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TitleGeneCARD-FISH: Detection of tceA and vcrA reductive dehalogenase genes in Dehalococcoides mccartyi by fluorescence in situ hybridization
AbstractDue to the direct involvement in the biodegradation of chlorinated solvents, reductive dehalogenase genes (RDase) are considered biomarkers of the metabolic potential of different strains of Dehalococcoides mccartyi (. Dhc). This is known to be the only microbe able to completely reduce toxic chlorinated solvents to harmless ethene. In the last years, several Molecular Biological Tools (MBTs) have been developed to optimize the detectability of Dhc cells and/or the RDase genes, with particular attention to the most important indicators of ethene formation, namely tceA and vcrA genes. Despite qPCR has been indicated as the MBT of choice, the use of CARD-FISH recently demonstrated to provide a more accurate quantification of Dhc cells in a wide concentration range, overcoming the drawbacks of loosing nucleic acids during the preparation of the sample associated with qPCR. CARD-FISH assays usually target 16S rRNA and up to date no protocol able to discriminate different Dhc strains by detecting RDase genes has been developed. This study reports the first evidence of in situ detection of tceA and vcrA genes into Dhc cells by applying a new procedure named geneCARD-FISH. Dhc strains carrying tceA and vcrA genes were identified and quantified in a PCE-to-ethene dechlorinating microbial enrichment and overall they represented 58.63%. ±. 2.45% and 40.46%. ±. 1.86% of the total Dhc cells, respectively. These values were markedly higher than those obtained by qPCR, which strongly underestimated the actual concentration of vcrA gene (0.08%. ±. 0.01% of Dhc 16S rRNA gene copies). The assay was successfully applied also for the analysis of environmental samples and remarkably strengthens the biomonitoring activities at field scale by providing the specific in situ discrimination of Dhc cells carrying the key-RDase genes.
SourceJournal of microbiological methods 110, pp. 27–32
KeywordsDehalococcoides mccartyiGenecard-FISHReductive dechlorinationReductive dehalogenaseTceAVcrA
JournalJournal of microbiological methods
EditorElsevier Science Ireland, Shannon, Paesi Bassi
Year2015
TypeArticolo in rivista
DOI10.1016/j.mimet.2015.01.005
AuthorsMatturro B.; Rossetti S.
Text300111 2015 10.1016/j.mimet.2015.01.005 Scopus 2 s2.0 84921418832 Dehalococcoides mccartyi Genecard FISH Reductive dechlorination Reductive dehalogenase TceA VcrA GeneCARD FISH Detection of tceA and vcrA reductive dehalogenase genes in Dehalococcoides mccartyi by fluorescence in situ hybridization Matturro B.; Rossetti S. Water Research Institute IRSA , National Research Council CNR , via Salaria, Monterotondo, RM, 00015, Italy Due to the direct involvement in the biodegradation of chlorinated solvents, reductive dehalogenase genes RDase are considered biomarkers of the metabolic potential of different strains of Dehalococcoides mccartyi . Dhc . This is known to be the only microbe able to completely reduce toxic chlorinated solvents to harmless ethene. In the last years, several Molecular Biological Tools MBTs have been developed to optimize the detectability of Dhc cells and/or the RDase genes, with particular attention to the most important indicators of ethene formation, namely tceA and vcrA genes. Despite qPCR has been indicated as the MBT of choice, the use of CARD FISH recently demonstrated to provide a more accurate quantification of Dhc cells in a wide concentration range, overcoming the drawbacks of loosing nucleic acids during the preparation of the sample associated with qPCR. CARD FISH assays usually target 16S rRNA and up to date no protocol able to discriminate different Dhc strains by detecting RDase genes has been developed. This study reports the first evidence of in situ detection of tceA and vcrA genes into Dhc cells by applying a new procedure named geneCARD FISH. Dhc strains carrying tceA and vcrA genes were identified and quantified in a PCE to ethene dechlorinating microbial enrichment and overall they represented 58.63%. ±. 2.45% and 40.46%. ±. 1.86% of the total Dhc cells, respectively. These values were markedly higher than those obtained by qPCR, which strongly underestimated the actual concentration of vcrA gene 0.08%. ±. 0.01% of Dhc 16S rRNA gene copies . The assay was successfully applied also for the analysis of environmental samples and remarkably strengthens the biomonitoring activities at field scale by providing the specific in situ discrimination of Dhc cells carrying the key RDase genes. 110 http //www.scopus.com/inward/record.url eid=2 s2.0 84921418832 partnerID=q2rCbXpz Articolo in rivista Elsevier Science Ireland 0167 7012 Journal of microbiological methods Journal of microbiological methods J. microbiol. methods Journal of microbiological methods. simona.rossetti ROSSETTI SIMONA bruna.matturro MATTURRO BRUNA KILL SPILL Integrated Biotechnological Solutions for Combating Marine Oil Spills