Dual Element (C/Cl) Isotope Analysis Indicates Distinct Mechanisms of Reductive Dehalogenation of Chlorinated Ethenes and Dichloroethane in Dehalococcoides mccartyi Strain BTF08 With Defined Reductive Dehalogenase Inventories

Abstract

Dehalococcoides mccartyi strain BTF08 has the unique property to couple complete dechlorination of tetrachloroethene and 1,2-dichloroethane to ethene with growth by using the halogenated compounds as terminal electron acceptor. The genome of strain BTF08 encodes 20 genes for reductive dehalogenase homologous proteins (RdhA) including those described for dehalogenation of tetrachloroethene (PceA, PteA), trichloroethene (TceA) and vinyl chloride (VcrA). Thus far it is unknown under which conditions the different RdhAs are expressed, what their substrate specificity is and if different reaction mechanisms are employed. Here we found by proteomic analysis from differentially activated batches that PteA and VcrA were expressed during dechlorination of tetrachloroethene to ethene, while TceA was expressed during 1,2-dichloroethane dehalogenation. Carbon and chlorine compound-specific stable isotope analysis suggested distinct reaction mechanisms for the dechlorination of (i) cis-dichloroethene and vinyl chloride versus (ii) tetrachloroethene. This differentiation was observed independent of the expressed RdhA proteins. Differently, two stable isotope fractionation patterns were observed for 1,2-dichloroethane transformation, for cells with distinct RdhA inventories. Conclusively, we could link specific RdhA expression with functions and provide an insight into the apparently substrate-specific reaction mechanisms in the pathway of reductive dehalogenation in D. mccartyi strain BTF08. Data are available via ProteomeXchange with identifiers PXD018558 and PXD018595.

Keywords: 1; 2-dichloroethane; Dehalococcoides mccartyi strain BTF08; cis-dichloroethene; compound-specific stable isotope analysis; nLC-MS/MS; reductive dehalogenation; tetrachloroethene; vinyl chloride.

FIGURE 1

Proposed mechanisms for reductive dehalogenation. (A) inner sphere two electron transfers, (B) inner sphere single electron transfers displaying different modes of bond cleavage, (C) outer sphere single electron transfer and (D) inner sphere single electron transfer for vicinally substituted compounds. Inner sphere two electron transfer (A) is further divided in nucleophilic-addition reaction (a), characterized by initial protonation followed by carbon-halogen bond cleavage, and nucleophilic elimination (b), possessing initial carbon-halogen bond cleavage with subsequent protonation of the remaining carbon (Heckel et al., 2018; Lihl et al., 2019). Inner sphere single electron transfers (B) are described as heterolytic (a) or homolytic (b,c) cleavage (Cooper et al., 2015; Payne et al., 2015). Two distinct mechanisms of homolytic cleavage are proposed leading either to abstraction of a cobalt-halogen complex (Cooper et al., 2015; Payne et al., 2015) (b) or association of the carbon backbone to the cobalt (Cooper et al., 2015; Payne et al., 2015) (c). Outer sphere single electron transfers (C) do not involve a carbon or halogen to cobalt interaction (Kunze et al., 2017). Inner sphere single electron transfer for vicinally substituted compounds (D) was proposed for concerted halogen removal (Payne et al., 2015). Displayed are the hydrocarbon backbone [R] and the oxidation states of the cobalt within the cobamid cofactor [Co^I–III].

FIGURE 2

Double-element-plots for stable carbon and chlorine isotope fractionation. Activity assays using resting cells of D. mccartyi strain BTF08 with electron acceptors (A) PCE, (B) 1,2-DCA, (C) cDCE or (D) VC in dependence of the electron acceptors used for growth in upscaled transfer 1. Linear fits are represented by dashed lines.