scholarly journals Characterization of a Dehalobacter Coculture That Dechlorinates 1,2-Dichloroethane to Ethene and Identification of the Putative Reductive Dehalogenase Gene

2009 ◽  
Vol 75 (9) ◽  
pp. 2684-2693 ◽  
Author(s):  
Ariel Grostern ◽  
Elizabeth A. Edwards
Keyword(s):  
Enrichment Culture ◽  
Growth Yield ◽  
Individual Species ◽  
Rrna Gene ◽  
Degenerate Primers ◽  
Reductive Dehalogenase ◽  
Gene Copies ◽  
Parent Culture ◽  
Dehalogenase Gene

ABSTRACT Dehalobacter and “Dehalococcoides” spp. were previously shown to be involved in the biotransformation of 1,1,2-trichloroethane (1,1,2-TCA) and 1,2-dichloroethane (1,2-DCA) to ethene in a mixed anaerobic enrichment culture. Here we report the further enrichment and characterization of a Dehalobacter sp. from this mixed culture in coculture with an Acetobacterium sp. Through a series of serial transfers and dilutions with acetate, H2, and 1,2-DCA, a stable coculture of Acetobacterium and Dehalobacter spp. was obtained, where Dehalobacter grew during dechlorination. The isolated Acetobacterium strain did not dechlorinate 1,2-DCA. Quantitative PCR with specific primers showed that Dehalobacter cells did not grow in the absence of a chlorinated electron acceptor and that the growth yield with 1,2-DCA was 6.9 (±0.7) × 107 16S rRNA gene copies/μmol 1,2-DCA degraded. PCR with degenerate primers targeting reductive dehalogenase genes detected three distinct Dehalobacter/Desulfitobacterium-type sequences in the mixed-parent culture, but only one of these was present in the 1,2-DCA-H2 coculture. Reverse transcriptase PCR revealed the transcription of this dehalogenase gene specifically during the dechlorination of 1,2-DCA. The 1,2-DCA-H2 coculture could dechlorinate 1,2-DCA but not 1,1,2-TCA, nor could it dechlorinate chlorinated ethenes. As a collective, the genus Dehalobacter has been show to dechlorinate many diverse compounds, but individual species seem to each have a narrow substrate range.

scholarly journals Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains

2012 ◽  
Vol 78 (18) ◽  
pp. 6630-6636 ◽  
Author(s):  
Jun Yan ◽  
Kirsti M. Ritalahti ◽  
Darlene D. Wagner ◽  
Frank E. Löffler
Keyword(s):  
Reductive Dechlorination ◽  
De Novo ◽  
Synthetic Medium ◽  
Geobacter Sulfurreducens ◽  
Rrna Gene ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Gene Copies ◽  
Pure Cultures

ABSTRACTDehalococcoides mccartyistrains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems.Dehalococcoideslacks the ability forde novocorrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B12) for growth. In contrast,Geobacter lovleyi, which dechlorinates tetrachloroethene tocis-1,2-dichloroethene (cis-DCE), and the nondechlorinating speciesGeobacter sulfurreducenshave 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. mccartyistrain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, andcis-DCE was dechlorinated to vinyl chloride and ethene concomitant withDehalococcoidesgrowth. In contrast, negligible increase inDehalococcoides16S rRNA gene copies and insignificant dechlorination occurred inG. sulfurreducens-D. mccartyistrain BAV1 or strain FL2 cocultures. Apparently,G. lovleyiproduces a cobamide that complementsDehalococcoides' nutritional requirements, whereasG. sulfurreducensdoes not. Interestingly,Dehalococcoidesdechlorination activity and growth could be restored inG. sulfurreducens-Dehalococcoidescocultures by adding 10 μM 5′,6′-dimethylbenzimidazole. Observations made with theG. sulfurreducens-Dehalococcoidescocultures suggest that the exchange of the lower ligand generated a cobalamin, which supportedDehalococcoidesactivity. These findings have implications forin situbioremediation and suggest that the corrinoid metabolism ofDehalococcoidesmust be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.

scholarly journals Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated “Dehalococcoides” and Methanospirillum Species

2008 ◽  
Vol 74 (21) ◽  
pp. 6709-6719 ◽  
Author(s):  
Annette R. Rowe ◽  
Brendan J. Lazar ◽  
Robert M. Morris ◽  
Ruth E. Richardson
Keyword(s):  
Gene Expression ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Population Distribution ◽  
Enrichment Culture ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Rrna Gene ◽  
Hydrogenase Gene

ABSTRACT This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming “Dehalococcoides ethenogenes” strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of “Dehalococcoides” and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

scholarly journals Characterization of an Isolate That Uses Vinyl Chloride as a Growth Substrate under Aerobic Conditions

2000 ◽  
Vol 66 (8) ◽  
pp. 3535-3542 ◽  
Author(s):  
Matthew F. Verce ◽  
Ricky L. Ulrich ◽  
David L. Freedman
Keyword(s):  
Vinyl Chloride ◽  
Suspended Solids ◽  
Specific Growth ◽  
Enrichment Culture ◽  
Initial Step ◽  
Aerobic Biodegradation ◽  
Utilization Rate ◽  
Rrna Gene ◽  
Donor Source

ABSTRACT An aerobic enrichment culture was developed by using vinyl chloride (VC) as the sole organic carbon and electron donor source. VC concentrations as high as 7.3 mM were biodegraded without apparent inhibition. VC use did not occur when nitrate was provided as the electron acceptor. A gram-negative, rod-shaped, motile isolate was obtained from the enrichment culture and identified based on biochemical characteristics and the sequence of its 16S rRNA gene asPseudomonas aeruginosa, designated strain MF1. The observed yield of MF1 when it was grown on VC was 0.20 mg of total suspended solids (TSS)/mg of VC. Ethene, acetate, glyoxylate, and glycolate also served as growth substrates, while ethane, chloroacetate, glycolaldehyde, and phenol did not. Stoichiometric release of chloride and minimal accumulation of soluble metabolites following VC consumption indicated that the predominant fate for VC is mineralization and incorporation into cell material. MF1 resumed consumption of VC after at least 24 days when none was provided, unlike various mycobacteria that lost their VC-degrading ability after brief periods in the absence of VC. When deprived of oxygen for 2.5 days, MF1 did not regain the ability to grow on VC, and a portion of the VC was transformed into VC-epoxide. Acetylene inhibited VC consumption by MF1, suggesting the involvement of a monooxygenase in the initial step of VC metabolism. The maximum specific VC utilization rate for MF1 was 0.41 μmol of VC/mg of TSS/day, the maximum specific growth rate was 0.0048/day, and the Monod half-saturation coefficient was 0.26 μM. A higher yield and faster kinetics occurred when MF1 grew on ethene. When grown on ethene, MF1 was able to switch to VC as a substrate without a lag. It therefore appears feasible to grow MF1 on a nontoxic substrate and then apply it to environments that do not exhibit a capacity for aerobic biodegradation of VC.

scholarly journals Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

2004 ◽  
Vol 70 (9) ◽  
pp. 5538-5545 ◽  
Author(s):  
Melanie Duhamel ◽  
Kaiguo Mo ◽  
Elizabeth A. Edwards
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Vinyl Chloride ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Enrichment Culture ◽  
Phylogenetic Analyses ◽  
Rrna Gene ◽  
Sufficient Information ◽  
Gene Copies ◽  
Gradient Gel Electrophoresis

ABSTRACT A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 108 16S rRNA gene copies/μmol of Cl− when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 108 16S rRNA gene copies/μmol of Cl−. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H2 enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.

scholarly journals Monitoring Abundance and Expression of “Dehalococcoides” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column

2008 ◽  
Vol 74 (18) ◽  
pp. 5695-5703 ◽  
Author(s):  
Sebastian Behrens ◽  
Mohammad F. Azizian ◽  
Paul J. McMurdie ◽  
Andrew Sabalowsky ◽  
Mark E. Dolan ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Reductive Dehalogenation ◽  
Gene Copy ◽  
Rrna Gene ◽  
Reductive Dehalogenase ◽  
Gene Copies ◽  
Donor And Acceptor ◽  
Rdase Genes

ABSTRACT We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “Dehalococcoides” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 106 (mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

scholarly journals Identification of Multiple Dehalogenase Genes Involved in Tetrachloroethene-to-Ethene Dechlorination in aDehalococcoides-Dominated Enrichment Culture

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Mohamed Ismaeil ◽  
Naoko Yoshida ◽  
Arata Katayama
Keyword(s):  
Amino Acid ◽  
Novel Species ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
The Other ◽  
Rrna Gene ◽  
Amino Acid Similarity ◽  
Reductive Dehalogenase ◽  
Per Gene ◽  
Complete Dechlorination

Chloroethenes (CEs) are widespread groundwater toxicants that are reductively dechlorinated to nontoxic ethene (ETH) by members ofDehalococcoides. This study established aDehalococcoides-dominated enrichment culture (designated “YN3”) that dechlorinates tetrachloroethene (PCE) to ETH with high dechlorination activity, that is, complete dechlorination of 800 μM PCE to ETH within 14 days in the presence ofDehalococcoidesspecies at5.7±1.9×107 copies of 16S rRNA gene/mL. The metagenome of YN3 harbored 18rdhAgenes (designatedYN3rdhA1–18) encoding the catalytic subunit of reductive dehalogenase (RdhA), four of which were suggested to be involved in PCE-to-ETH dechlorination based on significant increases in their transcription in response to CE addition. The predicted proteins for two of these four genes, YN3RdhA8 and YN3RdhA16,showed 94% and 97% of amino acid similarity with PceA and VcrA, which are well known to dechlorinate PCE to trichloroethene (TCE) and TCE to ETH, respectively. The other twordhAs,YN3rdhA6andYN3rdhA12,which were never proved asrdhAfor CEs, showed particularly high transcription upon addition of vinyl chloride (VC), with75±38and16±8.6mRNA copies per gene, respectively, suggesting their possible functions as novel VC-reductive dehalogenases. Moreover, metagenome data indicated the presence of three coexisting bacterial species, including novel species of the genusBacteroides, which might promote CE dechlorination byDehalococcoides.

scholarly journals Reductive Dechlorination of Tetrachloroethene to Ethene by a Two-Component Enzyme Pathway

1998 ◽  
Vol 64 (4) ◽  
pp. 1270-1275 ◽  
Author(s):  
Jon K. Magnuson ◽  
Robert V. Stern ◽  
James M. Gossett ◽  
Stephen H. Zinder ◽  
David R. Burris
Keyword(s):  
Enrichment Culture ◽  
Reductive Dehalogenation ◽  
Reductive Dehalogenase ◽  
Reversible Inhibition ◽  
Isolation And Characterization ◽  
Dehalococcoides Ethenogenes ◽  
Membrane Bound ◽  
Catalytic Mechanisms ◽  
Insight Into

ABSTRACT Two membrane-bound, reductive dehalogenases that constitute a novel pathway for complete dechlorination of tetrachloroethene (perchloroethylene [PCE]) to ethene were partially purified from an anaerobic microbial enrichment culture containing Dehalococcoides ethenogenes 195. When titanium(III) citrate and methyl viologen were used as reductants, PCE-reductive dehalogenase (PCE-RDase) (51 kDa) dechlorinated PCE to trichloroethene (TCE) at a rate of 20 μmol/min/mg of protein. TCE-reductive dehalogenase (TCE-RDase) (61 kDa) dechlorinated TCE to ethene. TCE,cis-1,2-dichloroethene, and 1,1-dichloroethene were dechlorinated at similar rates, 8 to 12 μmol/min/mg of protein. Vinyl chloride and trans-1,2-dichloroethene were degraded at rates which were approximately 2 orders of magnitude lower. The light-reversible inhibition of TCE-RDase by iodopropane and the light-reversible inhibition of PCE-RDase by iodoethane suggest that both of these dehalogenases contain Co(I) corrinoid cofactors. Isolation and characterization of these novel bacterial enzymes provided further insight into the catalytic mechanisms of biological reductive dehalogenation.

scholarly journals Characterization of six clinical isolates of Chimaeribacter gen. nov., a novel genus related to the Yersiniaceae family and the three species Chimaeribacter arupi sp. nov., Chimaeribacter coloradensis sp. nov, and Chimaeribacter californicus sp. nov.

2020 ◽  
Vol 70 (4) ◽  
pp. 2703-2712 ◽  
Author(s):  
Alessandro Rossi ◽  
Mark A. Fisher
Keyword(s):  
Type Strain ◽  
Single Species ◽  
Individual Species ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Gram Negative ◽  
Novel Genus ◽  
Content Type ◽  
Link Type

Eight genetically related, Gram-negative bacterial strains, isolated from clinical specimens between 2012 and 2016, were submitted to arup Laboratories for species identification. The lack of species- or genus-level matches in curated 16S rRNA gene databases prompted us to undertake the polyphasic characterization of these so far undescribed organisms. Six isolates available for additional testing were oxidase negative, catalase positive, pleomorphic, Gram-negative rods displaying temperature-dependent motility and producing yellow-pigmented colonies with three distinct morphotypes: medium-sized shiny, large mucoid and agar-pitting. Biochemical reactions and sugar fermentation patterns were most similar to members of the genus Serratia . Fatty acid profiles were highly similar across all six organisms, with the major components being: C16 : 0; C17 : 0 cyclo; C14 : 0 3-OH/iso-C16 : 1 I; C18 : 1 ω7c; and C16 : 1 ω7c/C16 : 1 ω6c. Whole-genome comparisons and multi locus sequence analysis (using the coding genes atpD, rpoB, gyrB and infB) suggest that the strains here described constitute three individual species within a novel genus related to the family Yersiniaceae . We propose for this novel taxon the name Chimaeribacter gen. nov., referring to the presentation of multiple characteristics typical of distinct Enterobacterales genera within a single organism. Four isolates are representative of a single species: Chimaeribacter arupi sp. nov (2016-Iso1, 2016-Iso2, type strain 2016-Iso3T=DSM 110101T=ATCC TSD-180T and 2013-Iso5). The remaining two isolates constitute the novel species Chimaeribacter coloradensis sp. nov. (type strain 2016-Iso4T=DSM 110102T=ATCC TSD-182T) and Chimaeribacter californicus sp. nov. (type strain 2015-Iso6T=DSM 110100T=ATCC TSD-181T). Our work provides the first formal characterization of the genus Chimaeribacter and forms the basis to study its taxonomic diversity.

scholarly journals Chlorinated electron acceptor availability selects for specificDehalococcoidespopulations in dechlorinating enrichment cultures and in groundwater

2017 ◽  
Author(s):  
A. Pérez-de-Mora ◽  
A. Lacourt ◽  
M.L. McMaster ◽  
X. Liang ◽  
S.M. Dworatzek ◽  
...  
Keyword(s):  
Electron Acceptor ◽  
Groundwater Remediation ◽  
Driving Forces ◽  
Major Influence ◽  
Rrna Gene ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Gene Copies ◽  
Field Samples ◽  
The Uk

AbstractIndividualDehalococcoides mccartyi (Dhc)strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA) genes contained within their respective genomes. While thousands ofrdhAgenes have been sequenced, the activity of the corresponding proteins have been identified in only a handful of cases. Most effort has focused on identifying the enzymes that dechlorinate substrates including trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC) relevant to groundwater remediation. The associatedrdhAgenes, namelytceA, bvcA,andvcrA, along with theD. mccartyi16S rRNA gene are often used to track growth and dechlorinating activity in DNA extracted from field samples. In this study, we augmented the typical suite of three characterizedrdhAgenes to include an additional 12 uncharacterizedrdhAsequences identified in the metagenome in the mixedDhc-containing culture KB-1 to track population shifts within the culture and at two bioaugmented field sites. Quantitative PCR assays were developed for the 15 selectedD. mccartyi rdhAgenes and evaluated using 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion ofrdhAgene copies relative toDhc16S gene copies indicated the presence of multiple distinctDhcpopulations in each culture. The specific electron acceptor amended to each culture had a major influence on the distribution ofD. mccartyipopulations and their associatedrdhAgenes. We also surveyed the abundance ofrdhAgenes in samples obtained from two bioaugmented field sites. Growth of the dominantD. mccartyipopulation in the KB-1 inoculum was detected in the UK site samples. At both field sites, the measurement of relativerdhAabundances revaled significantD. mccartyipopulation shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene, indicating that the selective pressure of the most abundant chlorinated electron acceptor that was observed in lab cultures was also occurring in the populations in the field. Understanding driving forces behindD. mccartyipopulation selection and activity is improving predictability of remediation performance at chlorinated solvent contaminated sites.

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