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Development of a Fluorescence-Activated Cell Sorting Method Coupled with Whole Genome Amplification To Analyze Minority and Trace Dehalococcoides Genomes in Microbial Communities

ABSTRACT: Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ~1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ~10% and ~1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

ORGANISM(S): Dehalococcoides mccartyi 195 Dehalococcoides mccartyi CBDB1 Dehalococcoides mccartyi VS Dehalococcoides mccartyi BAV1

PROVIDER: GSE54040 | GEO | 2015/01/31

SECONDARY ACCESSION(S): PRJNA234435

REPOSITORIES: GEO

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Development of a Fluorescence-Activated Cell Sorting Method Coupled with Whole Genome Amplification To Analyze Minority and Trace Dehalococcoides Genomes in Microbial Communities

Project description:Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 106 cells or M-bM-^HM-<1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing M-bM-^HM-<10% and M-bM-^HM-<1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low. The genomic DNA (gDNA) of each culture or sorted and amplifed DNA was analyzed in triplicate.

2015-01-31 | E-GEOD-54040 | biostudies-arrayexpress

Part2: Dual element (C/Cl) isotope analysis indicates distinct mechanisms of reductive dehalogenation of chlorinated ethenes and dichloroethane in Dehalococcoides mccartyi strain BTF08 with defined reductive dehalogenase inventories

Project description:Dehalococcoides mccartyi strain BTF08 has the unique property to couple complete dechlorination of tetrachloroethene and 1,2-dichloroethane to ethene with growth by using the halogenated compounds as terminal electron acceptor. The genome of strain BTF08 encodes 20 genes for reductive dehalogenase homologous proteins (RdhA) including those described for dehalogenation of tetrachloroethene (PceA, PteA), trichloroethene (TceA) and vinyl chloride (VcrA). Thus far it is unknown under which conditions the different RdhAs are expressed, what their substrate specificity is and if different reaction mechanisms are employed. Here we found by proteomic analysis from differentially activated batches that PteA and VcrA were expressed during dechlorination of tetrachloroethene to ethene, while TceA was expressed during 1,2-dichloroethane dehalogenation. Carbon and chlorine compound-specific stable isotope analysis suggested distinct reaction mechanisms for the dechlorination of (i) cis-dichloroethene and vinyl chloride and (ii) tetrachloroethene. This differentiation was observed independent of the expressed RdhA proteins. Differently, two stable isotope fractionation patterns were observed for 1,2-dichloroethane transformation, for cells with distinct RdhA inventories. Conclusively, we could link specific RdhA expression with functions and provide an insight into the apparently substrate-specific reaction mechanisms in the pathway of reductive dehalogenation in D. mccartyi strain BTF08.

2020-06-25 | PXD018595 | Pride

Dual element (C/Cl) isotope analysis indicates distinct mechanisms of reductive dehalogenation of chlorinated ethenes and dichloroethane in Dehalococcoides mccartyi strain BTF08 with defined reductive dehalogenase inventories

2020-06-10 | PXD018558 | Pride

Isolation of two new Dehalococcoides mccartyi strains with dissimilar dechlorination functions and their characterization by comparative genomics via microarray analysis

Project description:Microbial reductive dechlorination of trichloroethene (TCE) in groundwater often results in the accumulation of dichloroethenes (DCEs). Dehalococcoides mccartyi (Dhc) are the only known bacteria capable of dechlorination beyond DCE to non-toxic ethene. In this study, two newly isolated Dhc strains (11a and 11a5) with dissimilar functional abilities are described. Strain 11a reductively dechlorinates TCE, 1,1-DCE, cis-DCE, trans-DCE, and vinyl chloride (VC) to ethene, while strain 11a5 dechlorinates TCE and all three DCE isomers only to VC. Each of these dechlorination reactions are coupled to growth by these strains. The VC dechlorination rate of strain 11a occurs at a rate of 258 nmol per min per mg of protein, about two times faster than previously reported stains. Strain 11a possesses the vcrA gene while strain 11a5 contains the tceA gene. Strains 11a and 11a5 share 100% 16S rRNA gene sequence identity with previously sequenced Dhc strains BAV1 and CBDB1, placing it within the Pinellas subgroup, and 85.4% and 89.5% of all genes present in the CBDB1 and BAV1 genomes were detected in strains 11a and 11a5, respectively, using a custom-designed microarray targeting four sequenced Dhc strains. Genes that were not detected in strains 11a and 11a5 are mostly within the high plasticity regions or integrated elements of the sequenced strains. This study reports the functional description and comparative genomics of two additional Dhc isolates and provides evidence that the observed functional incongruence between the activity and core genome phylogenies of Dhc strains is likely driven by the horizontal transfer of key reductive dehalogenase-encoding genes.

2013-03-01 | GSE27611 | GEO

Immunomagnetic enrichment and flow cytometric isolation of breast cancer circulating tumor cells (CTCs) for genomic characterization

2012-12-31 | GSE27511 | GEO

Respiratory vinyl chloride reductive dechlorination to ethene in TceA-expressing Dehalococcoides mccartyi

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.

2020-10-09 | MSV000086254 | MassIVE

Bifunctional dehalogenases in Dehalococcoides mediate complete debromination of penta- and tetra-brominated diphenyl ethers

Project description:Polybrominated diphenyl ethers (PBDEs) are persistent, highly toxic, and widely distributed environmental pollutants. The microbial populations and functional reductive dehalogenases (RDases) responsible for PBDEs debromination in anoxic systems remain poorly understood, which confounds bioremediation of PBDE-contaminated sites. Here we report a PBDE-debrominating enrichment culture dominated by a previously undescribed Dehalococcoides mccartyi population. A D. mccartyi strain, designated TZ50, whose genome contains 25 putative RDase encoding genes was isolated from the debrominating enrichment culture. Strain TZ50 dehalogenated a mixture of penta- and tetra-BDE congeners (total BDEs 1.48 uM) to diphenyl ether within two weeks (0.58 uM Br- /d) via ortho- and meta- bromine elimination; strain TZ50 also dechlorinated tetrachloroethene (PCE) to vinyl chloride and ethene (260.2 M Cl- /d). Native-PAGE, proteomic profiling, and in vitro enzymatic activity assays implicated the involvement of three RDases in PBDEs and PCE dehalogenation. Two RDases, TZ50_0172 (PteATZ50) and TZ50_1083 (TceATZ50), were responsible for debromination of penta- and tetra-BDEs to di-BDE. TZ50_0172 and TZ50_1083 were also implicated in dechlorination of PCE to TCE and of TCE to vinyl chloride/ethene, respectively. The other expressed dehalogenase, TZ50_0090, was associated with debromination of di-BDE to diphenyl ether, but its role in PCE dechlorination was unclear. Comparatively few RDases are known to be involved in PBDE debromination and the identification of PteATZ50, TceATZ50, and TZ50_0090 provides additional information for evaluating debromination potential at contaminated sites. Moreover, the bifunctionality of the PteATZ50 and TceATZ50 in both PBDEs and PCE dehalogenation makes strain TZ50 a suitable candidate for remediation of co-contaminated sites.

2021-06-16 | PXD024983 | Pride

Immunomagnetic enrichment and flow cytometric isolation of breast cancer circulating tumor cells (CTCs) for genomic characterization

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