f Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

1,1,1-Trichloroethane (1,1,1-TCA) is a common groundwater contaminant that can be reductively dechlorinated to 1,1-dichloroethane (1,1-DCA) and monochloroethane, and can support the growth of certain dehalorespiring strains of Dehalobacter We used reductive dehalogenase cell-free extract assays (with reduced methyl viologen) and whole cell suspension dechlorination assays (with hydrogen) and a Dehalobacter-containing enrichment culture to explore the kinetics of l,1,1-TCA and 1,1-DCA reductive dechlorination in the presence of the common co-contaminants trichloroethene (TCE), cis-dichloroethene (cDCE), and vinyl chloride (VC). These chlorinated ethenes were most significant inhibitors of 1,1,1-TCA dechlorination in cell-free extracts, indicating direct effects on the reductive dehalogenase enzyme(s). The inhibition was present but less pronounced in whole cell suspension assays. None of the chlorinated ethenes inhibited 1,1-DCA dechlorination in cell-free extract assays, yet cDCE and particularly VC were inhibitors in whole cell assays, indicating an effect on Dehalobacter, but not on the dehalogenase enzyme(s). Marked differences in kinetic parameters for 1,1,1-TCA and 1,1-DCA, and an uncoupling of these two activities in cultures grown on 1,1-DCA compared to those grown on 1,1,1-TCA was strong evidence for the existence of distinct 1,1,1-TCA and 1,1-DCA reductive dehalogenase enzymes.

Mixtures of chlorinated ethenes and ethanes are often found at contaminated sites. In this study, we undertook a systematic investigation of the inhibitory effects of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) on chlorinated ethene dechlorination in three distinct Dehalococcoides-containing consortia. To focus on inhibition acting directly on the reductive dehalogenases, dechlorination assays used cell-free extracts prepared from cultures actively dechlorinating trichloroethene (TCE) to ethene. The dechlorination assays were initiated with TCE, cis-1,2-dichloroethene (cDCE), or vinyl chloride (VC) as substrates and either 1,1,1-TCA or 1,1-DCA as potential inhibitors. 1,1,1-TCA inhibited VC dechlorination similarly in cell suspension and cell-free extract assays, implicating an effect on the VC reductases associated with the dechlorination of VC to nontoxic ethene. Concentrations of 1,1,1-TCA in the range of 30-270 μg/L reduced VC dechlorination rates by approximately 50% relative to conditions without 1,1,1-TCA. 1,1,1-TCA also inhibited reductive dehalogenases involved in TCE and cDCE dechlorination. In contrast, 1,1-DCA had no pronounced inhibitory effects on chlorinated ethene reductive dehalogenases, indicating that removal of 1,1,1-TCA via reductive dechlorination to 1,1-DCA is a viable strategy to relieve inhibition.

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