Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog

Abstract

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (>or=95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3x10(7) and 1.1x10(7) cells g-1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

FIG. 1.

16S rRNA gene-based dendrogram showing the phylogenetic relationship of 24 peat clones (boldface) to the peat bog isolate MPL3 and to representatives of the phylum Acidobacteria. The classification into subdivisions follows the proposal by Hugenholtz et al. (15). Bootstrap values (1,000 data resamplings) of >50% are shown. The root was determined by using the 16S rRNA gene sequence of Chlorobium limicola Y10643 as an outgroup. The scale bar represents 0.1 substitution per nucleotide position.

FIG. 2.

16S rRNA gene-based dendrogram showing the phylogenetic relationship of 23 peat clones (boldface) to peat bog isolates MP03 and PN-29 and to representatives of the classes Alphaproteobacteria and Deltaproteobacteria. Bootstrap values (1,000 data resamplings) of >50% are shown. The root was determined by using the 16S rRNA gene sequence of Pseudomonas fluorescens AF094730 as an outgroup. The scale bar represents 0.1 substitution per nucleotide position.

FIG. 3.

(A) Specific detection of planctomycete microcolonies in Sphagnum peat by FISH. Epifluorescent micrographs of in situ hybridizations with Cy3-labeled probes Pla46 and Pla886 (left), DAPI staining (center), and the phase-contrast image (right) are shown. Bar, 5 μm. (B) 16S rRNA gene-based dendrogram showing the phylogenetic relationship of three peat clones (boldface) to peat bog isolates A10 and MPL7 and to representatives of the phylum Planctomycetes. Bootstrap values (1,000 data resamplings) of >50% are shown. The root was determined by using the 16S rRNA gene sequence of Chlorobium limicola Y10643 as an outgroup. The scale bar represents 0.1 substitution per nucleotide position.

FIG. 4.

Depth distribution of bacterial cells that were detected in Sphagnum peat bog Bakchar with probes specific for the Alphaproteobacteria, Planctomycetes, Acidobacteria, or Verrucomicrobia. Error bars indicate standard errors.