Showing papers on "Dehalococcoides published in 2005"

Genome Sequence of the PCE-Dechlorinating Bacterium Dehalococcoides ethenogenes

Abstract: 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.

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

Abstract: 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.

Isolation and characterization of Dehalococcoides sp. strain FL2, a trichloroethene (TCE)- and 1,2-dichloroethene-respiring anaerobe.

Abstract: A strictly anaerobic bacterium was isolated from tetrachloroethene (PCE)-to-ethene dechlorinating microcosms established with river sediment without prior exposure to chlorinated solvents. The isolation procedure included the addition of 2-bromoethanesulfonate to select against methanogenic archaea, >50 consecutive 1-2% (v/v) transfers to reduced mineral salts medium amended with trichloroethene (TCE), acetate, and hydrogen, the addition of ampicillin, and the dilution-to-extinction principle. Culture-dependent and 16S rRNA gene-targeted approaches suggested culture purity. Microscopic examination revealed a homogeneous culture of an organism with a distinct, disc-shaped morphology. The isolate shared >99% 16S rRNA gene sequence similarity with members of the Pinellas group of the Dehalococcoides cluster, and was designated Dehalococcoides sp. strain FL2. Strain FL2 could be propagated with TCE, cis-1,2-dichloroethene (cis-DCE), or trans-DCE as the electron acceptors, acetate as the carbon source, and hydrogen as the electron donor in defined, completely synthetic medium. No other growth-supporting redox couples were identified. Trichloroethene, cis-DCE and trans-DCE were dechlorinated at rates of 27.5, 30.4 and 18.8 micromol l-1 day-1 respectively. Quantitative real-time polymerase chain reaction (PCR) with a fluorescently labelled linear hybridization probe confirmed growth with these electron acceptors, and suggested that strain FL2 captures energy from both the TCE-to-cis-DCE and 1,2-DCE-to-VC dechlorination steps. Tetrachloroethene and vinyl chloride (VC) were slowly and cometabolically dechlorinated in the presence of a growth-supporting chloroethene, but ethene formation was incomplete, even after prolonged incubation. At room temperature, strain FL2 grew with a doubling time of 2.4 days, and yielded 166.1+/-10.2 mg of protein per mole of chloride released. In the presence of excess electron acceptor, strain FL2 consumed hydrogen to a concentration of 0.061+/-0.016 nM. Dechlorination ceased following the addition of 0.5 mM sulfite, whereas sulfate (10 mM) and nitrate (5 mM) had no inhibitory effects.

Multiple reductive-dehalogenase-homologous genes are simultaneously transcribed during dechlorination by dehalococcoides-containing cultures

Abstract: Degenerate primers were used to amplify 14 distinct reductive-dehalogenase-homologous (RDH) genes from the Dehalococcoides-containing mixed culture KB1. Most of the corresponding predicted proteins were highly similar (97 to >99% amino acid identity) to previously reported Dehalococcoides reductive dehalogenases. To examine the differential transcription of these RDH genes, KB1 was split into five subcultures amended with either trichloroethene, cis-1,2-dichloroethene, vinyl chloride, 1,2-dichlorethane, or no chlorinated electron acceptor. Total RNA was extracted following the onset of reductive dechlorination, and RDH transcripts were reverse transcribed and amplified using degenerate primers. The results indicate that the transcription of RDH genes requires the presence of a chlorinated electron acceptor, and for all treatments, multiple RDH genes were simultaneously transcribed, with transcripts of two of the genes being present under all four electron-accepting conditions. Two of the transcribed sequences were highly similar to reported vinyl chloride reductase genes, namely, vcrA from Dehalococcoides sp. strain VS and bvcA from Dehalococcoides sp. strain BAV1. These findings suggest that multiple RDH genes are induced by a single chlorinated substrate and that multiple reductive dehalogenases contribute to chloroethene degradation in KB1.

Phylogenetic Characterization of a Polychlorinated-Dioxin- Dechlorinating Microbial Community by Use of Microcosm Studies

Abstract: Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.

Enrichment, cultivation, and detection of reductively dechlorinating bacteria

Abstract: Strategies and procedures for enriching, isolating, and cultivating reductively dechlorinating bacteria that use chloroorganic compounds as metabolic electron acceptors from environmental samples are described. Further, nucleic acid-based approaches used to detect and quantify dechlorinator (i.e., Dehalococcoides)-specific genes are presented.

Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

Abstract: Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.

Transcriptional Expression of the tceA Gene in a Dehalococcoides-Containing Microbial Enrichment

Abstract: Dynamic changes in the transcriptional expression of the tceA gene, which encodes a trichloroethene reductive dehalogenase, were characterized in a Dehalococcoides-containing microbial enrichment culture. Expression was quantified by real-time PCR as the number of tceA transcripts per tceA gene. Expression of tceA increased 40-fold after chlorinated ethene-starved cells were exposed to trichloroethene (TCE), cis-dichloroethene (DCE), or 1,1-DCE but did not increase after exposure to tetrachloroethene or vinyl chloride. Surprisingly, tceA expression also increased 30-fold after cellular exposure to the nonmetabolic substrate trans-DCE, indicating that expression of tceA is induced by both growth-supporting and non-growth-supporting chlorinated ethenes. Additional experiments revealed that the level of tceA expression was independent of the concentration of chlorinated ethenes (sum concentrations of TCE and DCEs of 2.2 to 333 μM), the concentration of the electron donor hydrogen (concentrations of 12 nM to 17 μM), and the presence of alternate bacterial electron acceptors (5 mM concentrations of fumarate, sulfate, sulfite, thiosulfate, nitrate, or nitrite) but was highly dependent on incubation temperature.

A PCR-based specific assay reveals a population of bacteria within the Chloroflexi associated with the reductive dehalogenation of polychlorinated biphenyls.

Abstract: Polychlorinated biphenyls (PCBs) accumulate and persist in sediments posing a risk to human health and the environment. Highly chlorinated PCBs are reductively dechlorinated in anaerobic sediments and two bacteria, designated o-17 and DF-1, from a novel phylogenetic group that reductively dechlorinate PCBs have recently been identified. However, there is a paucity of knowledge about the distribution, diversity and ecology of PCB-dechlorinating bacteria due to difficulty in obtaining pure cultures and the lack of detection by universal PCR 16S rRNA gene primer sets in sediments. A specific PCR primer was developed and optimized for detection of o-17/DF-1 and other closely related bacteria in the environment. Using this primer set it was determined that bacteria of this group were enriched in sediment microcosms from Baltimore Harbour concurrent with active dechlorination of 2,2′,3,4,4′,5′-hexachlorobiphenyl. Additional 16S rRNA gene sequences that had high levels of similarity to described PCB dechlorinators were detected in sediments from the Elizabeth River tributary of Chesapeake Bay, which had confirmed PCB-dechlorinating activities. Phylogenetic comparison of these detected 16S rRNA gene sequences revealed a relatively diverse group of organisms within the dehalogenating Chloroflexi that are distinct from the Dehalococcoides spp. Results from this study indicate that reductive PCB dechlorination activity may be catalysed by a previously undescribed group of micro-organisms that appear to be prevalent in PCB-impacted sites.

Reductive dechlorination of tetrachloroethene to trans-dichloroethene and cis-dichloroethene by PCB-dechlorinating bacterium DF-1.

Abstract: Polychlorinated biphenyls (PCBs) and chlorinated ethenes (CEs) are known to pollute sediment, soil, and groundwater. The anaerobic dechlorination of these compounds is an integral part of their biodegradation in polluted environments. We report for the first time the dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) by bacterium DF-1. This PCB and chlorobenzene dechlorinating bacterium dechlorinated PCE to TCE, which was then converted into trans-1,2-dichloroethene (trans-DCE) and cis-1,2-dichloroethene (cis-DCE). The ratio of trans-DCE to cis-DCE produced by the culture had a range of 1.2-1.7. Bacterium DF-1 has been enriched in co-culture with a desulfovibrio-like microorganism. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA genes of the co-culture demonstrated that DF-1 was enriched during the dechlorination of PCE, PCB, and chlorobenzene. DF-1 was not detected in the absence of PCE dechlorination and the desulfovibrio-like organism, isolated in pure culture, did not dechlorinate PCE. This is the first identification of a microorganism capable of producing high amounts of trans-DCE from PCE and indicates that microorganisms such as DF-1 are a possible biological source of trans-DCE in the environment.

Phospholipid Furan Fatty Acids and Ubiquinone-8: Lipid Biomarkers That May Protect Dehalococcoides Strains from Free Radicals

Abstract: Dehalococcoides species have a highly restricted lifestyle and are only known to derive energy from reductive dehalogenation reactions. The lipid fraction of two Dehalococcoides isolates, strains BAV1 and FL2, and a tetrachloroethene-to-ethene-dechlorinating Dehalococcoides-containing consortium were analyzed for neutral lipids and phospholipid fatty acids. Unusual phospholipid modifications, including the replacement of unsaturated fatty acids with furan fatty acids, were detected in both Dehalococcoides isolates and the mixed culture. The following three furan fatty acids are reported as present in bacterial phospholipids for the first time: 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6), 9-(5-butyl-2-furyl)-nonanoate (Fu17:2ω5), and 8-(5-pentyl-2-furyl)-octanoate (Fu17:2ω6). The neutral lipids of the Dehalococcoides cultures contained unusually large amounts of benzoquinones (i.e., ubiquinones [UQ]), which is unusual for anaerobes. In particular, the UQ-8 content of Dehalococcoides was 5- to 20-fold greater than that generated in aerobically grown Escherichia coli cultures relative to the phospholipid fatty acid content. Naphthoquinone isoprenologues (MK), which are often found in anaerobically grown bacteria and archaea, were also detected. Dehalococcoides shows a difference in isoprenologue pattern between UQ-8 and MK-5 that is atypical of other bacteria capable of producing both quinone types. The difference in UQ-8 and MK-5 isoprenologue patterns strongly suggests a special function for UQ in Dehalococcoides, and Dehalococcoides may utilize structural modifications in its lipid armamentarium to protect against free radicals that are generated in the process of reductive dechlorination.

Biotransformation of Polychlorinated Dioxins and Microbial Community Dynamics in Sediment Microcosms at Different Contamination Levels

Abstract: Semi-anaerobic microcosms containing different levels of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed by seeding with different mass ratios of lake sediment and dioxin-contaminated soil and incubating with organic medium for 1 year. In all microcosms, PCDD/Fs were reduced as a first-order reaction with similar removal rate coefficients, and only trace amounts of less chlorinated congeners were produced as the intermediate and end products. This apparent complete dechlorination of PCDD/Fs seemed to be due to a combination of reductive dechlorination of PCDD/Fs and oxidative degradation of the dechlorinated products. Total cell counting, 16S rRNA gene clone library analyses and quinone profiling showed that the microcosms contained relatively constant total populations with members of the phyla Bacteroidetes, Firmicutes and Proteobacteria (especially “Deltaproteobacteria”) as the major constituents, independent of pollution levels. Quantitative real-time PCR with a specific primer set showed that the population density of “Dehalococcoides” and its phylogenetic relatives was highly correlated with the concentration of PCDD/Fs present. Some “Dehalococcoides” strains were isolated from the microcosms by repeated enrichment with chloroaromatics as the terminal electron acceptor. However, these isolates did not match with the major “Dehalococcoides”-related clones directly PCR-amplified. The results of this study suggest that PCDD/Fs in natural environments under given conditions are transformed with similar half-reduction rates independent of their concentrations, and a wide variety of “Dehalococcoides”-related bacteria play the primary role in this process.

Estimation of "Dehalococcoides" Populations in Lake Sediment Contaminated with Low Levels of Polychlorinated Dioxins

Abstract: Vertical profiles of polychlorinated dioxins and microbial biomass including "Dehalococcoides" populations in cores of sediment of Lake Suwa, Japan, were investigated. The core samples were analyzed in 3-cm intervals at 0-15 cm and 50 cm, where a sharp gradient of Eh from 5 to −110 mV with depth occurred. The concentration of polychlorinated dioxins was relatively constant at 0-15 cm, ranging from 7.6 to 8.3 ng (7.0-9.2 pg-TEQ [toxic equivalent]) g-1 dry wt, but decreased sharply at 50 cm. The total bacterial count was in the order of 108 to 109 g-1 dry wt, being highest at 3-6 cm and decreasing in the deeper sediment. A similar vertical profile was found for respiratory quinones with larger amounts of ubiquinones than menaquinones at 0-15 cm. Quantitative real-time PCR with a specific primer set showed that "Dehalococcoides" and its phylogenetic relatives occurred in the order of 104 g -1 (dry wt) at 0-12 cm but were absent at 50 cm. The amplified clones showed 91-100% similarity (mostly <94%) in sequence to a well-known dioxin-dechlorinating organism, "Dehalococcoides" sp. strain CBDB1. These results suggested that the surface sediment up to a depth of 12 cm provided favorable conditions for the growth and activity of both aerobic and anaerobic microorganisms including "Dehalococcoides". It is likely that a wide variety of "Dehalococcoides" and phylogenetic relatives thereof are omnipresent even in sediment with low levels of dioxins and play the primary role in dechlorinating organohalorides over a relatively wide range of Eh.

In-situ Bioremediation of a Chlorinated Solvent Residual Source in Unconsolidated Sediments and Bedrock Using Bioaugmentation

Abstract: The Caldwell Trucking Superfund Site is located in Essex County, NJ, and covers approximately 15 acres. Groundwater in glacial deposits as well as fractured basalt bedrock is contaminated with chlorinated ethenes, ethanes, and methanes up to approximately 4,000 ft (1,200 m) downgradient of the site. Trichloroethene (TCE ) concentrations in the source area were as high as 700,000 μg/L (about 60% of TCE solubility). Natural biodegradation is present over much of the site; however some areas, particularly near the source, appeared to be substrate limited. Following microcosm studies which demonstrated that complete degradation of the contaminants could be achieved, a comprehensive groundwater remedy was proposed that included bioremediation of the source area. A field pilot test of in-situ enhanced bioremediation in the source area was initiated in 2001. The layout included six nutrient injection wells screened in glacial deposits and fractured bedrock, and seven monitoring wells. Injection wells were bioaugmented with a culture of naturally occurring microorganisms (KB-1 Culture including Dehalococcoides ethenogenes) in March 2001. In over 30 months of operation, the system was optimized by adjustment of the amendment composition and the injection frequency. Gene probe techniques were used to verify initial and continued survival and propagation of the Dehalococcoides ethenogenes organisms.

Microbial reductive dehalogenation of vinyl chloride

Abstract: Compositions and methods are provided that relate to the bioremediation of chlorinated ethenes, particularly the bioremediation of vinyl chloride by Dehalococcoides-like organisms. An isolated strain of bacteria, Dehalococcoides sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.

Indication for horizontal transfer of reductive dehalogenase genes

Abstract: Tetra- (PCE) and trichloroethene (TCE) are major groundwater pollutants due to their extensive industrial use. Several anaerobic bacteria have been isolated using chloroethenes as terminal electron acceptor. Most of these bacteria dechlorinate PCE and TCE to cis-1,2-dichloroethene (cis-1,2-DCE). A few Dehalococcoides strains are able to dechlorinate cis-1,2-DCE and vinyl chloride (VC) to the ethene. Identification of the key enzyme, the reductive dehalogenase, has revealed a new class of enzymes containing a corrinoid and two iron-sulfur clusters as cofactors. The PCE reductive dehalogenase (PceAB) of Dehalobacter restrictus and Desulfitobacterium hafniense strain TCE1 showed 100% sequence identity which raised the question of a possible horizontal gene transfer. The flanking regions of the reductive dehalogenase genes (pceAB) revealed the presence of a composite transposon (named Tn-Dha1) in strain TCE1 bordered with two identical insertion sequences ISDha1 and containing besides the already characterized pceAB, two genes (pceCT) related to members of the o-chlorophenol reductive dehalogenase gene cluster of Desulfitobacterium dehalogenans. In contrast, only the pceABCT gene cluster (i.e. without the transposon structure) was present in Dehalobacter restrictus. Various circular molecules of Tn-Dha1 indicated that Tn-Dha1 is an active mobile genetic element. The genome of Dehalococcoides ethenogenes was shown to contain eighteen copies of putative reductive dehalogenase genes. A genomic signature of D. ethenogenes was obtained by calculating the frequency of 4-letter DNA words along the genome. Local disruptions of the genomic signature were observed, corresponding to DNA, which may have been acquired by horizontal gene transfer. Fifteen putative reductive dehalogenase genes were located in such atypical regions. Moreover, several genes encoding for recombinases (transposase, integrase) were found within these atypical regions, strongly indicating that these may have been acquired horizontally.