Grantee Research Project Results
Final Report: Characterization and Applications of Reductive Dehalogenase Genes in Enhancement and Monitoring of Biodegradation of Chlorinated Pollutants
EPA Grant Number: R830250Title: Characterization and Applications of Reductive Dehalogenase Genes in Enhancement and Monitoring of Biodegradation of Chlorinated Pollutants
Investigators: Tiedje, James M. , Davis, John M.G. , Hashsham, Syed
Institution: Michigan State University
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2002 through August 31, 2004
Project Amount: $249,842
RFA: Futures Research in Natural Sciences (2001) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Land and Waste Management , Hazardous Waste/Remediation
Objective:
Chlorinated compounds including chlorophenols, chloroethylenes (tetrachloroethene [PCE], trichloroethene [TCE], cis-dichloroethene [DCE], vinyl chloride [VC], and 1,1,1-trichloroethane (TCA) are major environmental pollutants. In anaerobic environments, chlorinated compounds have the potential to be dechlorinated by bacteria as a terminal electron acceptor in an alternate form of respiration known as halorespiration. The process of halorespiration and the distribution and ecology of halorespiring bacteria are poorly understood. These bacteria are potentially important for in situ bioremediation of chlorinated compounds, but it is difficult to predict the potential for decontamination when the distribution and function of the genes are not known. We proposed to develop a DNA microarray of dehalogenase genes to detect and monitor dehalogenase potential and activity at contaminated sites. The objectives of the research project were to: (1) clone and sequence reductive dehalogenase genes from bacteria that perform reductive dehalogenation of different pollutant chemicals; (2) isolate and characterize microbes that dechlorinate TCA (a pollutant at more than half of the Superfund sites) for which biodegradation is not yet discovered and to isolate the responsible dehalogenase; and (3) build a DNA microarray using these and our previously cloned dehalogenase genes as probes and evaluate the array's ability to detect specific dehalogenase genes in contaminated sites. We tested if the microarray can be used to detect messenger RNA molecules transcribed in response to exposure of dehalogenators to various chlorinated compounds. This would aid the assignment of function to those dehalogenase genes of unknown function. The assignment of function, discovery of new functions, and understanding of the ecology of these organisms would aid in the removal of these pollutants from the environment.
Summary/Accomplishments (Outputs/Outcomes):
TCA is a ubiquitous environmental pollutant because of its widespread use as an industrial solvent, its improper disposal, and its substantial emission to the atmosphere. We report the isolation of an anaerobic bacterium, strain TCA1, that reductively dechlorinates TCA to 1,1-dichloroethane (DCA) and chloroethane (CA); strain TCA1 required hydrogen (H2) as an electron donor and TCA as an electron acceptor for growth indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that strain TCA1 is related to gram-positive bacteria with a low DNA G+C content and its closest relative is Dehalobacter restrictus, an obligate H2-oxidizing, chloroethene-respiring bacterium. We also identified a reductive dehalogenase (RDase) gene in TCA1 that resembled PCE RDase gene of Desulfitobacterium sp. Y51.
The strictly anaerobic bacterium Desulfitobacterium hafniense DCB-2 grows by pyruvate fermentation and respiration using a wide range of electron acceptors including sulfur compounds, chlorinated compounds, and oxidized metals. This metabolic agility makes D. hafniense DCB-2 useful in dechlorination, metal reduction, and bioremediation studies. The sequence of the genome has been determined and seven open reading frames similar to the cprA (chlorophenol RDase) gene of D. dehalogenans have been detected. To identify the RDase genes induced during alternate respiration with 3-chloro-4-hydroxybenzoate (3C4HBA), 3,5-dichlorophenol (DCP), or ortho-bromophenol (o-BP), Xeotron® microarrays of the genome of DCB-2 were prepared along with a total of 20 unique probes for known and suspected RDase genes from other dehalogenators. Competitive hybridization of cDNA prepared from cultures grown by pyruvate fermentation and three dehalorespiration conditions indicated that three RDase genes (designated as MENN, MFRS, and MSGV) were induced by 3C4HBA, and two genes (MSSA and VKMN) were induced by both DCP and o-BP. Also induced were genes within putative RDase operons, ABC transporter genes, and genes involved in electron transport systems. Genes expressed uniquely or commonly by the substrates were identified, which suggests the presence of branched metabolic pathways. Probes for the 20 RDase genes were highly specific with one exception. Reverse Transcription-PCR targeting the seven cprA homologs revealed the same patterns of RDase gene expression. Northern hybridization assays targeting selected RDase genes showed a single mRNA species transcribed from MENN (1.8 kb) and VKMN (2 kb) that is long enough to contain genetic information for two linked genes, cprA and cprB (docking protein gene). Northern hybridization, however, was not adequate as confirmation of the microarray results because of the variable levels of cross-hybridization between the phylogenetically related RDase genes. We also cloned six of the seven RDase genes (except for MKGL gene) in Escherichia coli with modifications in their promoters and ribosome binding sites to facilitate their gene expression in gram-negative host cells. Although their protein products were observed in the E. coli clones, functional activity of the proteins remains to be seen.
Conclusions:
Anaerobic bacteria that are able to conserve metabolic energy from the dechlorination of chlorinated compounds such as chlorophenols, chloroethylenes (PCE, TCE, DCE, VC), and TCA have gained a lot of attention because of their role in bioremediation of contaminated sites and the novel respiration pathways they possess. We discovered and isolated an anaerobic dehalorespiring Dehalobacter that couples reductive dechlorination of TCA to growth, along with identification of a potential RDase gene. This discovery will enhance a better understanding of the physiology and phylogeny of dehalorespiring bacteria and also will provide a strategy for bioremediation of TCA in soils and ground waters. Multiple RDase genes of D. hafniense DCB-2 were cloned and expressed in E. coli allowing us to study their biochemical properties and potential application as biocatalysts for transformation of the pollutants in situ. Microarray studies of the genome of DCB-2 including other known RDase genes provided us not only with a better understanding of the function and regulation of the RDase genes, but also with a basis for the detection of RDase genes specific for particular pollutants at contaminated sites.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 7 publications | 5 publications in selected types | All 3 journal articles |
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Denef VJ, Park J, Rodrigues JLM, Tsoi TV, Hashsham SA, Tiedje JM. Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environmental Microbiology 2003;5(10):933-943. |
R830250 (Final) |
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Hashsham SA, Wick LM, Rouillard JM, Gulari E, Tiedje JM. Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs. Biosensors & Bioelectronics 2004;20(4):668-683. |
R830250 (Final) |
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Sun B, Griffin BM, Ayala-del-Rio HL, Hashsham SA, Tiedje JM. Microbial dehalorespiration with 1,1,1-trichloroethane. Science 2002;298(5595):1023-1025. |
R830250 (Final) |
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Supplemental Keywords:
desulfitobacterium, dehalospirillum, dehalobacter, dehalococcoides, desulfomonile, desulfovibrio, perchloroethylene, trichloroethylene, chloropheno, chlorobenzoate,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Restoration, Environmental Monitoring, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Futures, Exp. Research/future, dechlorination, biodiversity, conservation, contaminant uptake, ecological pollutants, exploratory research, reductive dehalogenase genes, environmental rehabilitation, environmental stressRelevant Websites:
None.
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.