Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments.
Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and beta-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, beta-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species.
Overmann
J
J
Paläomikrobiologie, Institut für Chemie und Biologie des Meeres, Universität Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany. j.overmann@palmikro.icbm.uni-oldenburg.de
Coolen
M J
MJ
Tuschak
C
C
D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D003901:Desulfovibrio / Q000235:genetics / Q000302:isolation & purification; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005618:Fresh Water; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012623:Seawater; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014871:Water Microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S