Dehalococcoides Ethenogenes Research Articles

A Case Study for Microbial Biodegradation: Anaerobic Bacterial Reductive Dechlorination of Polychlorinated Biphenyls—From Sediment to Defined Medium

The history of anaerobic microbial polychlorinated biphenyl (PCB) dechlorination is traced over 20 years using a case study of PCB dechlorination in the Housatonic River (Massachusetts) as an example. The history progresses from the characterization of the PCBs in the sediment, to cultivation in sediment microcosms, to the identification of four distinct types of PCB dechlorination, to a successful field test, to the cultivation in defined medium of the organisms responsible for extensive dechlorination of Aroclor 1260, and finally to the identification of a Dehalococcoides population that links its growth to the dechlorination of Aroclor 1260. Other PCB dechlorinators have also been identified. Two bacterial strains, o-17 and DF-1, that link their growth to the dechlorination of several PCB congeners belong to a novel clade of putative dechlorinating bacteria within the phylum Chloroflexi. Dehalococcoides ethenogenes strain 195 also dechlorinates several PCB congeners when grown on chlorinated ethenes. Evidence is mounting that Dehalococcoides and other dechlorinating Chloroflexi may play a significant role in the dechlorination of commercial PCBs in situ.

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

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.

Biostimulation and bioaugmentation to enhance dechlorination of polychlorinated dibenzo-p-dioxins in contaminated sediments

Dechlorination of spiked 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) was investigated in sediment microcosms from three polychlorinated dibenzo-p-dioxin and dibenzofuran (CDD/F)-contaminated sites: River Kymijoki, Finland; Gulf Island Pond, Maine; and Lake Roosevelt, Washington. Dechlorination was stimulated by addition of electron donor and halogenated priming compounds, and bioaugmentation by a mixed culture containing Dehalococcoides ethenogenes strain 195. Amendment with 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) promoted rapid dechlorination of 1,2,3,4-TeCDD to 2-monochlorodibenzo-p-dioxin (2MCDD) in Gulf Island Pond and River Kymijoki sediments, however, only slow dechlorination to 1,4-dichlorodibenzo-p-dioxin was observed in Lake Roosevelt sediments. The dechlorination pathway in 1,2,3,4-TeCB-amended microcosms proceeded mainly via 1,3-dichlorodibenzo-p-dioxin, with less production of 2,3-dichlorodibenzo-p-dioxin in comparison with other treatments. Microbial community analyses indicated that Dehalococcoides-like bacteria were enriched with 1,2,3,4-TeCB. Quantitative real-time PCR analysis of Dehalococcoides-specific 16S rRNA genes and the D. ethenogenes strain 195 dehalogenase gene, tceA, showed at least an order of magnitude higher gene copy numbers in the bioaugmented than in the nonbioaugmented microcosms. An active-dechlorinating population is present in the River Kymijoki and biostimulation may enhance both native Dehalococcoides spp. and the bioaugmented D. ethenogenes strain 195.

Dehalococcoides’ Gene Transcripts As Quantitative Bioindicators of Tetrachloroethene, Trichloroethene, and cis-1,2-Dichloroethene Dehalorespiration Rates

Gene transcripts corresponding to 16S rRNA, the hydrogenase (H2ase) Hup, a sequence annotated asformate dehydrogenase (Fdh) and reductive dehalogenases (RDases) TceA, PceA, DET1559, and DET1545 in Dehalococcoides ethenogenes strain 195 (DET) hold promise as potential bioindicators of the dehalorespiration of chlorinated ethenes. Here, we present quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) data taken from DET-containing mixed culture microcosms (4.4 x 10(8) DET 16S rRNA gene copies/mL) operated under continuous-feed conditions, with the aim of clarifying the relationships between pseudosteady-state abundances of bioindicator transcripts and respiration rate of various substrates: tetrachloroethene (PCE), trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). Results from PCE-fed microcosms suggested an induction threshold for transcription of some bioindicator genes between chloroethene respiration rates of 2.1 and 9.5 microeeq/L/hr. Putative RDase genes DET1559 and DET1545, however, were up-regulated at low PCE respiration rates and may be functionally significant when substrate levels are low. Data from PCE-fed microcosms operated at saturation kinetics indicated that a high respiration rate was not necessarily associated with a correspondingly high bioindicator transcript abundance. From these microcosms we calculated an approximate yield value of 1.6 x 10(8) 16S rRNA gene copies (cells) per micromol Cl- released and estimated a kmax of PCE respiration of 3 x 10(-9) micromol Cl- per 16S rRNA gene copy per day. TCE- and cDCE-fed microcosm studies indicated that Fdh, Hup, and TceA were the most abundant transcripts and could make suitable choices as bioindicators of activity for these substrates. Hup transcripts could be positively correlated to respiration rate (between approximately 8 and 45 microeeq/L/hr) regardless of chloroethene substrate, with transcript levels predicted to increase by 1.8 x 10(9) copies/mL culture for every/eeq/ L/hr increase in respiration rate (R2 = 0.90). Although RDase transcripts may provide information on substrate range, H2ase transcripts may be better indicators of per cell respiration rates.

Correlation of Respiratory Gene Expression Levels and Pseudo-Steady State PCE Respiration Rates in Dehalococcoides ethenogenes

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrelation of Respiratory Gene Expression Levels and Pseudo-Steady State PCE Respiration Rates in Dehalococcoides ethenogenesBrian G. Rahm and Ruth E. Richardson*Cite this: Environ. Sci. Technol. 2008, 42, 11, 4236Publication Date (Web):April 24, 2008Publication History Received9 March 2008Accepted10 March 2008Published online24 April 2008Published inissue 1 June 2008https://doi.org/10.1021/es800696vCopyright © 2008 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views295Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (92 KB) Get e-AlertsSUBJECTS:Genetics,Power conversion efficiency Get e-Alerts

Comparative Genomics of “ Dehalococcoides ethenogenes ” 195 and an Enrichment Culture Containing Unsequenced “ Dehalococcoides ” Strains

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.

Temporal Transcriptomic Microarray Analysis of “ Dehalococcoides ethenogenes ” Strain 195 during the Transition into Stationary Phase

"Dehalococcoides" bacteria can reductively dehalogenate a wide range of halogenated organic pollutants. In this study, DNA microarrays were used to monitor dynamic changes in the transcriptome as "Dehalococcoides ethenogenes" strain 195 transitioned from exponential growth into stationary phase. In total, 415 nonredundant genes were identified as differentially expressed. As expected, genes involved with translation and energy metabolism were down-regulated while genes involved with general stress response, transcription, and signal transduction were up-regulated. Unexpected, however, was the 8- to 10-fold up-regulation of four putative reductive dehalogenases (RDases) (DET0173, DET0180, DET1535, and DET1545). Another unexpected finding was the up-regulation of a large number of genes located within integrated elements, including a putative prophage and a multicopy transposon. Finally, genes encoding the dominant hydrogenase-RDase respiratory chain of this strain (Hup and TceA) were expressed at stable levels throughout the experiment, providing molecular evidence that strain 195 can uncouple dechlorination from net growth.

Variability in microbial carbon isotope fractionation of tetra- and trichloroethene upon reductive dechlorination

The variability of stable carbon isotope fractionation upon reductive dechlorination of tetra- and trichloroethene by several microbial strains was investigated to examine the uncertainties related to the in situ application of compound specific isotope analysis (CSIA) of chlorinated ethenes. Carbon isotope fractionation was investigated with a set of microorganisms representative for the currently known diversity of dehalorespirers: Dehalococcoides ethenogenes strain 195, Desulfitobacterium sp. strain Viet1, Desulfuromonas michiganensis and Geobacter lovleyi sp. strain SZ and compared to the previous reports using Sulfurospirillum spp. and Desulfitobacterium sp. strain PCE-S. Carbon isotope fractionation of tetrachloroethene (PCE) and trichlorethene (TCE) was highly variable ranging from the absence of significant fractionation to carbon isotope fractionation (εC) of 16.7 and 3.5–18.9 for PCE and TCE, respectively. Fractionation of both compounds by D. ethenogenes strain 195 (PCE: εC = 6.0; TCE: εC = 13.7) was similar to the literature data for mixed cultures containing Dehalococcoides spp. D. michiganensis (PCE: no significant fractionation; TCE: εC = 3.5) and G. lovleyi sp. strain SZ (PCE no significant fractionation; TCE: εC = 8.5) generated the lowest fractionation of all studied strains. Desulfitobacterium sp. strain Viet1 (PCE: εC = 16.7) gave the highest enrichment factor for PCE.

Dechlorination and Detoxification of 1,2,3,4,7,8-Hexachlorodibenzofuran by a Mixed Culture Containing Dehalococcoides ethenogenes Strain 195

Toxic polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) with chlorines substituted at the lateral 2, 3, 7, and 8 positions are of great environmental concern. We investigated the dechlorination of 1,2,3,4,7,8-hexachlorodibenzofuran (1,2,3,4,7,8-HxCDF) and 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD) by a mixed culture containing Dehalococcoides ethenogenes strain 195. The 1,2,3,4,7,8-HxCDF was dechlorinated to 1,3,4,7,8-pentachlorodibenzofuran and 1,2,4,7,8-pentachlorodibenzofuran and further to two tetrachlorodibenzofuran congeners, which were identified as 1,3,7,8-tetrachlorodibenzofuran and 1,2,4,8-tetrachlorodibenzofuran. Because no 2,3,7,8-substituted congeners were formed as dechlorination products from 1,2,3,4,7,8-HxCDF, this dechlorination represents a detoxification reaction. Tetrachloroethene (PCE) and 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) were added as additional halogenated substrates to enhance the degree of 1,2,3,4,7,8-HxCDF dechlorination. The 1,2,3,4-TeCB enhanced the extent of dechlorination of 1,2,3,4,7,8-HxCDF approximately 3-fold compared to PCE or no additional substrate amendment. No dechlorination products were detected from OCDD. Bioremediation of PCDD/Fs by bacterial reductive dechlorination should address the pathway of dechlorination to ensure detoxification.

Stable Carbon Isotope Fractionation of Chloroethenes by Dehalorespiring Isolates

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 structure differences among the chloroethenes. For TCE reduction, S. multivorans and D. restrictus strain PER-K23 exhibited enrichment factors of -16.4 +/- 1.5 and -3.3 +/- 0.3, respectively. While all of the organisms studied here utilize RDases that require corrinoid cofactors, the biotic TCE enrichment factors varied widely from those reported for the abiotic cobalamin-catalyzed reaction, indicating that additional factors affect the extent of fractionation in these biological systems. The enrichment factors measured for the Dehalococcoides-containing enrichment culture did not match well with those from any of the isolates, demonstrating the inherent difficulties in predicting fractionation factors of undefined communities. Although compound-specific isotope fractionation is a powerful tool for evaluating the progress of in situ bioremediation in the field, given the wide range of enrichment factors associated with functionally similar and phylogenetically diverse organisms, caution must be exercised when applying enrichment factors for the interpretation of dechlorination data.

Temporal expression of respiratory genes in an enrichment culture containing Dehalococcoides ethenogenes.

Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (+/- 7.5)-, 23 (+/- 6.7)-, 16 (+/- 3.3)-, and 13 (+/- 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.

Increasing biohydrogen production by metabolic engineering

Two types of enzymes can catalyze the reduction of protons to H 2, namely nitrogenase and hydrogenase. Although much progress has been made in the elucidation of gene expression, structure and regulation of these key enzymes, no practical and economically competitive process for the continuous production of biological H 2 (biohydrogen) has, as yet, been put on the market. One of the difficulties is due to the fact that H 2 output represents an energy loss for the cell and that microbial metabolic network has evolved for rationalization of energy use and optimization of specific growth rate. The study of the physiology of genetically modified photosynthetic microorganisms has shown that electron flux could be redirected to the bidirectional hydrogenase in a ndhB mutant of Synechocystis and that a change in carbon metabolism in mutants of Rhodobacter capsulatus unable to grow photoautotrophically could affect the flow of reducing equivalent from organic substrates to nitrogenase. Increasing the flux through an existing pathway or redirecting enzyme-catalyzed reactions is an approach referred to as metabolic engineering. Various “naturally engineered” organisms are found in Nature. An example is provided by the bacterium Dehalococcoides ethenogenes, which is the only bacterium known to reductively dechlorinate the ground water pollutants tetrachloroethene and trichloethene to ethene. D. ethenogenes exhibits an unusual metabolic specialization; it uses only H 2 as an electron donor and chlorinated compounds as electron acceptors to support growth. In accordance, the sequence of its genome has revealed the presence of five hydrogenase complexes.

Transcription and mass‐spectroscopic proteomic studies of electron transport oxidoreductases in Dehalococcoides ethenogenes

Besides 19 potential reductive dehalogenase genes, the genome of Dehalococcoides ethenogenes strain 195 contains over 60 genes annotated as encoding oxidoreductases, including five hydrogenase complexes and a formate dehydrogenase (Fdh). Using quantitative reverse transcriptase polymerase chain reaction, we found that genes encoding a periplasmic Hup hydrogenase and the Fdh were the most highly expressed in batch-grown pure cultures, in which the H2 partial pressure was >0.1 atm, and in butyrate/tetrachloroethene-mixed cultures, in which H2 partial pressures were 10(-4)-10(-5) atm. Shotgun electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry were used to identify multiple peptides in pure culture membrane-enriched fractions matching several highly expressed respiratory enzymes, including three hydrogenases, two reductive dehalogenases, Fdh and DET1407, a 105.5-kDa protein we propose to be part of an S-layer cell wall. Both transcript and mass spectrometric approaches indicated that the putative Fdh was an important oxidoreductase in these cells; nevertheless, D. ethenogenes cultures could not use formate as an electron donor for reductive dechlorination. Analysis of the gene encoding the large subunit of Fdh indicated that while it was related to other Fdh proteins, its sequence encodes serine rather than cysteine or selenocysteine at a critical position, casting doubt on its function. Overall, genomic and proteomic approaches have provided novel insights into the metabolism of this difficult to culture organism.

A Modeling Study and Implications of Competition between Dehalococcoides ethenogenes and Other Tetrachloroethene- Respiring Bacteria

Bioaugmentation with cultures containing Dehalococcoides strains that dechlorinate cis-1,2-dichloroethene (cDCE) to ethene is often recommended at sites where indigenous populations dechlorinate tetrachloroethene (PCE) to cDCE. In these cases, Dehalococcoides populations may have to compete with other dehalorespirers for chlorinated ethenes and/or electron donors. A continuous-flow stirred tank reactor model was used to describe the substrate interactions in three conceptual models of competition between PCE-respiring populations under natural attenuation and engineered bioremediation scenarios. Model simulations were used to evaluate the effects of other chlorinated ethene respirers on substrate utilization by, and growth of, a Dehalococcoides strain (Dhc. ethenogenes strain 195) and identify the key factors influencing the outcome of competition among different dehalorespirers. The results suggest that, under natural attenuation conditions, Dhc. ethenogenes is unlikely to be the dominant population if a dehalorespirer that can compete for limiting amounts of reducing equivalents is present. Engineered bioremediation conditions resulted in greater enrichment of Dhc. ethenogenes than of competing dehalorespirers. Under several conditions, Dhc. ethenogenes coexisted with a PCE-to-cDCE (or PCE-to-TCE) dehalorespirer, primarily by functioning as a cDCE- (or TCE)-to-ethene dechlorinating specialist. From a bioremediation standpoint, maintenance of multiple dehalorespiring specialists appears ideal because it may result in the fastest and most extensive chlorinated ethene transformations. Thus, to improve our ability to successfully implement bioremediation, it may be helpful to characterize the indigenous PCE-to-cDCE respiring populations and the nature and distribution of electron donors used by these dehalorespirers at contaminated sites. Further understanding of these interactions requires more accurate information on the kinetics of known dehalorespiring populations.

Bacterial diversity of an acidic Louisiana groundwater contaminated by dense nonaqueous-phase liquid containing chloroethanes and other solvents

Bacterial concentration and diversity was assessed in a moderately acidic (pH 5.1) anaerobic groundwater contaminated by chlorosolvent-containing DNAPL at a Superfund site located near Baton Rouge, Louisiana. Groundwater analysis revealed a total aqueous-phase chlorosolvent concentration exceeding 1000 mg L(-1), including chloroethanes, vinyl chloride, 1,2-dichloropropane, and hexachloro-1,3-butadiene as the primary contaminants. Direct counting of stained cells revealed more than 3 x 10(7) cells mL(-1) in the groundwater, with 58% intact and potentially viable. Universal and 'Dehalococcoides'-specific 16S rRNA gene libraries were created and analyzed. Universal clones were grouped into 18 operational taxonomic units (OTUs), which were dominated by low-G+C Gram-positive bacteria (62%) and included several as yet uncultured or undescribed organisms. Several unique 16S rRNA gene sequences closely related to Dehalococcoides ethenogenes were detected. Anaerobically grown isolates (168 in total) were also sequenced. These were phylogenetically grouped into 18 OTUs, of which only three were represented in the clone library. Phylogenetic analysis of isolates and the clone sequences revealed close relationships with dechlorinators, fermenters, and hydrogen producers. Despite acidic conditions and saturation or near-saturation chlorosolvent concentrations, the data presented here demonstrate that large numbers of novel bacteria are present in groundwater within the DNAPL source zone, and the population appears to contain bacterial components necessary to carry out reductive dechlorination.

Characterization of the Biosynthetic Pathway of Glucosylglycerate in the Archaeon Methanococcoides burtonii

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50 degrees C. The maximal activity of GpgP was at 50 degrees C with GPG as the substrate and at 60 degrees C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

Genome sequence of the chlorinated compound–respiring bacterium Dehalococcoides species strain CBDB1

Dehalococcoides species are strictly anaerobic bacteria, which catabolize many of the most toxic and persistent chlorinated aromatics and aliphatics by reductive dechlorination and are used for in situ bioremediation of contaminated sites. Our sequencing of the complete 1,395,502 base pair genome of Dehalococcoides strain CBDB1 has revealed the presence of 32 reductive-dehalogenase-homologous (rdh) genes, possibly conferring on the bacteria an immense dehalogenating potential. Most rdh genes were associated with genes encoding transcription regulators such as two-component regulatory systems or transcription regulators of the MarR-type. Four new paralog groups of rdh-associated genes without known function were detected. Comparison with the recently sequenced genome of Dehalococcoides ethenogenes strain 195 reveals a high degree of gene context conservation (synteny) but exceptionally high plasticity in all regions containing rdh genes, suggesting that these regions are under intense evolutionary pressure.

Genome Sequence of the PCE-Dechlorinating BacteriumDehalococcoides ethenogenes

Dehalococcoides ethenogenes is the only bacterium known to reductively dechlorinate the groundwater pollutants, tetrachloroethene (PCE) and trichloroethene, to ethene. Its 1,469,720-base pair chromosome contains large dynamic duplicated regions and integrated elements. Genes encoding 17 putative reductive dehalogenases, nearly all of which were adjacent to genes for transcription regulators, and five hydrogenase complexes were identified. These findings, plus a limited repertoire of other metabolic modes, indicate that D. ethenogenes is highly evolved to utilize halogenated organic compounds and H2. Diversification of reductive dehalogenase functions appears to have been mediated by recent genetic exchange and amplification. Genome analysis provides insights into the organism's complex nutrient requirements and suggests that an ancestor was a nitrogen-fixing autotroph.

Long-term tetrachlorethene degradation sustained by endogenous cell decay

The role of endogenous decay in the reductive dechlorination of tetrachlorethene (PCE) was studied in a sand column containing an anaerobic microbial consortium that included Dehalococcoides ethenogenes. The column consortium had continuously transformed PCE to vinyl chloride (<5%) and ethene (>95%) with the addition of ethanol and yeast extract for a 3 year period. The column was operated for a further period of 1 year with the addition of 60 µM PCE only. In the absence of external electron donors, PCE dechlorination in the column initially slowed but continued at a relatively constant rate for 1 year. The conversion to vinyl chloride (VC) and ethene (ETH) was significantly reduced with cis-dichloroethylene (cis-DCE) becoming the major (90%) end product of dechlorination. Microcosm studies, with no external electron donor added, indicated that reductive dechlorination was sustained by reducing equivalents derived from endogenous decay products. Measures of cellular adenosine triphosphate (ATP) along the length of the column indicated that even after 1 year of only PCE addition, an active biological community was sustained. Key words: tetrachloroethene (PCE), ethanol, anaerobic biodegradation, endogenous decay, column study.

Enrichment of anaerobic polychlorinated biphenyl dechlorinators from sediment with iron as a hydrogen source

Little is known about anaerobic polychlorinated biphenyl (PCB) dechlorination, although it is believed that some microorganisms are capable of respiring PCBs, gaining energy for growth from PCB dechlorination. If this is the case, the amendment of appropriate electron donors to contaminated sediment should stimulate dechlorination. The effect of elemental iron (Fe0) addition, an easily amended electron donor, on the microbial dechlorination of the PCB congeners 3,4,5-trichlorobiphenyl (3,4,5-CB) and 2,2',3,4,4',5,5'-heptachlorobiphenyl (2,2',3,4,4',5,5'-CB) was investigated in microcosms containing estuarine sediment from Baltimore Harbor. Results showed that the addition of 0.1 g Fe0/g sediment reduced the lag time for removal of doubly flanked para chlorines by approximately 100 days. Because Fe0 is a source of cathodic hydrogen (H2), the effect of direct H2 addition to sediment microcosms was also tested. The addition of 0.001 atm H2 in the headspace generated the same dechlorination activity and reduction in lag time as the addition of 0.1g Fe0/g. Higher concentrations of Fe0 or H2 increased the lag prior to dechlorination. Additional results showed that an alkaline pH (> or = 7.5), high [Fe2+] (3.3 g/L), or HS- (0.1 mg/L total sulfide) inhibited dechlorination. Elevated concentrations of Fe2+, OH-, and HS- are products of Fe0 oxidation or increased microbial activity (methanogenesis, homoacetogenesis, and sulfate reduction), both of which would result from the amendment of large quantities of Fe0 or H2 to sediment. This research shows that not only can PCB dechlorination be stimulated through the addition of electron donor, but implies that the dechlorinators are enriched by the continuous addition of low concentrations of H2, similar to other known dechlorinators, such as the dehalorespirer Dehalococcoides ethenogenes. These results suggest that the direct addition of controlled amounts of Fe0 to sediments may be an effective remediation tool to reduce the lag period prior to dechlorination at PCB-impacted sites. They also suggest that PCB dechlorinators may be enriched using techniques similar to those used with known dehalorespirers.