Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring.

Desulfomonile tiedjei and Desulfitobacterium dehalogenans were chosen as model bacteria to demonstrate the introduction of an anaerobic microbia reductive dechlorination activity into nonsterile soil slurry microcosms by inoculation. De novo 3-chlorobenzoate dechlorination activity was established with the bacterium D. tiedjei in microcosms normally devoid of this dechlorination capacity. The addition of D. tiedjei to microcosms supplemented with 20 mM pyruvate as the cosubstrate resulted in total biotransformation of 1.5 mM 3-chlorobenzoate within 7 days. The introduction of the bacterium Desulfitobacterium dehalogenans into nonsterile microcosms resulted in a shortening of the period required for dechlorination activity to be established. In microcosms inoculated with Desulfitobacterium dehalogenans, total degradation of 6 mM 3-chloro-4-hydroxy phenoxyacetic acid (3-Cl-4-OHPA) was observed after 4 days in contrast to the result in noninoculated microcosms, where the total degradation of 3-Cl-4-OHPA by indigenous microorganisms was observed after 11 days. Both externally introduced bacterial strains were detected in soil slurry microcosms by a nested-PCR methodology.     

Anaerobic dehalogenation of hydroxylated polychlorinated biphenyls by Desulfitobacterium dehalogenans

Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, we report here on the rapid reductive dehalogenation of para-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium Desulfitobacterium dehalogenans completely dehalogenates all flanking chlorines (chlorines in ortho position to the para-hydroxyl group) from congeners such as 3,3',5, 5'-tetrachloro-4,4'-dihydroxybiphenyl.     

Reduction of humic substances by halorespiring, sulphate-reducing and methanogenic microorganisms

Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone-2,6-disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate-reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

Enzymes involved in the anoxic utilization of phenyl methyl ethers by <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> DCB2 and <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> PCE-S

Phenyl methyl ethers are utilized by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S; the methyl group derived from the O-demethylation of these substrates can be used as electron donor for anaerobic fumarate respiration or dehalorespiration. The activity of all enzymes involved in the oxidation of the methyl group to carbon dioxide via the acetyl-CoA pathway was detected in cell extracts of both strains. In addition, a carbon monoxide dehydrogenase activity could be detected. Activity staining of this enzyme indicated that the enzyme is a bifunctional CO dehydrogenase/acetyl-CoA synthase.     

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.     

Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes

Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases. For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used. For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method. Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp. strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases. In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S. multivorans, pceA of D. restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria. Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 10(2) copies per reaction.     

Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.     

Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 10(3) to 10(4) transformants per microg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30 degrees C, whereas the replicon was not functional at 40 degrees C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG+host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 x 10(-4) per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.     

Complete genome sequence of the dehalorespiring bacterium Desulfitobacterium hafniense Y51 and comparison with Dehalococcoides ethenogenes 195

Desulfitobacterium strains have the ability to dechlorinate halogenated compounds under anaerobic conditions by dehalorespiration. The complete genome of the tetrachloroethene (PCE)-dechlorinating strain Desulfitobacterium hafniense Y51 is a 5,727,534-bp circular chromosome harboring 5,060 predicted protein coding sequences. This genome contains only two reductive dehalogenase genes, a lower number than reported in most other dehalorespiring strains. More than 50 members of the dimethyl sulfoxide reductase superfamily and 30 paralogs of the flavoprotein subunit of the fumarate reductase are encoded as well. A remarkable feature of the genome is the large number of O-demethylase paralogs, which allow utilization of lignin-derived phenyl methyl ethers as electron donors. The large genome reveals a more versatile microorganism that can utilize a larger set of specialized electron donors and acceptors than previously thought. This is in sharp contrast to the PCE-dechlorinating strain Dehalococcoides ethenogenes 195, which has a relatively small genome with a narrow metabolic repertoire. A genomic comparison of these two very different strains allowed us to narrow down the potential candidates implicated in the dechlorination process. Our results provide further impetus to the use of desulfitobacteria as tools for bioremediation.     

Microbial anaerobic demethylation and dechlorination of chlorinated hydroquinone metabolites synthesized by basidiomycete fungi

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.     

The mosaic genome of Anaeromyxobacter dehalogenans strain 2CP-C suggests an aerobic common ancestor to the delta-proteobacteria

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta-Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb(3) oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.