Improved PCR-DGGE for high resolution diversity screening of complex sulfate-reducing prokaryotic communities in soils and sediments

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 -