Chlorobenzenes are toxic, highly persistent and ubiquitously distributed environmental contaminants that accumulate in the food chain. The only known microbial transformation of 1,2,3,5-tetrachlorobenzene (TeCB) and higher chlorinated benzenes is the reductive dechlorination to lower chlorinated benzenes under anaerobic conditions observed with mixed bacterial cultures. The lower chlorinated benzenes can subsequently be mineralized by aerobic bacteria. Here we describe the isolation of the oxygen-sensitive strain CBDB1, a pure culture capable of reductive dechlorination of chlorobenzenes. Strain CBDB1 is a highly specialized bacterium that stoichiometrically dechlorinates 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, 1,2,3,4-TeCB, 1,2,3,5-TeCB and 1,2,4,5-TeCB to dichlorobenzenes or 1,3,5-TCB. The presence of chlorobenzene as an electron acceptor and hydrogen as an electron donor is essential for growth, and indicates that strain CBDB1 meets its energy needs by a dehalorespiratory process. According to their 16S rRNA gene sequences, strain CBDB1, Dehalococcoides ethenogenes and several uncultivated bacteria form a new bacterial cluster, of which strain CBDB1 is the first, so far, to thrive on a purely synthetic medium.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) are among the most notorious environmental pollutants. Some congeners, particularly those with lateral chlorine substitutions at positions 2, 3, 7 and 8, are extremely toxic and carcinogenic to humans. One particularly promising mechanism for the detoxification of PCDDs and PCDFs is microbial reductive dechlorination. So far only a limited number of phylogenetically diverse anaerobic bacteria have been found that couple the reductive dehalogenation of chlorinated compounds--the substitution of a chlorine for a hydrogen atom--to energy conservation and growth in a process called dehalorespiration. Microbial dechlorination of PCDDs occurs in sediments and anaerobic mixed cultures from sediments, but the responsible organisms have not yet been identified or isolated. Here we show the presence of a Dehalococcoides species in four dioxin-dechlorinating enrichment cultures from a freshwater sediment highly contaminated with PCDDs and PCDFs. We also show that the previously described chlorobenzene-dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 (ref. 3) is able to reductively dechlorinate selected dioxin congeners. Reductive dechlorination of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) demonstrates that environmentally significant dioxins are attacked by this bacterium.

Dehalococcoides strains reductively dechlorinate a wide variety of halogenated compounds including chlorinated benzenes, biphenyls, naphthalenes, dioxins, and ethenes. Recent genome sequencing of the two Dehalococcoides strains CBDB1 and 195 revealed the presence of 32 and 18 reductive dehalogenase homologous genes, respectively, and therefore suggested an even higher dechlorinating potential than previously anticipated. Here, we demonstrate reductive dehalogenation of chlorophenol congeners by Dehalococcoides strains CBDB1 and 195. Strain CBDB1 completely converted 2,3-dichlorophenol, all six trichlorophenols, all three tetrachlorophenols, and pentachlorophenol to lower chlorinated phenols. Observed dechlorination rates in batch cultures with cell numbers of 10(7) mL(-1) amounted up to 35 microM day(-1). Chlorophenols were preferentially dechlorinated in the ortho position, but also doubly flanked and singly flanked meta- or para-chlorine substituents were removed. We used a newly designed computer-assisted direct cell counting protocol and quantitative PCR to demonstrate that strain CBDB1 uses chlorophenols as electron acceptors for respiratory growth. The growth yield of strain CBDB1 with 2,3-dichlorophenol was 7.6 x 10(13) cells per mol of Cl- released, and the growth rate was 0.41 day(-1). For strain 195, fast ortho dechlorination of 2,3-dichlorophenol, 2,3,4-trichlorophenol, and 2,3,6-trichlorophenol was detected, with only the ortho chlorine removed. Because chlorinated phenolic compounds are widely distributed as natural components in anaerobic environments, our results reveal one mode in which the Dehalococcoides species could have survived through earth history.

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene-and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.