Browsing by Author "Gossett, James M."

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Microcosm Assessment of a DNA Probe Applied to Aerobic Degradation of cis-1,2-Dichloroethene by Polaromonas sp. Strain JS666
(Wiley, 2010-05) Giddings, Clolle G.S.; Jennings, Laura K.; Gossett, James M.
A molecular biological tool based on an organism-specific DNA sequence does not necessarily indicate in situ activity but serves important functions of evaluating the potential for biodegradation and mapping the distribution of an organism. Currently, DNA-based probes are accepted as evaluative tools for site assessment. However, these techniques are far from standardized, and information on precision is usually lacking. Here, we present the development and evaluation of a DNA probe for Polaromonas sp. strain JS666, a bacterium that couples growth to aerobic oxidation of cis-1,2-dichloroethene (cDCE), and is therefore a promising candidate for bioaugmentation at sites where cDCE has accumulated in aerobic zones. The DNA probe was used in conjunction with quantitative polymerase chain reaction to track the abundance of JS666 in microcosms. This series of studies has allowed explicit resolution of the accuracy and precision of the probe and its correlation with variations in microcosm performance. We determined that the method is sufficient to monitor distribution of JS666 at bioaugmented sites. We found within environmental, mixed cultures, that the DNA target does not persist long after cell death, demonstrating that positive result from the probe is a strong indicator that degradation can occur in suitable environmental conditions. Finally, in the absence of suspected predation, the probe accurately and precisely tracks growth. Collectively, the studies appear to validate the utility of the molecular probe for site assessment in a bioaugmentation context.
Proteomic and transcriptomic analyses reveal genes upregulated by cis-Dichloroethene in Polaromonas sp. strain JS666
(2009-04) Jennings, Laura K.; Chartrand, Michelle M.; Lacrampe-Couloume, Georges; Lollar, Barbara S.; Spain, Jim C.; Gossett, James M.
Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from â€“17.4 to â€“22.4â€° . Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C=C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.
Proteomic and Transcriptomic Analyses Reveal Genes Upregulated by cis-Dichloroethene in Polaromonas sp. Strain JS666
(American Society for Microbiology, 2009-06) Jennings, Laura K.; Chartrand, Michelle M. G.; Lacrampe-Couloume, Georges; Sherwood Lollar, Barbara; Spain, Jim C.; Gossett, James M.
Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from −17.4 to −22.4‰. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C═C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.
Proteomic and Transcriptomic Analyses Reveal Genes Upregulated by cis-Dichloroethene in Polaromonas sp. Strain JS666
(American Society for Microbiology, 2009-06) Jennings, Laura; Chartrand, Michelle; Lacrampe-Couloume, Georges; Sherwood Lollar, Barbara; Spain, Jim C.; Gossett, James M.
Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from −17.4 to −22.4‰. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C═C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.