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Appl Environ Microbiol, March 1998, p. 871-879, Vol. 64, No. 3
Wageningen Agricultural University,
Department of Biomolecular Sciences, Laboratory of Microbiology,
Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
Received 30 September 1997/Accepted 17 December 1997
The main bacteria in peaty, acid grassland soils in the Netherlands
were investigated by ribosome isolation, temperature gradient gel
electrophoresis, hybridization, cloning, and sequencing. Instead of
using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active
bacteria. After direct ribosome isolation from soil, a partial
amplicon of bacterial 16S rRNA was generated by
reverse transcription-PCR. The sequence-specific
separation by temperature gradient gel electrophoresis yielded
soil-specific fingerprints, which were compared to signals from a clone
library of genes coding for 16S rRNA. Cloned 16S rDNA sequences
matching with intense bands in the fingerprint were sequenced. The
relationships of the sequences to those of cultured organisms of known
phylogeny were determined. Most of the amplicons originated from
organisms closely related to Bacillus species. Such
sequences were also detected by direct dot blot hybridization on soil
rRNA: a probe specific for Firmicutes with low G+C content
counted for about 50% of all bacterial rRNA. The bacterial activity in
Drentse A grassland soil could be estimated by direct dot blot
hybridization and sequencing of clones; it was found that about 65% of
all the bacterial ribosomes originated from Firmicutes. The
most active bacteria apparently were Bacillus species, from
which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely
related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the
alpha subclass.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Phylogeny of the Main Bacterial 16S rRNA Sequences
in Drentse A Grassland Soils (The Netherlands)
*
Corresponding author. Present address: Instituto de
Recursos Naturales y Agrobiologia, C.S.I.C., Apartado 1052, 41080 Seville, Spain. Phone: Tel.: 34 5 4624711 ext. 131. Fax: 34 5 4624002. E-mail: Andreas{at}cica.es.
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