Dehalococcoides sp. strain BAV1 couples growth with the reductive dechlorination of vinyl chloride (VC) to ethene. Degenerate primers targeting conserved regions in reductive dehalogenase (RDase) genes were designed and used to PCR amplify putative RDase genes from strain BAV1. Seven unique RDase gene fragments were identified. Transcription analysis of VC-grown BAV1 cultures suggested that bvcA was involved in VC reductive dechlorination, and the complete sequence of bvcA was obtained. bvcA was absent in Dehalococcoides isolates that failed to respire VC, yet was detected in four of eight VC-respiring mixed cultures.

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.

A chlorobenzene reductive dehalogenase of the anaerobic dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 was identified. Due to poor biomass yields, standard protein isolation procedures were not applicable. Therefore, cell extracts from cultures grown on trichlorobenzenes were separated by native polyacrylamide gel electrophoresis and analyzed directly for chlorobenzene reductive dehalogenase activity within gel fragments. Activity was found in a single band, even though electrophoretic separation was performed under aerobic conditions. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and nano-liquid chromatography-MALDI MS analysis of silver-stained replicas of the active band on native polyacrylamide gels identified a protein product of the cbdbA84 gene, now called cbrA. The cbdbA84 gene is one of 32 reductive dehalogenase homologous genes present in the genome of strain CBDB1. The chlorobenzene reductive dehalogenase identified in our study represents a member of the family of corrinoid/iron-sulfur cluster-containing reductive dehalogenases. No orthologs of cbdbA84 were found in the completely sequenced genomes of Dehalococcoides sp. strains 195 and BAV1 nor among the genes amplified from Dehalococcoides sp. strain FL2 or mixed cultures containing Dehalococcoides. Another dehalogenase homologue (cbdbA80) was expressed in cultures that contained 1,2,4-trichlorobenzene, but its role is unclear. Other highly expressed proteins identified with our approach included the major subunit of a protein annotated as formate dehydrogenase, transporter subunits, and a putative S-layer protein.Dehalococcoides sp. strain CBDB1 belongs to a phylogenetically isolated cluster of strictly anaerobic bacteria that use chlorinated compounds in their energy metabolism by coupling reductive dehalogenation to electron transport phosphorylation (2, 22). Strain CBDB1 uses polychlorinated benzenes, phenols, and dibenzodioxins as growth-supporting electron acceptors (2, 3, 5, 13). Among the chlorobenzenes, 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, all tetrachlorobenzene (TeCB) isomers, and penta-and hexachlorobenzene are dechlorinated (2, 10, 13). Reductive dechlorination of chlorinated benzenes was also demonstrated for the Dehalococcoides ethenogenes strain 195 (7), a strain that was originally cultivated with chlorinated ethenes as electron acceptors (22), and for Dehalococcoides-like bacterium DF-1, identified in a mixed culture (38).Several enzymes catalyzing the respiratory reductive dechlorination of chloroaromatics have been isolated and characterized, e.g., the chlorophenol dehalogenases of Desulfitobacterium spp. (6, 15, 36) and the 3-chlorobenzoate dehalogenase of Desulfomonile tiedjei (30). Attempts to isolate chlorobenzene reductive dehalogenase from Dehalococcoides sp. strain CBDB1 have been hampered by poor biomass yields (10). However, the characterization of chlorobenzene reductive dehalogenase activity in cell extracts of strain CBDB1 showed that this enzyme shared several properties wit...

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 ...