induced lacZ activity. 1,2-trans DCE was unique among the BMO substrates tested in it's ability to induce expression of lacZ, 2-fold above background, in the reporter strain. A wide range of concentrations induced lacZ activities (10 to 100 μM), and low levels of 1,2-trans DCE achieved high levels of induction after 4 hrs. However, lacZ activities were limited to an induction of about four-fold above background and this limit allowed lower concentrations of 1,2-trans DCE to eventually produce equal levels of beta-galactosidease. These data provide proof-of- concept that BMO-dependent cometabolism can occur independently of butane as an inducer and electron donor for BMO gene expression and activity.
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