Improved PCR-DGGE for high resolution diversity screening of complex sulfate-reducing prokaryotic communities in soils and sediments.
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.
Miletto
Marzia
M
Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Wetland Ecology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands. m.miletto@nioo.knaw.nl
Bodelier
Paul L E
PL
Laanbroek
Hendrikus J
HJ
D044822:Biodiversity; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D020564:Deltaproteobacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019015:Geologic Sediments / Q000382:microbiology*; D050885:Hydrogensulfite Reductase / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D004269:DNA, Bacterial; D050885:Hydrogensulfite Reductase