Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 108 cells (g soil, dry wt.)–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 108 cells (g soil, dry wt.)–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 108 cells (g soil, dry wt.)–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments.

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