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

Abstract

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 × 10 8 cells (g soil, dry wt.) -1 , while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 10 8 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 × 10 8 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.

Abundance, composition, diversity and novelty of soil Proteobacteria

“…Although such larger proportion of Proteobacteria in pyrosequencing-based studies might be a true reflection of the communities analyzed, it might also indicate the existence of a cloning bias or that classification based on small 16S rRNA gene fragments could lead to different taxonomic assignments than classification based on near to full-length sequences, as suggested earlier (Elshahed et al, 2008). Nevertheless, Proteobacteria remains the most abundant soil phylum, regardless of the utilized approach, which aside from PCR-based clone libraries and pyrosequencing has included metagenomics (Liles et al, 2003;Tringe et al, 2005), fluorescent in situ hybridization (Zarda et al, 1997) and microarray analysis (Yergeau et al, 2009).…”

Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary

“…The hybridization solution contained 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 0.01% sodium dodecyl sulfate, and the concentration of formamide determined to achieve specificity for the bacterial groups targeted by the different probes (Zarda et al 1997;Eilers et al 2000b). After hybridization, the sample was transferred to a wash solution containing 20 mM Tris-HCl (pH 7.4), 5 mM edatic acid (EDTA), 0.01% sodium dodecyl sulfate, and a concentration of NaCl appropriate for the probe (Zarda et al 1997;Eilers et al 2000b). The sample was then rinsed with water, air dried, and mounted with oil on a glass slide with the cells facing away from the slide.…”

Long-Term Population Dynamics of Phototrophic Sulfur Bacteria in the Chemocline of Lake Cadagno, Switzerland

“…After hybridization, the slides were washed in buffer containing 20 mM Tris-HCl, pH 7.2; 10 mM EDTA; 0.01% SDS; and either 450, 318, 159, 112, 80, or 56 mM NaCl depending on the formamide concentration during hybridization (10,20,25,30,35, and 40%, respectively) for 20 min at 48°C. They were subsequently rinsed with distilled water and air dried (37). The slides were mounted with Citifluor AF1 (Citifluor Ltd., London, United Kingdom) and examined by epifluorescence microscopy using filter sets F31 (AHF Analysentechnik, Tübingen, Germany; D360/40, 400DCLP, and D460/50 for DAPI) and F41 (AHF Analysentechnik; HQ535/50, Q565LP, and HQ610/75 for Cy3).…”