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.

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated ␤-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

The capability of anaerobic bacterial consortia from different environmental sources including soils, sewage sludges, and sediment of the river Saale (Germany) to dehalogenate chlorinated dioxins was compared using 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) as the model compound. The inocula were amended with mineral medium and organic acids and spiked with a high concentration (50 µM) of 1,2,3,4-TCDD to stimulate microbial dehalogenating activity. Reductive dechlorination was observed to 1,3dichlorodibenzo-p-dioxin (1,3-DCDD) as the main product and to minor amounts of the 1,2,4-and 1,2,3-trichlorodibenzop-dioxins (TrCDD) using incubations with Saale River sediment. No reaction was observed in the controls and in incubations with soils or sewage sludges. The dechlorination of 1,2,4-and 1,2,3-TrCDD was analyzed in separate subcultures. Reductive dechlorination of 1,2,4-TrCDD was a relative fast process (about 6 µM converted within 58 days) and yielded only one product (1,3-DCDD). 1,2,3-TrCDD was slowly dechlorinated to equal amounts of 1,3-and 2,3-DCDD. These observations suggest that the main dechlorination route of 1,2,3,4-TCDD to 1,3-DCDD proceeds primarily via the removal of a lateral chlorine atom with 1,2,4-TrCDD as the intermediate.

The transcription of reductive dehalogenase homologous (rdh) genes of "Dehalococcoides" sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3-and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of the transcription of 29 rdhA genes was indicated in response to 1,2,3-and 1,2,4-TCB added after a substrate depletion period of 72 h. The transcription of the remaining three rdhA genes additionally was detected using specific primers. While most rdhA genes were upregulated similarly in cultures after induction with 1,2,3-TCB or 1,2,4-TCB, three rdhA genes responded differentially to 1,2,3-and 1,2,4-TCB, as revealed by the comparison of t-RFLP profiles. The enhanced transcription of cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TCB, while the transcription of cbdbA1624 was strongly induced by 1,2,4-TCB. Comparison of t-RFLP profiles obtained from cDNA and genomic DNA indicated a particularly high induction of the transcription of cbrA (‫؍‬cbdbA84) by both TCBs. As indicated by reverse transcription-quantitative PCR, the transcription of these plus two other rdhA genes (cbdbA1588 and cbdbA1618) increased within 48 to 72 h by one or two orders of magnitude. Subsequently, transcript levels slowly decreased and approached initial transcript levels several days after complete dehalogenation. Finally, cbrA was transcribed to a level of 22 transcripts per cbrA gene, suggesting that cbrA mRNA could be an appropriate biomarker for the investigation of the natural dechlorination potential at chlorobenzene-contaminated sites.Highly chlorinated aromatic compounds, like polychlorinated benzenes, dibenzo-p-dioxins and -furans, and polychlorinated biphenyls (PCB) persist in the environment for long periods, tend to accumulate in food chains, and are ubiquitously distributed. Many freshwater and marine sediments worldwide have been polluted by high concentrations of these compounds (4, 7, 43). Under reducing conditions often prevailing in sediments, reductive dehalogenation is considered the main biological process for the natural attenuation of highly chlorinated compounds. Several anaerobic bacteria are known to grow by respiratory dehalogenation with chlorinated compounds (for a review, see reference 40). Physiological studies with pure cultures of "Dehalococcoides" spp. and related bacteria of subphylum II of the Chloroflexi have shown that they are highly specialized in using reductive dechlorination as a respiratory process (3,12,13,30,41). The genome sequences of "Dehalococcoides ethenogenes" strain 195 and Dehalococcoides sp. strain CBDB1 confirmed their metabolic restriction to respiratory dehalogenation (24, 38) and revealed the presence of 17 and 32 reductive dehalogenase homologous (rdh) genes, respectively, some...

The genomes of two novel Dehalococcoides mccartyi strains, DCMB5 and BTF08, enriched from the heavily organohalide-contaminated megasite around Bitterfeld (Germany), were fully sequenced and annotated. Although overal lsimilar, the genome sequences of the two strains reveal remarkable differences in their genetic content, reflecting a specific adaptation to the contaminants at the field sites from which they were enriched. The genome of strain BTF08 encodes for 20 reductive dehalogenases, and is the first example of a genome containing all three enzymes that are necessary to couple the complete reductive dechlorination of PCE to ethene to growth. The genes encoding trichloroethene and vinyl chloride reductive dehalogenases, tceA and vcrA, are located within mobile genetic elements, suggesting their recent horizontal acquisition.The genome of strain DCMB5 contains 23 reductive dehalogenase genes,including cbrA, which encodes a chlorobenzene reductive dehalogenase, and a gene cluster encoding arsenic resistance proteins, both corresponding to typical pollutants at its isolation site.