TY - JOUR
T1 - Improved PCR-DGGE for high resolution diversity screening of complex sulfate-reducing prokaryotic communities in soils and sediments
AU - Miletto, M.
AU - Bodelier, P.L.E.
AU - Laanbroek, R.
N1 - Reporting year: 2007
Metis note: 4036;CL; MWE; file:///C:/pdfs/Pdfs2007/Miletto_ea_4036.pdf
PY - 2007
Y1 - 2007
N2 - In this study we evaluated a high resolution PCR-DGGE strategy for the characterization of complex sulfate-reducing microbial communities inhabiting natural environments. dsrB fragments were amplified with a two-step nested PCR protocol using combinations of primers targeting the dissimilatory (bi)sulfite reductase genes. The PCR-DGGE conditions were initially optimized using a dsrAB clone library obtained from a vegetated intertidal riparian soil along the river Rhine (Rozenburg, the Netherlands). Partial dsrB were successfully amplified from the same environmental DNA extracts used to construct the library, DGGE-separated and directly sequenced. The two approaches were in good agreement: the phylogenetic distribution of clones and DGGE-separated dsrB was comparable, suggesting the presence of sulfate-reducing prokaryotes (SRP) belonging to the families ‘Desulfobacteraceae,' ‘Desulfobulbaceae’ and ‘Syntrophobacteraceae,' and to the Desulfomonile tiedjei- and Desulfobacterium anilini-groups. The nested PCR-DGGE was also used to analyze sediment samples (Appels, Belgium) from a series of microcosms subjected to a tidal flooding regime with water of different salinity, and proved to be a valid tool also to monitor the SRP community variation over time and space as a consequence of environmental changes.
AB - In this study we evaluated a high resolution PCR-DGGE strategy for the characterization of complex sulfate-reducing microbial communities inhabiting natural environments. dsrB fragments were amplified with a two-step nested PCR protocol using combinations of primers targeting the dissimilatory (bi)sulfite reductase genes. The PCR-DGGE conditions were initially optimized using a dsrAB clone library obtained from a vegetated intertidal riparian soil along the river Rhine (Rozenburg, the Netherlands). Partial dsrB were successfully amplified from the same environmental DNA extracts used to construct the library, DGGE-separated and directly sequenced. The two approaches were in good agreement: the phylogenetic distribution of clones and DGGE-separated dsrB was comparable, suggesting the presence of sulfate-reducing prokaryotes (SRP) belonging to the families ‘Desulfobacteraceae,' ‘Desulfobulbaceae’ and ‘Syntrophobacteraceae,' and to the Desulfomonile tiedjei- and Desulfobacterium anilini-groups. The nested PCR-DGGE was also used to analyze sediment samples (Appels, Belgium) from a series of microcosms subjected to a tidal flooding regime with water of different salinity, and proved to be a valid tool also to monitor the SRP community variation over time and space as a consequence of environmental changes.
U2 - 10.1016/j.mimet.2007.03.015
DO - 10.1016/j.mimet.2007.03.015
M3 - Article
SN - 0167-7012
VL - 70
SP - 103
EP - 111
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -