Dehalorespiration with hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1

The gene cassette (camA ؉ camB ؉ camC) encoding a cytochrome P-450 cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol · mg (dry weight)؊1 · h ؊1, and intermediate pentachlorophenol was also identified.Hexachlorobenzene (C 6 Cl 6 ; HCB) was listed as 1 of the 12 persistent organic pollutants in the Stockholm Convention for its tendency to accumulate along the food chain and its recalcitrance to degradation, together with its harmful effects on human beings and the environment (1). Microbial degradation is a promising effective way to bioremediate environmental pollutants, including persistent organic pollutants. However, a pure culture capable of completely catabolizing HCB has not yet been isolated. Therefore, constructing an HCB-degrading strain via metabolic engineering may be a practical alternative to eliminate HCB in the environment.It had been demonstrated that the F87W/Y96F/L244A/ V247L mutant of cytochrome P-450 cam (CYP101) can oxidize HCB to pentachlorophenol (PCP) (4), which can be completely degraded by many microorganisms in the environment. In particular, the catabolic pathway of PCP in Sphingobium chlorophenolicum ATCC 39723 has been characterized thoroughly both biochemically and genetically (2).In this study, we report the conversion of the PCP utilizer ATCC 39723 to an HCB degrader by introducing a genetic segment (camA ϩ camB ϩ camC), which codes for the mutant of CYP101, into the target gene pcpM through homologous recombination.Construction of recombinant plasmids and strains. The plasmids, strains, and the primers used are listed in Table 1. DNA isolations and manipulations were performed according to established methods (11). The construction of the targeting vector for disruption of the genomic pcpM gene is illustrated in Fig. 1A. pcpM was amplified from strain ATCC 39723 with primers MF-1 and MR-1. The resulting 916-bp PCR product was then digested with XbaI and HindIII before being ligated into pBluescript II SK(ϩ) to create pZWY001. The functional expression cassette, including the Ptac-tac-camA ϩ B ϩ C transcriptional terminator, was PCR amplified from plasmid pCWSGBcamC using primers A5 and A6. The amplified 3,386-bp fragment was then digested with EcoRI and SacI before being ligated into pBluescript II SK(ϩ) to generate pZWY002. The neomycin phosphotransferase II gene (nptII) was PCR amplified from pTnMod-OKm (5) with primers KF-1 (containing SacI and EcoRI sites in tandem) and KR-1 (containing a SacI site). The amplified 1,179-bp fragment was digested with SacI before being ligated into pBluescript II SK(ϩ) to generate pZWY003. The nptII gene cassette was then excised from pZWY003 with SacI and cloned into pZWY002 to generate pZWY004. The insert from pZWY004 was released with EcoRI and cloned into pZWY001 to generate pZWY005. All constructs were confirmed by DNA sequencing (GiKang, Shanghai, Chin...

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“…This is in contrast to previous studies on the reductive dechlorination of HCB, which led to accumulation of toxic intermediates (3,6,7,14).…”

Section: and Intermediate Pentachlorophenol Was Also Identifiedcontrasting

confidence: 99%

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

Ding

Liu

Zhou

2006

Appl Environ Microbiol

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“…Strain BAV1, however, respires vinyl chloride (VC) to ethene (13), whereas strains FL2 and 195 are unable to use VC as a metabolic electron acceptor (12,32). Reductive dechlorination of chlorinated benzenes and PCDDs which had previously been shown for strain CBDB1 (2,9,18) was also recently demonstrated for strain 195 (11). However, different patterns of chlorobenzene dechlorination (3) and differences in the utilization of chlorobenzene and PCDD congeners have been observed for strain CBDB1 (2,9,17,18) and strain 195 (11).…”

mentioning

confidence: 84%

“…Several anaerobic bacteria use chlorinated compounds in their energy metabolism, coupling reductive dehalogenation to electron transport phosphorylation (16,25). The Dehalococcoides group forms a bacterial cluster that is phylogenetically distant from other dechlorinating bacteria (2,31) and uses a spectrum of chlorinated compounds as growth-supporting electron acceptors, including chlorinated ethenes (13,24,31), chlorinated benzenes (2,18), and polychlorinated dibenzodioxins (PCDDs) (9). Four members of the Dehalococcoides group have been isolated.…”

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confidence: 99%

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Hölscher

Krajmalnik‐Brown²,

Ritalahti³

et al. 2004

Appl Environ Microbiol

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128

122

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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.

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“…1). Similar population densities were also observed in Dehalococcoides-containing enrichment cultures and isolated strains (13,22,23,31). Besides absolute 16S rRNA gene copy numbers, rRNA relative abundance (Dehalococcoides species 16S rRNA copies/total Bacteria 16S rRNA copies) was used to relate the growth of the species to the transformation of HCB.…”

Section: Resultsmentioning

confidence: 57%

Role of “Dehalococcoides” spp. in the Anaerobic Transformation of Hexachlorobenzene in European Rivers

Taş

Eekert

Wagner

et al. 2011

Appl Environ Microbiol

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The diffuse pollution by chlorinated organic compounds in river basins is a concern, due to their potential adverse effects on human health and the environment. Organohalides, like hexachlorobenzene (HCB), are recalcitrant to aerobic microbial degradation, and "Dehalococcoides" spp. are the only known microorganisms capable of anaerobic transformation of these compounds coupled to their growth. In this study, sediments from four European rivers were studied in order to determine their HCB dechlorination capacities and the role of Dehalococcoides spp. in this process. Only a weak correlation was observed between Dehalococcoides species abundance and HCB transformation rates from different locations. In one of these locations, in the Ebro River sediment, HCB dechlorination could be linked to Dehalococcoides species growth and activity by 16S rRNAbased molecular methods. Furthermore, HCB dechlorination activity in this sediment was found over the full range of ambient temperatures that this sediment can be exposed to during different seasons throughout the year. The sediment contained several reductive dehalogenase (rdh) genes, and analysis of their transcription revealed the dominance of cbrA, previously shown to encode a trichlorobenzene reductive dehalogenase. This study investigated the role of Dehalococcoides spp. in HCB dechlorination in river sediments and evaluated if the current knowledge of rdh genes could be used to assess HCB bioremediation potential.Production of hexachlorobenzene (HCB)-containing pesticides is banned in most of the world due to HCB's toxic and carcinogenic nature. However, production and emission of the compound still occurs as an intermediate in chemical processes and from natural sources, such as volcanoes (5, 17). In aquatic environments, HCB is mainly deposited in the sediment due to its hydrophobicity. Bacterial reductive dechlorination plays an important role in the degradation of chlorinated aromatic contaminants like HCB in these anaerobic environments (15,19). Under anaerobic conditions, HCB is used by some bacteria in their energy metabolism by coupling reductive dehalogenation to electron transport phosphorylation (36). This is the only known pathway, so far, for the microbial transformation of HCB that is linked to microbial growth. Reductive dechlorination of HCB and its lesser chlorinated derivatives was previously reported in several river sediments (8,11,18,19). Nevertheless, in none of these studies were the microorganisms responsible for the process characterized.Three strains of bacteria capable of degrading HCB via reductive dechlorination have been isolated so far. "Dehalococcoides" sp. strain CBDB1 and "Dehalococcoides ethenogenes" strain 195 dechlorinate HCB to 1,3-dichlorobenzene (DCB), 1,4-DCB, and 1,3,5-trichlorobenzene (TCB) (3,14). Additionally, production of 1,2-DCB was also observed with strain 195. A distant relative of Dehalococcoides spp., "Dehalobium chlorocoercia" DF-1, dechlorinates HCB only to 1,3,5-TCB (48). Besides HCB, strain CBDB1 can also degr...

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Dehalorespiration with hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1

Cited by 100 publications

References 20 publications

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Role of “Dehalococcoides” spp. in the Anaerobic Transformation of Hexachlorobenzene in European Rivers

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