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Frank E Löffler - One of the best experts on this subject based on the ideXlab platform.

  • guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity
    Philosophical Transactions of the Royal Society B, 2013
    Co-Authors: Jun Yan, Frank E Löffler, Yi Yang
    Abstract:

    Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l−1 CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l−1 CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8–9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l−1 extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p -cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides ' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5′,6′-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides ' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

  • functional characterization of reductive dehalogenases by using blue native polyacrylamide gel electrophoresis
    Applied and Environmental Microbiology, 2013
    Co-Authors: Shuiquan Tang, Frank E Löffler, Elizabeth A. Edwards, Jana Seifert, Xiaoming Liang, Winnie W M Chan, Kelly E Fletcher, Lorenz Adrian
    Abstract:

    Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

  • Unexpected specificity of interspecies cobamide transfer from Geobacter spp. to organohalide-respiring Dehalococcoides mccartyi strains.
    Applied and environmental microbiology, 2012
    Co-Authors: Jun Yan, Kirsti M Ritalahti, Darlene D. Wagner, Frank E Löffler
    Abstract:

    Dehalococcoides mccartyi strains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems. Dehalococcoides lacks the ability for de novo corrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B12) for growth. In contrast, Geobacter lovleyi, which dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE), and the nondechlorinating species Geobacter sulfurreducens have complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium. G. lovleyi-D. mccartyi strain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, and cis-DCE was dechlorinated to vinyl chloride and ethene concomitant with Dehalococcoides growth. In contrast, negligible increase in Dehalococcoides 16S rRNA gene copies and insignificant dechlorination occurred in G. sulfurreducens-D. mccartyi strain BAV1 or strain FL2 cocultures. Apparently, G. lovleyi produces a cobamide that complements Dehalococcoides' nutritional requirements, whereas G. sulfurreducens does not. Interestingly, Dehalococcoides dechlorination activity and growth could be restored in G. sulfurreducens-Dehalococcoides cocultures by adding 10 μM 5′,6′-dimethylbenzimidazole. Observations made with the G. sulfurreducens-Dehalococcoides cocultures suggest that the exchange of the lower ligand generated a cobalamin, which supported Dehalococcoides activity. These findings have implications for in situ bioremediation and suggest that the corrinoid metabolism of Dehalococcoides must be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.

  • localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides
    PLOS Genetics, 2009
    Co-Authors: Paul J. Mcmurdie, Kirsti M Ritalahti, Susan Holmes, Sebastian Behrens, Jochen A Muller, Jonathan Goke, Ryan Wagner, Eugene Goltsman, Alla Lapidus, Frank E Löffler
    Abstract:

    Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

  • Spatial and temporal distributions of Geobacter lovleyi and Dehalococcoides spp. during bioenhanced PCE-NAPL dissolution.
    Environmental science & technology, 2009
    Co-Authors: Benjamin K Amos, Kurt D Pennell, Eric J. Suchomel, Frank E Löffler
    Abstract:

    The spatial and temporal distributions of multiple reductively dechlorinating bacteria were simultaneously assessed in a one-dimensional sand column containing a tetrachloroethene (PCE) nonaqueous phase liquid (NAPL) source and associated plume zones. The column was uniformly inoculated with a PCE-to-ethene dechlorinating microbial consortium that contained Dehalococcoides spp., Dehalobacter spp., and Geobacter lovleyi strain SZ. Geobacter and Dehalococcoides populations grew and colonized the column material, including the mixed-NAPL (0.25 mol/mol PCE in hexadecane) source zone. In contrast, Dehalobacter cells did not colonize the porous column material, and planktonic Dehalobacter cell titers remained below the detection limit of ca. 2.6 x 10(2) cells/mL throughout the experiment. Significant PCE dechlorination was observed and resulted in bioenhanced NAPL dissolution up to 21-fold (maximum) and 5.2-fold (cumulative) relative to abiotic dissolution. cis-1,2-Dichloroethene (cis-DCE) wasthe primary dechlorination product although vinyl chloride (VC) was also formed throughout the experiment. Ethene production occurred after significant depletion of PCE from the NAPL and when cis-DCE concentrations dropped below 6 microM. Data obtained after increasing the column residence time from 1.1 to 2.8 days and introducing a VC pulse to the column indicated that both the residence time and cis-DCE inhibition limited significant VC and ethene production. Although both Geobacter and Dehalococcoides cells were present and active in the mixed-NAPL source zone and plume region, Geobacter cell numbers were typically more than 1 order of magnitude higher than Dehalococcoides cell numbers, which is consistent with the production of predominantly cis-DCE. Analysis of both liquid- and solid-phase samples indicated that Geobacter cells grew and remained attached to the porous medium within the source zone but were largely planktonic in the plume region. In contrast Dehalococcoides cell were attached throughoutthecolumn,and Dehalococcoides cell titers increased by 1 to 2 orders of magnitude over the length of the column, correlating to increases in VC concentrations. The results from this study highlight that bioenhanced dissolution is governed by a complex interplay between resident dechlorinators, contaminant concentrations, and other aquifer-specific characteristics (e.g., hydrology).

Lisa Alvarez-cohen - One of the best experts on this subject based on the ideXlab platform.

  • ORIGINAL ARTICLE Metagenomic analysis of a stable trichloroethene-degrading microbial community
    2016
    Co-Authors: Vanessa L Brisson, Susannah G. Tringe, Kimberlee A West, Patrick Kh Lee, Eoin L Brodie, Lisa Alvarez-cohen
    Abstract:

    Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductiv

  • Metagenomic analysis of a stable trichloroethene-degrading microbial community
    The ISME Journal, 2012
    Co-Authors: Vanessa L Brisson, Susannah G. Tringe, Kimberlee A West, Patrick Kh Lee, Eoin L Brodie, Lisa Alvarez-cohen
    Abstract:

    Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides , Desulfovibrio , Methanobacterium , Methanospirillum , as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides . Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.

  • Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray
    The ISME Journal, 2011
    Co-Authors: Patrick Kh Lee, Stephen H Zinder, Kimberlee A West, Eoin L Brodie, Dan Cheng, Gary L Andersen, Gregory J Dick, Lisa Alvarez-cohen
    Abstract:

    Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis- dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans- DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions ( pceA , tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides -containing samples to provide insights into their gene content and dechlorination functions.

  • Quantifying Genes and Transcripts to Assess the In Situ Physiology of Dehalococcoides spp. in a Trichloroethene-Contaminated Groundwater Site
    Applied and environmental microbiology, 2008
    Co-Authors: Patrick Kh Lee, Tamzen W. Macbeth, Kent S. Sorenson, Rula A. Deeb, Lisa Alvarez-cohen
    Abstract:

    Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of "Dehalococcoides" spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

  • Stable carbon isotope fractionation of chloroethenes by dehalorespiring isolates.
    Environmental Science & Technology, 2007
    Co-Authors: Mark E Conrad, Lisa Alvarez-cohen
    Abstract:

    Stable carbon isotope fractionation during the reductive dechlorination of chloroethenes by two bacterial strains that dechlorinate to ethene, Dehalococcoides ethenogenes 195 and Dehalococcoides sp. strain BAV1 as well as Sulfurospirillum multivorans and Dehalobacter restrictus strain PER-K23, isolates that do not dechlorinate past DCE, are reported. Fractionation by a Dehalococcoides-containing enrichment culture is also measured for comparison to the isolates. All data adequately fit the Rayleigh model and results are presented as enrichment factors. For strain 195, the measured enrichment factors were −9.6 ± 0.4, −21.1 ± 1.8, and −5.8 ± 0.5 when degrading TCE, cDCE, and 1,1-DCE, respectively. Strain BAV1 exhibited enrichment factors of −16.9 ± 1.4, −8.4 ± 0.3, −21.4 ± 0.9, and −24.0 ± 2.0 for cDCE, 1,1-DCE, tDCE, and VC, respectively. The surprisingly large differences in enrichment factors caused by individual reductases (RDases) reducing different chloroethenes is likely the result of chemical struct...

Elizabeth A. Edwards - One of the best experts on this subject based on the ideXlab platform.

  • sustained dechlorination of vinyl chloride to ethene in Dehalococcoides enriched cultures grown without addition of exogenous vitamins and at low ph
    bioRxiv, 2019
    Co-Authors: Luz Puentes A Jacome, Olivia Molenda, Pohsiang Wang, Ahsanul M Islam, Elizabeth A. Edwards
    Abstract:

    Trichloroethene (TCE) is a ubiquitous groundwater pollutant. Successful TCE bioremediation has been demonstrated at field sites using specialized microbial consortia harboring TCE-respiring Dehaloccocoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins in the medium and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of more toxic vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In cultures grown without exogenous vitamins or cobalamin, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are the dominant acetogens. At neutral pH, a growing Acetobacterium population supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in the amount of measured cobalamin (~20-fold). Sustained dechlorination of VC to ethene was achieved at a pH as low as 5.5, yet at low pH Acetobacterium is less abundant, potentially affecting the production of DMB and/or cobalamin. However, dechlorination activity at very low pH (< 5.0) was not stimulated by DMB supplementation, but was restored by raising pH to neutral. Assays in cell extracts revealed that vinyl chloride reductase (VcrA) activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the roles of and interplay between vitamin-producing populations and pH in microbial dechlorinating communities, and their importance for successful chlorinated ethenes bioremediation at field sites.

  • Eight new genomes of organohalide-respiring Dehalococcoides mccartyi reveal evolutionary trends in reductive dehalogenase enzymes
    2018
    Co-Authors: Olivia Molenda, Shuiquan Tang, Line Lomheim, Elizabeth A. Edwards
    Abstract:

    Background: Bioaugmentation is now a well-established approach for attenuating toxic groundwater and soil contaminants, particularly for chlorinated ethenes and ethanes. The KB-1 and WBC-2 consortia are cultures used for this purpose. These consortia contain organisms belonging to the Dehalococcoidia, including strains of Dehalococcoides mccartyi in KB-1 and of both D. mccartyi and Dehalogenimonas in WBC-2. These tiny anaerobic bacteria couple respiratory reductive dechlorination to growth and harbour multiple reductive dehalogenase genes (rdhA) in their genomes, the majority of which have yet to be characterized. Results: Using a combination of Illumina mate-pair and paired-end sequencing we closed the genomes of eight new strains of Dehalococcoides mccartyi found in three related KB-1 sub-cultures that were enriched on trichloroethene (TCE), 1,2-dichloroethane (1,2-DCA) and vinyl chloride (VC), bringing the total number of genomes available in NCBI to 24. A pangenome analysis was conducted on 24 Dehalococcoides genomes and five Dehalogenimonas genomes (2 in draft) currently available in NCBI. This Dehalococcoidia pangenome generated 2875 protein families comprising of 623 core, 2203 accessory, and 49 unique protein families. In Dehalococcoides mccartyi the complement of reductive dehalogenase genes varies by strain, but what was most surprising was how the majority of rdhA sequences actually exhibit a remarkable degree of synteny across all D. mccartyi genomes. Several homologous sequences are also shared with Dehalogenimonas genomes. Nucleotide and predicted protein sequences for all reductive dehalogenases were aligned to begin to decode the evolutionary history of reductive dehalogenases in the Dehalococcoidia. Conclusions: The conserved synteny of the rdhA genes observed across Dehalococcoides genomes indicates that the major differences between strain rdhA gene complement has resulted from gene loss rather than recombination. These rdhA have a long evolutionary history and trace their origin in the Dehalococcoidia prior to the speciation of Dehalococcoides and Dehalogenimonas. The only rdhA genes suspected to have been acquired by lateral gene transfer are protein-coding rdhA that have been identified to catalyze dehalogenation of industrial pollutants. Sequence analysis suggests that evolutionary pressures resulting in new rdhA genes involve adaptation of existing dehalogenases to new substrates, mobilization of rdhA between genomes or within a genome, and to a lesser degree manipulation of regulatory regions to alter expression.

  • a switch of chlorinated substrate causes emergence of a previously undetected native dehalobacter population in an established Dehalococcoides dominated chloroethene dechlorinating enrichment culture
    FEMS Microbiology Ecology, 2017
    Co-Authors: Luz Puentes A Jacome, Elizabeth A. Edwards
    Abstract:

    Chlorobenzenes are soil and groundwater pollutants of concern that can be reductively dehalogenated by organohalide-respiring bacteria from the genera Dehalococcoides and Dehalobacter. The bioaugmentation culture KB-1® harbours Dehalococcoides mccartyi spp. that reductively dehalogenate trichloroethene to ethene. It contains more than 30 reductive dehalogenase genes; some of them are highly similar to genes found in the chlorobenzene-respiring Dehalococcoides mccartyi strain CBDB1. We explored the chlorobenzene dehalogenation capability of the KB-1 enrichment culture using 1,2,4-trichlorobenzene (1,2,4-TCB). We achieved adaptation of KB-1 to 1,2,4-TCB that is dehalogenated to a mixture of dichlorobenzenes, and subsequently to monochlorobenzene and benzene. Surprisingly, a native Dehalobacter population, and not a Dehalococcoides population, couples the dechlorination of 1,2,4-TCB to growth achieving an average yield of 1.1 ± 0.6 × 1013 cells per mole of Cl- released. Interestingly, the dechlorination of 1,2,4-TCB occurs alongside the complete dechlorination of trichloroethene to ethene in cultures fed both electron acceptors. Dehalobacter was not previously identified as a major player in KB-1, but its ecological niche was favoured by the introduction of 1,2,4-TCB. Based on 16S rRNA phylogeny, Dehalobacter populations seem to cluster into specialised clades, and are likely undergoing substrate specialisation as a strategy to reduce competition for electron acceptors.

  • 1,2-Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium.
    Environmental science & technology, 2016
    Co-Authors: Koshlan Mayer-blackwell, Elizabeth A. Edwards, Maeva Fincker, Olivia Molenda, Susan Holmes, Benjamin J. Callahan, Holly L. Sewell, Alfred M. Spormann
    Abstract:

    Bioremediation of groundwater contaminated with chlorinated aliphatic hydrocarbons such as perchloroethene and trichloroethene can result in the accumulation of the undesirable intermediate vinyl chloride. Such accumulation can either be due to the absence of specific vinyl chloride respiring Dehalococcoides mccartyi or to the inhibition of such strains by the metabolism of other microorganisms. The fitness of vinyl chloride respiring Dehalococcoides mccartyi subpopulations is particularly uncertain in the presence of chloroethene/chloroethane cocontaminant mixtures, which are commonly found in contaminated groundwater. Therefore, we investigated the structure of Dehalococcoides populations in a continuously fed reactor system under changing chloroethene/ethane influent conditions. We observed that increasing the influent ratio of 1,2-dichloroethane to trichloroethene was associated with ecological selection of a tceA-containing Dehalococcoides population relative to a vcrA-containing Dehalococcoides popu...

  • Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes
    Applied and environmental microbiology, 2015
    Co-Authors: Xiaoming Liang, Shuiquan Tang, Olivia Molenda, Elizabeth A. Edwards
    Abstract:

    Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

Patrick Kh Lee - One of the best experts on this subject based on the ideXlab platform.

  • ORIGINAL ARTICLE Metagenomic analysis of a stable trichloroethene-degrading microbial community
    2016
    Co-Authors: Vanessa L Brisson, Susannah G. Tringe, Kimberlee A West, Patrick Kh Lee, Eoin L Brodie, Lisa Alvarez-cohen
    Abstract:

    Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductiv

  • Metagenomic analysis of a stable trichloroethene-degrading microbial community
    The ISME Journal, 2012
    Co-Authors: Vanessa L Brisson, Susannah G. Tringe, Kimberlee A West, Patrick Kh Lee, Eoin L Brodie, Lisa Alvarez-cohen
    Abstract:

    Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides , Desulfovibrio , Methanobacterium , Methanospirillum , as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides . Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.

  • Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray
    The ISME Journal, 2011
    Co-Authors: Patrick Kh Lee, Stephen H Zinder, Kimberlee A West, Eoin L Brodie, Dan Cheng, Gary L Andersen, Gregory J Dick, Lisa Alvarez-cohen
    Abstract:

    Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis- dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans- DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions ( pceA , tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides -containing samples to provide insights into their gene content and dechlorination functions.

  • comparative genomics of Dehalococcoides ethenogenes 195 and an enrichment culture containing unsequenced Dehalococcoides strains
    Applied and Environmental Microbiology, 2008
    Co-Authors: Kimberlee A West, Stephen H Zinder, Patrick Kh Lee, Eoin L Brodie, David R. Johnson, Todd Z Desantis, Helene Feil, Gary L Andersen, Lisa Alvarezcohen
    Abstract:

    Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by some “Dehalococcoides” organisms. A Dehalococcoides-organism-containing microbial consortium (referred to as ANAS) with the ability to degrade TCE to ethene, an innocuous end product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of “Dehalococcoides ethenogenes” 195. This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray results revealed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, cobalamin-salvaging system, nitrogen fixation pathway, and five hydrogenase complexes, are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase, tceA, was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria, which is essential in developing effective strategies for the bioremediation of PCE and TCE in the environment.

  • Quantifying Genes and Transcripts to Assess the In Situ Physiology of Dehalococcoides spp. in a Trichloroethene-Contaminated Groundwater Site
    Applied and environmental microbiology, 2008
    Co-Authors: Patrick Kh Lee, Tamzen W. Macbeth, Kent S. Sorenson, Rula A. Deeb, Lisa Alvarez-cohen
    Abstract:

    Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of "Dehalococcoides" spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.

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  • Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.
    Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2013
    Co-Authors: Christian J. Schipp, Jana Seifert, Ernest Marco-urrea, Anja Kublik, Lorenz Adrian
    Abstract:

    Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.

  • functional characterization of reductive dehalogenases by using blue native polyacrylamide gel electrophoresis
    Applied and Environmental Microbiology, 2013
    Co-Authors: Shuiquan Tang, Frank E Löffler, Elizabeth A. Edwards, Jana Seifert, Xiaoming Liang, Winnie W M Chan, Kelly E Fletcher, Lorenz Adrian
    Abstract:

    Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

  • Identification and Characterization of a Re-Citrate Synthase in Dehalococcoides Strain CBDB1
    Journal of bacteriology, 2011
    Co-Authors: Ernest Marco-urrea, Steffanie Paul, Viola Khodaverdi, Jana Seifert, Martin Von Bergen, Utta Kretzschmar, Lorenz Adrian
    Abstract:

    The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.

  • ERC-group microflex: microbiology of Dehalococcoides-like Chloroflexi
    Reviews in Environmental Science and Bio Technology, 2009
    Co-Authors: Lorenz Adrian
    Abstract:

    The Microflex project, funded within the first call of the European Research Council, focuses on a specific group of bacteria, the Dehalococcoides-like Chloroflexi. This group of bacteria deeply rooting in the phylogenetic tree is formed by several cultivated strains of the proposed genus “Dehalococcoides” and many sequences of uncultivated organisms mostly from marine sediments or terrestrial subsurface locations. The project compares cultivated Dehalococcoides species growing by organohalide respiration using halogenated compounds as electron acceptors with marine Chloroflexi populations. For this comparison a wide array of different approaches and techniques are used including cultivation, biochemical analyses, molecular tools and isotopic fractionation measurements. The project aims at contributing to the understanding of the physiology of Dehalococcoides-like Chloroflexi in deep marine sediments and their mode of living. A second aim of the project is the further understanding of the physiology and biochemistry of dehalogenating Dehalococcoides species and how these bacteria can be used efficiently for bioremediation of contaminated subsurface environments.

  • identification of a chlorobenzene reductive dehalogenase in Dehalococcoides sp strain cbdb1
    Applied and Environmental Microbiology, 2007
    Co-Authors: Lorenz Adrian, Jan Rahnenfuhrer, Johan Gobom, Tina Holscher
    Abstract:

    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.