Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K iE and K iES . These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41917/1/203-171-5-301_91710301.pd

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis -dichloroethylene ( cis -DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans -dichloroethylene ( trans -DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans -DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis -DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans -DCE, cis -DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans -DCE oxidation products were also toxic but to a lesser extent. cis -DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41918/1/203-172-6-393_91720393.pd

Identification of intermediates of in vivo trichloroethylene oxidation by the membrane-associated methane monooxygenase

The rate and products of trichloroethylene (TCE) oxidation by Methylomicrobium album BG8 expressing membrane-associated methane monooxygenase (pMMO) were determined using 14 C radiotracer techniques. [ 14 C]TCE was degraded at a rate of 1.24 nmol (min mg protein) −1 with the initial production of glyoxylate and then formate. Radiolabeled CO 2 was also found after incubating M. album BG8 for 5 h with [ 14 C]TCE. Experiments with purified pMMO from Methylococcus capsulatus Bath showed that TCE could be mineralized to CO 2 by pMMO. Oxygen uptake studies verified that M. album BG8 could oxidize glyoxylate and that pMMO was responsible for the oxidation based on acetylene inactivation studies. Here we propose a pathway of TCE oxidation by pMMO-expressing cells in which TCE is first converted to TCE-epoxide. The epoxide then spontaneously undergoes HCl elimination to form glyoxylate which can be further oxidized by pMMO to formate and CO 2 .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74667/1/j.1574-6968.2000.tb09090.x.pd

Substrate oxidation by methanotrophs expressing particulate methane monooxygenase (pMMO): A study of whole -cell oxidation of trichloroethylene and its potential use for environmental remediation.

Many studies have examined rapid trichloroethylene (TCE) co-metabolism by methanotrophs expressing soluble methane monooxygenase (sMMO). Most known methanotrophs, however, can not express sMMO and rely on particulate methane monooxygenase (pMMO). Furthermore, as sMMO is only expressed at low copper/biomass ratios, pMMO may predominate in the environment. Therefore, the goal of this research is to provide more information on TCE degradation by methanotrophs expressing pMMO to better understand methanotrophic-mediated TCE bioremediation. Addition of copper enhanced TCE oxidation by Methylosinus trichosporium OB3b expressing pMMO but did not dramatically affect Methylomicrobium album BG8. As M. album BG8 can express only pMMO, it may have a better copper uptake system than M. trichosporium OB3b which can express both forms of MMO. Addition of formate as an external source of reducing equivalents increased TCE oxidation by both strains. Therefore, these results suggest to provide high copper and formate concentrations to enhance TCE bioremediation by methanotrophs expressing pMMO. The products of TCE oxidation by in vivo pMMO of M. album BG8 were determined using radiotracer techniques. TCE was suspected to be oxidized into TCE-epoxide which was spontaneously hydrolyzed to form glyoxylate as the primary aqueous product. Glyoxylate was subsequently oxidized by pMMO to form formate and CO2. Formate could also be further oxidized to CO2 by formate dehydrogenase in whole-cells. Unlike sMMO-mediated TCE oxidation, chloral and dichloroacetate were not detected. In experiments of simultaneous oxidation of methane and chlorinated hydrocarbons, TCE and dichloromethane (DCM) did not significantly affect methane oxidation by M. trichosporium OB3b expressing pMMO in the absence of formate. If formate was added, TCE acted as a non-competitive inhibitor while DCM acted as a competitive inhibitor of methane oxidation by M. trichosporium OB3b expressing pMMO. Therefore, a simple competitive inhibition model can not be applied to accurately predict the time required in achieving the goal of TCE bioremediation by methanotrophs expressing pMMO. As in vivo pMMO and sMMO activities show different characteristics, an assay to distinguish them is needed to optimize bioremediation. Phenylacetylene inhibited in vivo sMMO activity at 100 muM and was shown to be a mechanism-based inactivator of sMMO. In vivo pMMO activity was affected at a similar level only in the presence of 1000 muM phenylacetylene. These studies provide the foundation for using phenylacetylene in environmental microbiology.Ph.D.Applied SciencesEnvironmental engineeringEnvironmental scienceHealth and Environmental SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132623/2/9977204.pd