Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

Abstract

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities. Microbial reductive dechlorination and organohalide respiration play a significant role in the natural and engineered attenuation of chlorinated ethenes, such as tetrachloroethene (PCE) and trichloroethene (TCE). However, microbial dechlorination via hydrogenolysis sometimes stalls at dichloroethene (DCE) and vinyl chloride (VC) which is problematic as these intermediates are more water-soluble and more toxic than PCE or TCE, particularly VC (1). Organisms that are known to be capable of respiring chlorinated ethenes to nontoxic ethene are restricted to some Dehalococcoides mccartyi strains (2-4). The genomes of two VC-respiring Dehalococcoides isolates were described previously (5), as well as the metagenome of the VC-respiring mixed culture KB-1 (6, 7). In addition to chlorinated ethenes, some D. mccartyi strains can also dechlorinate 1,2-dichloroethane (1,2-DCA) (8), 1,2-dichloropropane (9), and a variety of chlorinated and brominated aromatic contaminants (10-12).Membrane-bound reductive dehalogenases (RDases) are key enzymes that mediate dechlorination reactions in organohalide respiring microorganisms. Many RDases and putative RDase sequences have been identified in D. mccartyi strains (13-21). To date, only a few RD...

Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Its trans -Dichloroethene Reductive Dehalogenase, TdrA

“…Most of the dechlorination activity was observed in band 4 and band 5, in the region of 242 kDa (see Fig. S2 in the supplemental material), at the same molecular mass where other dehalogenases have been identified in previous studies (25,26). Cell extract samples and corresponding gel slices from the tDCE enrichment culture showed higher activities with tDCE as the substrate than those from the VC enrichment culture ( Table 1).…”

“…This region is likely the most informative with respect to the substrate identity for these RdhA enzymes. Ortholog groups show promise in their ability to group dehalogenases with functional relevance, as was the case here with WBC-2 VcrA, whose sequence has 97% identity to the sequences in the known VcrA-containing ortholog group 8, which has a substrate-forming channel identical to that of OG8 members, and which demonstrates dechlorinating activity similar to that of other already characterized VcrA enzymes from this group (14,23,25,26) (see Table S6 in the supplemental material; see the shading showing putative substrate-binding residues on pages 21 to 25 in the supplemental material).…”

“…Protein concentrations in samples of the cell extracts (50 l) were measured using the Bradford assay (Bio-Rad). The ImageJ program (http://imagej.nih.gov/ij/) was used to estimate the amount of protein found in gel slices, as described elsewhere (26).…”

“…These studies have revealed differences in the substrate preferences for the chlorinated ethene dehalogenases VcrA, TceA, and BvcA. VcrA has a preference for VC, cis-dichloroethene (cDCE), 1,1-dichloroethene (1,1-DCE), and TCE (14,25) as well as 1,2-dichloroethane (1,2-DCA) (23,26); TceA has activity on TCE, cDCE, tDCE, 1,1-DCE, VC, 1,2-DCA, and several brominated aliphatic acids (13); and BvcA has activity on VC, cDCE, 1,2-DCA, 1,1-DCE, and tDCE (25). Although BvcA and TceA do have some activity on tDCE, no other chloroethene RDase with primarily tDCE activity has been identified to date.…”

Chlorinated electron acceptor availability selects for specificDehalococcoidespopulations in dechlorinating enrichment cultures and in groundwater

“…To this set of 10 uncharacterized genes, we added five more genes of interest: KB1-1 (OG10), KB1-5 (OG15), KB1_14/ vcrA (OG8), KB1_6/ bvcA (OG28) and KB1_27/ tceA (OG5). KB1-1 and KB1-5 were selected because their corresponding proteins have previously been detected in KB-1 (54, 55). Furthermore, KB1-5 (OG 15) is orthologous to DET1545 from Strain 195, which is expressed upon starvation (55, 56).…”