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.

The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield ( Y) of 2.1+/-0.24 g protein/mol Cl(-) released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9+/-0.15 g/mol Cl(-). Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.

Bacteria which utilize the xenobiotic compounds chloridazon, antipyrin, and pyramidon as sole carbon sources were isolated from various soil samples. The 22 strains isolated are similar with respect to morphological, physiological, biochemical, serological, and genetic properties. These bacteria are aerobic gram-negative rods or coccal rods (0.7 to 1.0 by 1.0 to 2.0 pm) that occur singly, ip pairs, or in short chains and are nonmotile and nonsporeforming. Physiological and biochemical characteristics and susceptibility to antibiotics were determined. The strains need vitamin BI2 as a growth factor; they are catalase positive and weakly oxidase positive and show slight H2S production. All of the other tests which we performed were negative. The nutritional spectrum is extraordinarily limited, with optimal growth on chloridazon, antipyrin, pyramidon, and L-phenylalanine. Most sugars, alcohols, amino and carboxylic acids, and ordinary complex media are not utilized. The bacteria are osmotically sensitive. They are a serologically uniform group of organisms, which are harmless to rats and rabbits. Their guanine-plus-cytosine contents range between 65 and 68.5 mol% . The chloridazon-degrading bacteria are characterized as a new genus, Phenylobacterium, with a single species, Phenylobacterium immobile. The type strain Phenylobacterium immobile strain E (= DSM 1986), is not closely related to any other gram-negative bacterium, as shown by a 16s ribosomal ribonucleic acid partial sequence analysis. This organism is a member of group I of the purple nonsulfur bacteria, but is phylogenetically isolated in this group. Phenylobacterium immobile is remotely related to Pseudomonas diminuta, Rhizobium leguminosarum, rhodopseudomonads, and Aquaspirillum itersonii. Like other members of this group, Phenylobacterium immobile contains 2,3-diamino-2,3-dideoxy-~-glucose in its lipopolysaccharide. The murein equals a normal murein from a gram-negative bacterium. All citric acid cycle enzymes are detectable in Phenylobacterium immobile.Herbicides, fungicides, and other agrochemicals are distributed in large amounts, and microorganisms which are able to degrade synthetic organic tnolecules play an important role in the elimination of these chemicals from soil and water. Chloridazon [5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone] is the active ingredient of the herbicide Pyramin, which has been used for more than 20 years for the control of weeds in sugar beet and beet root culture.

Enzymatic reductive dehalogenation of tri-, tetra-, penta-, and hexachlorobenzenes was demonstrated in cell extracts with low protein concentration (0.5 to 1 g of protein/ml) derived from the chlorobenzene-respiring anaerobe Dehalococcoides sp. strain CBDB1. 1,2,3-trichlorobenzene dehalogenase activity was associated with the membrane fraction. Light-reversible inhibition by alkyl iodides indicated the presence of a corrinoid cofactor.

In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membranebound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with D-mannitol, D-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with D-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQdependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.The acetic acid bacterium Gluconobacter oxydans is characterized by its ability to incompletely oxidize various sugars and alcohols by using membrane-associated dehydrogenases that contain pyrroloquinoline quinone (PQQ) as a cofactor (33). Examples are the quinoprotein glucose dehydrogenase (3) and the quinoprotein glycerol dehydrogenase, which also oxidizes D-gluconate, D-mannitol, D-sorbitol, and other polyols (34). The oxidation reactions take place in the periplasmic space and are coupled to the respiratory chain (19,33). Several oxidation reactions carried out by G. oxydans quinoproteins are of industrial importance, e.g., the conversion of D-gluconate to 5-D-ketogluconate, a precursor for the production of L-tartaric acid, and the formation of L-sorbose, an intermediate in the synthesis of vitamin C (reviewed in reference 9). PQQ has invoked considerable attention due to its positive physiological effects in mammals (47). A possible role of PQQ as a vitamin in mammals has been suggested but is controversially debated (13,28,40).Genes involved in PQQ synthesis have been characterized for several bacteria, including Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens AM1, and Pseudomonas sp. (reviewed in reference 19). In Klebsiella pneumoniae, the PQQ biosynthetic genes are clustered in the pqqABCDEF operon (35). In Pseudomonas aeruginosa, the pqqABCDE operon is separated from the pqqF operon (18,48). Methylobacterium extorquens AM1 contains a ...