Six obligately anaerobic bacterial isolates (195^T, CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3–1 µm in diameter and 0.1–0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B₁₂was omitted from the medium. Addition of sterile cell-free supernatant ofDehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15–35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195^Tand CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the nameDehalococcoides mccartyigen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100 = JCM 16839 = KCTC 5957), FL2 ( = ATCC BAA-2098 = DSM 23585 = JCM 16840 = KCTC 5959), GT ( = ATCC BAA-2099 = JCM 16841 = KCTC 5958), CBDB1, 195^T( = ATCC BAA-2266^T = KCTC 15142^T) and VS are considered strains ofDehalococcoides mccartyi, with strain 195^Tas the type strain. The new class Dehalococcoidia classis nov., orderDehalococcoidalesord. nov. and familyDehalococcoidaceaefam. nov. are described to accommodate the new taxon.

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene ( vcrA ) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB , encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC , encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism. Dehalococcoides are free-living sediment and subsurface bacteria with remarkably small, streamlined genomes and an unusual degree of niche specialization. These strictly anaerobic bacteria gain metabolic energy exclusively through a novel type of respiration that results in reductive elimination of chlorides from organochlorines, many of which are priority pollutants. In this article, we compare the first complete genome sequences of Dehalococcoides strains that grow via respiration of vinyl chloride (VC), a human carcinogen and abundant groundwater pollutant. Our work provides novel insights into Dehalococcoides chromosome organization and evolution, identifies specific positions in the chromosomes where new genes—like the genes responsible for growth on VC—are integrated, and generates clues how these dechlorinating bacteria adapt to anthropogenic contamination. This information sheds new light on Dehalococcoides biology and ecology, with implications for enhanced bioremediation to protect dwindling drinking water reservoirs.