Putative Reductive Dehalogenase Research Articles

Complete Reductive Dechlorination of 4-Hydroxy-chlorothalonil by Dehalogenimonas Populations.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TePN) is one of the most widely used fungicides all over the world. Its major environmental transformation product 4-hydroxy-chlorothalonil (4-hydroxy-2,5,6-trichloroisophthalonitrile, 4-OH-TPN) is more persistent, mobile, and toxic and is frequently detected at a higher concentration in various habitats compared to its parent compound TePN. Further microbial transformation of 4-OH-TPN has never been reported. In this study, we demonstrated that 4-OH-TPN underwent complete microbial reductive dehalogenation to 4-hydroxy-isophthalonitrile via 4-hydroxy-dichloroisophthalonitrile and 4-hydroxy-monochloroisophthalonitrile. 16S rRNA gene amplicon sequencing demonstrated that Dehalogenimonas species was enriched from 6% to 17-22% after reductive dechlorination of 77.24 μmol of 4-OH-TPN. Meanwhile, Dehalogenimonas copies increased by one order of magnitude and obtained a yield of 1.78 ± 1.47 × 108 cells per μmol Cl- released (N = 6), indicating that 4-OH-TPN served as the terminal electron acceptor for organohalide respiration of Dehalogenimonas species. A draft genome of Dehalogenimonas species was assembled through metagenomic sequencing, which harbors 30 putative reductive dehalogenase genes. Syntrophobacter, Acetobacterium, and Methanosarcina spp. were found to be the major non-dechlorinating populations in the microbial community, who might play important roles in the reductive dechlorination of 4-OH-TPN by the Dehalogenimonas species. This study first reports that Dehalogenimonas sp. can also respire on the seemingly dead-end product of TePN, paving the way to complete biotransformation of the widely present TePN and broadening the substrate spectrum of Dehalogenimonas sp. to polychlorinated hydroxy-benzonitrile.

Dehalococcoides-Containing Enrichment Cultures Transform Two Chlorinated Organophosphate Esters.

Although chlorinated organophosphate esters (Cl-OPEs) have been reported to be ubiquitously distributed in various anoxic environments, little information is available on their fate under anoxic conditions. In this study, we report two Dehalococcoides-containing enrichment cultures that transformed 3.88 ± 0.22 μmol tris(2-chloroethyl) phosphate (TCEP) and 2.61 ± 0.02 μmol tris(1-chloro-2-propyl) phosphate (TCPP) within 10 days. Based on the identification of the transformed products and deuteration experiments, we inferred that TCEP may be transformed to generate bis(2-chloroethyl) phosphate and ethene via one-electron transfer (radical mechanism), followed by C-O bond cleavage. Ethene was subsequently reduced to ethane. Similarly, TCPP was transformed to form bis(1-chloro-2-propyl) phosphate and propene. 16S rRNA gene amplicon sequencing and quantitative polymerase chain reaction analysis revealed that Dehalococcoides was the predominant contributor to the transformation of TCEP and TCPP. Two draft genomes of Dehalococcoides assembled from the metagenomes of the TCEP- and TCPP-transforming enrichment cultures contained 14 and 15 putative reductive dehalogenase (rdh) genes, respectively. Most of these rdh genes were actively transcribed, suggesting that they might contribute to the transformation of TCEP and TCPP. Taken together, this study provides insights into the role of Dehalococcoides during the transformation of representative Cl-OPEs.

Genome Sequence of "Candidatus Dehalogenimonas etheniformans" Strain GP, a Vinyl Chloride-Respiring Anaerobe.

"Candidatus Dehalogenimonas etheniformans" strain GP couples growth with the reductive dechlorination of vinyl chloride and several polychlorinated ethenes. The genome sequence comprises a circular 2.07-Mb chromosome with a G+C content of 51.9% and harbors 50 putative reductive dehalogenase genes.

Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions.

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood-Ljungdahl pathway, electron bifurcation, and ferredoxin-dependent transport-coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO2 reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H2 consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.

Metagenomes from Coastal Marine Sediments Give Insights into the Ecological Role and Cellular Features of Loki- and Thorarchaeota.

The genomes of Asgard Archaea, a novel archaeal proposed superphylum, share an enriched repertoire of eukaryotic signature genes and thus promise to provide insights into early eukaryote evolution. However, the distribution, metabolisms, cellular structures, and ecology of the members within this superphylum are not well understood. Here we provide a meta-analysis of the environmental distribution of the Asgard archaea, based on available 16S rRNA gene sequences. Metagenome sequencing of samples from a salt-crusted lagoon on the Baja California Peninsula of Mexico allowed the assembly of a new Thorarchaeota and three Lokiarchaeota genomes. Comparative analyses of all known Lokiarchaeota and Thorarchaeota genomes revealed overlapping genome content, including central carbon metabolism. Members of both groups contained putative reductive dehalogenase genes, suggesting that these organisms might be able to metabolize halogenated organic compounds. Unlike the first report on Lokiarchaeota, we identified genes encoding glycerol-1-phosphate dehydrogenase in all Loki- and Thorarchaeota genomes, suggesting that these organisms are able to synthesize bona fide archaeal lipids with their characteristic glycerol stereochemistry.IMPORTANCE Microorganisms of the superphylum Asgard Archaea are considered to be the closest living prokaryotic relatives of eukaryotes (including plants and animals) and thus promise to give insights into the early evolution of more complex life forms. However, very little is known about their biology as none of the organisms has yet been cultivated in the laboratory. Here we report on the ecological distribution of Asgard Archaea and on four newly sequenced genomes of the Lokiarchaeota and Thorarchaeota lineages that give insight into possible metabolic features that might eventually help to identify these enigmatic groups of archaea in the environment and to culture them.

Discovered by genomics: putative reductive dehalogenases with N-terminus transmembrane helixes.

ABSTRACTAttempts for bioremediation of toxic organohalogens resulted in the identification of organohalide-respiring bacteria harbouring reductive dehalogenases (RDases) enzymes. RDases consist of the catalytic subunit (RdhA, encoded by rdhA) that does not have membrane-integral domains, and a small putative membrane anchor (RdhB, encoded by rdhB) that (presumably) locates the A subunit to the outside of the cytoplasmic membrane. Recent genomic studies identified a putative rdh gene in an uncultured deltaproteobacterial genome that was not accompanied by an rdhB gene, but contained transmembrane helixes in N-terminus. Therefore, rather than having a separate membrane anchor protein, this putative RDase is likely a hybrid of RdhA and RdhB, and directly connected to the membrane with transmembrane helixes. However, functionality of the hybrid putative RDase remains unknown. Further analysis showed that the hybrid putative rdh genes are present in the genomes of pure cultures and uncultured members of Bacteriodetes and Deltaproteobacteria, but also in the genomes of the candidate divisions. The encoded hybrid putative RDases have cytoplasmic or exoplasmic C-terminus localization, and cluster phylogenetically separately from the existing RDase groups. With increasing availability of (meta)genomes, more diverse and likely novel rdh genes are expected, but questions regarding their functionality and ecological roles remain open.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes.

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per μmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila.

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. Inthe sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

Genomic characterization of Dehalococcoides mccartyi strain 11a5 reveals a circular extrachromosomal genetic element and a new tetrachloroethene reductive dehalogenase gene.

Dehalococcoides mccartyi exhibits versatile capabilities to respire halogenated compounds under anaerobic conditions. In this study, we report the assembly and annotation of the complete genome of a chloroethene dechlorinating D. mccartyi strain 11a5. Bearing a 1461 973 base-pair chromosome, strain 11a5 distinguishes itself from other D. mccartyi strains by possessing a 5940 base-pair circular extrachromosomal genetic element which contains a reductive dehalogenase homolog. The whole genome of strain 11a5 harbors 31 putative reductive dehalogenase genes. Through transcriptional and proteomic analyses, we identified a new tetrachloroethene (PCE) reductive dehalogenase, PteA (11a5_1355), which catalyzes reductive dechlorination from PCE to trichloroethene (TCE) and shares only 38% similarity in amino acid sequence with its closest relative PceA in D. mccartyi strain 195. The acquisition of the genome of strain 11a5 enlarged the database of D. mccartyi and enriched our understanding of this unique species, among which, the identification of a new PCE reductive dehalogenase can assist in understanding PCE dechlorination process. In addition, the discovery of the circular extrachromosomal genetic element in strain 11a5 may provide insights to investigate how reductive dehalogenase homologous genes are transferred and carried by organohalide respiring bacteria.

Reductive dehalogenation activity of indigenous microorganism in sediments of the Hackensack River, New Jersey

Organohalogen pollutants are of concern in many river and estuarine environments, such as the New York-New Jersey Harbor estuary and its tributaries. The Hackensack River is contaminated with various metals, hydrocarbons and halogenated organics, including polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins. In order to examine the potential for microbial reductive dechlorination by indigenous microorganisms, sediment samples were collected from five different estuarine locations along the Hackensack River. Hexachlorobenzene (HCB), hexabromobenzene (HBB), and pentachloroaniline (PCA) were selected as model organohalogen pollutants to assess anaerobic dehalogenating potential. Dechlorinating activity of HCB and PCA was observed in sediment microcosms for all sampling sites. HCB was dechlorinated via pentachlorobenzene (PeCB) and trichlorobenzene (TriCB) to dichlorobenzene (DCB). PCA was dechlorinated via tetrachloroaniline (TeCA), trichloroanilines (TriCA), and dichloroanilines (DCA) to monochloroaniline (MCA). No HBB debromination was observed over 12 months of incubation. However, with HCB as a co-substrate slow HBB debromination was observed with production of tetrabromobenzene (TeBB) and tribromobenzene (TriBB). Chloroflexi specific 16S rRNA gene PCR-DGGE followed by sequence analysis detected Dehalococcoides species in sediments of the freshwater location, but not in the estuarine site. Analysis targeting 12 putative reductive dehalogenase (rdh) genes showed that these were enriched concomitant with HCB or PCA dechlorination in freshwater sediment microcosms.

Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates.

The genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete heme biosynthesis pathway is present in the five Dehalobacter genomes. This pathway corresponds to a newly described alternative heme biosynthesis route first identified in Archaea. This analysis of organohalide-respiring Firmicutes and Chloroflexi reveals profound evolutionary differences despite very similar niche-specific metabolism and function.

Genomic Characterization of Dehalococcoides mccartyi Strain JNA That Reductively Dechlorinates Tetrachloroethene and Polychlorinated Biphenyls.

Dehalococcoides mccartyi strain JNA detoxifies highly chlorinated polychlorinated biphenyl (PCB) mixtures via 85 distinct dechlorination reactions, suggesting that it has great potential for PCB bioremediation. However, its genomic and functional gene information remain unknown due to extremely slow growth of strain JNA with PCBs. In this study, we used tetracholorethene (PCE) as an alternative electron acceptor to grow sufficient biomass of strain JNA for subsequent genome sequencing and functional gene identification. Analysis of the assembled draft genome (1 462 509 bp) revealed the presence of 29 putative reductive dehalogenase (RDase) genes. Among them, JNA_RD8 and JNA_RD11 genes were highly transcribed in both PCE- and PCB-fed cultures. Furthermore, in vitro assays with crude cell lysate from PCE grown cells revealed dechlorination activity against both PCE and 2,2',3,4,4',5,5'-heptachlorobiphenyl. These data suggest that both JNA_RD8 and JNA_RD11 may be bifunctional PCE/PCB RDases. This study deepens the knowledge of organohalide respiration of PCBs and facilitates in situ PCB-bioremediation with strain JNA.

Dehalococcoides mccartyi strain DCMB5 Respires a broad spectrum of chlorinated aromatic compounds.

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Enriching for microbial reductive dechlorination of polychlorinated dibenzo-p-dioxins and dibenzofurans

Anaerobic enrichment cultures derived from contaminated Kymijoki River sediments dechlorinated 1,2,3,4-tetrachlorodibenzofuran (1,2,3,4-tetra-CDF), octachlorodibenzofuran (octa-CDF) and 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-tetra-CDD). 1,2,3,4-tetra-CDF was dechlorinated via 1,2,3-, 2,3,4-, and 1,3,4/1,2,4-tri-CDFs to 1,3-, 2,3-, and 2,4-di-CDFs and finally to 4-mono-CDF. The dechlorination rate of 1,2,3,4-tetra-CDF was generally slower than that of 1,2,3,4-tetra-CDD. The rate and extent of 1,2,3,4-tetra-CDD dechlorination was enhanced by addition of pentachloronitrobenzene (PCNB) as a co-substrate. Dechlorination of spiked octa-CDF was observed with the production of hepta-, hexa-, penta- and tetra-CDFs over 6 months. Two major phylotypes of the Chloroflexi community showed an increase, one of which was identical to the Dehalococcoides mccartyi Pinellas subgroup. A set of twelve putative reductive dehalogenase (rdh) genes increased in abundance with addition of 1,2,3,4-tetra-CDF, 1,2,3,4-tetra-CDD and/or PCNB. This information will aid in understanding how indigenous microbial communities impact the fate of PCDFs and in developing strategies for bioremediation of PCDD/F contaminated sediments.

Functional genotyping of Sulfurospirillum spp. in mixed cultures allowed the identification of a new tetrachloroethene reductive dehalogenase.

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed.

Identification of a reductive tetrachloroethene dehalogenase in Shewanella sediminis.

The genome sequence of psychrophilic Shewanella sediminis revealed the presence of five putative reductive dehalogenases (Rdhs). We found that cell extracts of pyruvate/fumarate-grown S. sediminis cells catalysed reduced methyl viologen-dependent reductive dechlorination of tetrachloroethene (PCE) to trichloroethene (TCE) at a specific activity of approximately 1 nmol TCE min(-1) (mg protein)(-1). Dechlorination of PCE followed Michaelis-Menten kinetics with an apparent Km of 120 μM PCE. No PCE dechlorination was observed with heat-denatured extract or when cyanocobalamin was omitted from the growth medium; however, the presence of PCE in the growth medium increased PCE transformation rates. Analysis of mutants carrying in-frame deletions of all five Rdhs encoding genes showed that only deletion of Ssed_3769 resulted in the loss of PCE dechlorination activity suggesting that Ssed_3769 is a functional Rdh. This is the first study to show reductive dechlorination activity of PCE in a sediment-dwelling Shewanella species that may be important for linking the flux of organohalogens to organic carbon via reductive dehalogenation in marine sediments.

Remediation of Polychlorinated Biphenyl Impacted Sediment by Concurrent Bioaugmentation with Anaerobic Halorespiring and Aerobic Degrading Bacteria

Bioremediation of sediments contaminated with commercial polychlorinated biphenyls (PCBs) is potentially achievable by the sequential activity of anaerobic halorespiration to convert higher chlorinated congeners to less chlorinated congeners that are susceptible to aerobic respiratory degradation. The efficacy of bioaugmentation with anaerobic halorespiring Dehalobium chlorocoercia DF1 and aerobic Burkholderia xenovorans LB400 added concurrently with granulated activated carbon (GAC) as a delivery system was determined in 2 L laboratory mesocosms containing weathered Aroclor-contaminated sediment from Baltimore Harbor, MD, USA. The greatest effect was seen in the mesocosm bioaugmented with both DF1 and LB400 together, which resulted in an 80% decrease by mass of PCBs, from 8 to <2 mg/kg after 120 days. There was no significant increase in lesser-chlorinated congeners, indicating that both anaerobic dechlorination by DF1 and aerobic degradation by LB400 occurred. In contrast, nonbioaugmented controls containing filtered culture supernatant showed only a 25% decrease in total levels of PCBs after 365 days, which was likely due to biostimulation of the indigenous population by the medium. Direct colony counts and molecular analysis targeting a putative reductive dehalogenase gene of D. chlorocoercia or the bphA gene of LB400 showed the presence of viable DF1 and LB400 in bioaugmented mesocosms after 365 days, indicating that both nonindigenous strains were sustainable within the indigenous microbial community. These results suggest that an in situ treatment employing the simultaneous application of anaerobic and aerobic microorganisms could be an effective and environmentally sustainable strategy to reduce PCBs levels in contaminated sediment.

Global gene expression of Dehalococcoides within a robust dynamic TCE‐dechlorinating community under conditions of periodic substrate supply

A microarray targeting four sequenced strains in the Dehalococcoides (Dhc) genus was used to analyze gene expression in a robust long-term trichloroethene (TCE)-degrading microbial community (designated ANAS) during feeding cycles that involve conditions of periodic substrate supply. The Dhc transcriptome was examined at three time-points throughout a batch feeding cycle: T1 (27 h) when TCE, dichloroethene (DCE), and vinyl chloride (VC) were present; T2 (54 h) when only VC remained; and T3 (13 days) when Dhc had been starved of substrate for 9 days. Ninety percent of the Dhc open reading frames (ORFs) that were detected in the ANAS DNA were found to be expressed as RNA sometime during the time course, demonstrating extraordinary utilization of the streamlined genome. Ninety-seven percent of these transcripts were differentially expressed during the time course indicating efficiency of transcription through regulation in Dhc. Most Dhc genes were significantly down-regulated at T3 , responding to a lack of substrate as would be expected. The tceA and vcrA genes, which code for proteins with known chlorinated ethene reduction functions, were highly expressed at both T1 and T2 , whereas two other putative reductive dehalogenase genes (DET0173 and DET1545) were most highly expressed at T2 , likely in response to the presence of VC. Hydrogenases were most highly expressed at T1 , reflecting their important role in accumulating electrons used to initiate reductive dechlorination and other biosynthesis pathways. Cobalamin transport genes were preferentially expressed at T2 , reflecting an increase in corrinoid transport as chloroethenes were degraded and a decrease in activity of the transport system after dehalogenation was complete. This is the first application of a microarray targeting a known genus, including both core genomes and identified strain-specific genes, to improve our understanding of transcriptional dynamics within an undefined microbial community.