ABSTRACT: Groundwater dechlorinating microbial communities from synthetic mineral medium in Toronto, Ontario, Canada - Site contaminated with chlorinated ethenes metagenome
Project description:Four stable and robust TCE-dechlorinating microbial communities were enriched from TCE-contaminated groundwater under four different conditions exploring two parameters, high and low methanogenic activity (Meth and NoMeth), with and without vitamin B12 supplement (MethB12 and NoMethB12, Meth and NoMeth, respectively). Identical amounts of lactate (2.7 mmol) and TCE (20 μl) were supplied as electron donor and electron acceptor. All four cultures were capable of reductively dechlorinating TCE to VC and ethene. Genomic DNA of the four enrichments was applied on a quad-Dhc-genome microarray in order to characterize the gene content of Dehalococcoides species present in the four enrichments
Project description:HiSpOD is a new efficient functional microarrays probe design algorithm especially dedicated for the microbial ecology and environmental studies. It was used to design 3392 probes targeting 21 genes involved in chlorinated solvent biodegradation pathways and synthesized on a nimblegen microarray. In order to test the probe specificity, the microarray was firstly hybridized to 6 M-BM-5g of labelled aRNA from sheep rumen content (background aRNA). Secondly, hybridization of 1011 copies of labelled aRNA derived from in vitro transcription of three synthetic genes (mmoC, vcrA and tceA) and mixed with 6 M-BM-5g of the same complex background material were performed to test their sensibility. Finally, the expression analysis of a contaminated groundwater sample was performed. A 3 chip study was realized. The first one is a negative control performed with a complex background material (labelled antisense mRNA from sheep rumen content). The second one is a positive control realized with labelled antisense RNA derived from in vitro transcription of three synthetic genes mixed the same complex background material. The third consists in the hybridization of antisense mRNA retrieved from a contaminated groundwater. Each probe (3392) was synthetized in triplicate, and a total of 8,863 random probes was used to determine the background noise.
Project description:A functional microarray targeting 24 genes involved in chlorinated solvent biodegradation pathways has been developed and used to monitor the gene expression in a contaminated site (site B) under ERD (enhanced reductive dechlorination) treatment. The microarray format provided by NimbleGen and used in this study is 12x135K. 4 µg of labelled antisense mRNA from 3 groundwater samples were hybridized on the microarray.
Project description:A functional microarray targeting 24 genes involved in chlorinated solvent biodegradation pathways has been developed and used to monitor the gene diversity present in four trichloroethylene (TCE) contaminated sites under ERD (enhanced reductive dechlorination) treatment. The microarray format provided by NimbleGen and used in this study is 12x135K. 2 µg of labelled gDNA from 30 groundwater samples were hybridized on the microarrays.
Project description:Organohalide-respiring Dehalococcoidia bacteria are one of the few microorganisms capable of transforming chlorinated solvents to benign ethene in anoxic environments. The tceA gene found in these bacteria, coding the trichloroethene-dechlorinating RDase TceA, is frequently detected in contaminated groundwater but not recognized as a biomarker for vinyl chloride detoxification. Here, we demonstrate that the tceA-carrying Dehalococcoides mccartyi (Dhc) strains FL2 and 195 grow with VC as electron acceptor when sufficient vitamin B12 is provided. Global proteomic profiling confirmed the predominant TceA expression in VC-grown Dhc FL2 cells, providing a line of evidence for the implication of TceA in respiratory VC reductive dechlorination.
Project description:Four stable and robust TCE-dechlorinating microbial communities were enriched from TCE-contaminated groundwater under four different conditions exploring two parameters, high and low methanogenic activity (Meth and NoMeth), with and without vitamin B12 supplement (MethB12 and NoMethB12, Meth and NoMeth, respectively). Identical amounts of lactate (2.7 mmol) and TCE (20 M-NM-<l) were supplied as electron donor and electron acceptor. All four cultures were capable of reductively dechlorinating TCE to VC and ethene. Genomic DNA of the four enrichments was applied on a quad-Dhc-genome microarray in order to characterize the gene content of Dehalococcoides species present in the four enrichments The genomic DNA of four enrichment cultures completely dechlorinated TCE to VC and ethene was used on the microarray to query Dehalococcoides species present in the mixed cultures.
Project description:HiSpOD is a new efficient functional microarrays probe design algorithm especially dedicated for the microbial ecology and environmental studies. It was used to design 3392 probes targeting 21 genes involved in chlorinated solvent biodegradation pathways and synthesized on a nimblegen microarray. In order to test the probe specificity, the microarray was firstly hybridized to 6 µg of labelled aRNA from sheep rumen content (background aRNA). Secondly, hybridization of 1011 copies of labelled aRNA derived from in vitro transcription of three synthetic genes (mmoC, vcrA and tceA) and mixed with 6 µg of the same complex background material were performed to test their sensibility. Finally, the expression analysis of a contaminated groundwater sample was performed.
Project description:A functional microarray targeting 24 genes involved in chlorinated solvent biodegradation pathways has been developed and used to monitor the gene expression in a contaminated site (site B) under ERD (enhanced reductive dechlorination) treatment. The microarray format provided by NimbleGen and used in this study is 12x135K. 4 M-BM-5g of labelled antisense mRNA from 3 groundwater samples were hybridized on the microarray. A 3-chip study was performed, each corresponding to hybridization with 4 M-BM-5g of labelled antisense mRNA retrieved from a monitoring well of a contaminated site (site B). Each probe (760nt) on the microarray was synthesized in eight replicates, and a total of 5,707 random probes was used to determine the background noise. Groundwater samples were collected from a contaminated site (site B) from three monitoring wells (P1, P2 and P3). P1: well located upstream to the contamination source. P2: well in the contamination source. P3 : well located downstream to the contamination source.
