Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries.

2.50
Hdl Handle:
http://hdl.handle.net/10033/12425
Title:
Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries.
Authors:
Junca, Howard ( 0000-0003-4546-6229 ) ; Pieper, Dietmar H
Abstract:
Developments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures and to monitor their dynamics under in situ conditions. However, there has been a distinct lack of emphasis on monitoring the functional diversity in the environment. Genes encoding catechol 2,3-dioxygenases (C23O), as key enzymes of various aerobic aromatic degradation pathways, were used as functional targets to assess the catabolic gene diversity in differentially BTEX contaminated environments by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs, and results closely matched with the information retrieved from random sequencing of PCR-DNA clone libraries. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish preselection or enrichment bias and to assess the function of predominant gene polymorphs. C23O abundance in isolates correlated with the levels of BTEX pollution in the soil samples analysed. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralizing phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralize benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. The PCR-SSCP technique is thus a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the functioning of microbial communities.
Citation:
Environ. Microbiol. 2004, 6(2):95-110
Issue Date:
1-Feb-2004
URI:
http://hdl.handle.net/10033/12425
PubMed ID:
14756875
Type:
Article
Language:
en
ISSN:
1462-2912
Appears in Collections:
publications of the research group microbial interactions and processes (MINP)

Full metadata record

DC FieldValue Language
dc.contributor.authorJunca, Howarden
dc.contributor.authorPieper, Dietmar Hen
dc.date.accessioned2007-06-25T12:03:09Zen
dc.date.available2007-06-25T12:03:09Zen
dc.date.issued2004-02-01en
dc.identifier.citationEnviron. Microbiol. 2004, 6(2):95-110en
dc.identifier.issn1462-2912en
dc.identifier.pmid14756875en
dc.identifier.urihttp://hdl.handle.net/10033/12425en
dc.description.abstractDevelopments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures and to monitor their dynamics under in situ conditions. However, there has been a distinct lack of emphasis on monitoring the functional diversity in the environment. Genes encoding catechol 2,3-dioxygenases (C23O), as key enzymes of various aerobic aromatic degradation pathways, were used as functional targets to assess the catabolic gene diversity in differentially BTEX contaminated environments by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Site specific PCR-SSCP fingerprints were obtained, showing that gene diversity experienced shifts correlated to temporal changes and levels of contamination. PCR-SSCP enabled the recovery of predominant gene polymorphs, and results closely matched with the information retrieved from random sequencing of PCR-DNA clone libraries. A new method for isolating strains capable of growing on BTEX compounds was developed to diminish preselection or enrichment bias and to assess the function of predominant gene polymorphs. C23O abundance in isolates correlated with the levels of BTEX pollution in the soil samples analysed. Isolates harbouring C23O genes, identical to the gene polymorph predominant in all contaminated sites analysed, showed an unexpected benzene but not toluene mineralizing phenotype whereas isolates harbouring a C23O gene variant differing by a single point mutation and observed in highly polluted sites only, were capable, among some other isolates, to mineralize benzene and toluene, indicating a catabolically determined sharing of carbon sources on-site. The PCR-SSCP technique is thus a powerful tool for assessing the diversity of functional genes and the identification of predominant gene polymorphs in environmental samples as a prerequisite to understand the functioning of microbial communities.en
dc.format.extent2594803 bytesen
dc.format.mimetypeapplication/pdfen
dc.language.isoenen
dc.titleFunctional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries.en
dc.typeArticleen
dc.format.digYESen

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