Analysis of Bacterial Community Structure in Bulk Soil by in Situ Hybridization

8/11/2019 Analysis of Bacterial Community Structure in Bulk Soil by in Situ Hybridization

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Abstract In situ hybridization with rRNA-targeted, fluo-rescent (Cy3-labeled) oligonucleotide probes was used to

analyze bacterial community structure in ethanol- orparaformaldehyde-fixed bulk soil after homogenization ofsoil samples in 0.1% pyrophosphate by mild ultrasonictreatment. In ethanol-fixed samples 37 7%, and inparaformaldehyde 41 8% of the 4, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with thebacterial probe Eub338. The yield could not be increasedby enzymatic and/or chemical pretreatments known to en-hance the permeability of bacterial cells for probes. How-ever, during storage in ethanol for 7 months, the de-tectability of bacteria increased in both ethanol- andparaformaldehyde-fixed samples to up to 47 8% due toan increase in the detection yield of members of the -

subdivision of Proteobacteria from 2 1% to 10 3%.Approximately half of the bacteria detected by probeEub338 could be affiliated to major phylogenetic groupssuch as the -, -, -, and -subdivisions of Proteobacte-ria, gram-positive bacteria with a high G+C DNA content,bacteria of the Cytophaga-Flavobacterium cluster of theCFB phylum, and the planctomycetes. The analysis re-vealed that bacteria of the - and -subdivision of Pro-teobacteria and the planctomycetes were predominant.Here, members of the -subdivision of Proteobacteria ac-counted for approximately 10 3% of DAPI-stainedcells, which corresponded to 44 16 108 cells (g soil,dry wt.)1, while members of the -subdivision of Pro-

teobacteria made up 4 2% of DAPI-stained cells [17 9 108 cells (g soil, dry wt.)1]. A large population of bac-

teria 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 planc-tomycetes in soil confirms previous reports on the occur-rence of planctomycetes in soil and indicates a yet un-known ecological significance of this group, which todate has never been isolated from terrestrial environ-ments.

Key words Fluorescent oligonucleotide probes Planctomycetes rRNA Whole-cell hybridization

Introduction

The fluorescent in situ hybridization technique withrRNA-targeted oligonucleotide probes has been used in-creasingly to analyze bacterial community structure invarious environments such as aquatic systems (Wagner etal. 1994b; Alfreider et al. 1996), sediments (Ramsing etal. 1993; Spring et al. 1993), and soils (Hahn et al. 1992;Fischer et al. 1995a). These studies have demonstrated theinadequacy of culture-dependent detection protocols, whichoften underestimate total numbers of bacteria and the di-versity of the bacterial community [for review, see Amannet al. (1995)].

In situ hybridization can most reliably be used onphysiologically active bacteria, e.g., syntrophic organisms

(Harmsen et al. 1996), or on bacterial endosymbionts(Amann et al. 1991). Good detection yields have alsobeen obtained in nutrient-rich environments such as acti-vated sludge or lake snow in which 89% (Wagner et al.1994b) or 55100% (Weiss et al. 1996) of the 4,6-di-amidino-2-phenylindole (DAPI)-stained bacteria couldbe detected, respectively. In bulk soil, however, only asmall fraction of the total bacterial community (1%) hasbeen detected by rRNA-targeted probes (Hahn et al.1992). Bulk soil is a nutritionally poor environment towhich bacteria may adapt by the formation of resting ordormant cells such as dwarf cells, cysts, or spores (Roszakand Colwell 1987). Since the signal intensity obtained by

Boris Zarda Dittmar Hahn Antonis Chatzinotas Wilhelm Schnhuber Alexander Neef

Rudolf I. Amann Josef Zeyer

Analysis of bacterial community structure in bulk soilby in situ hybridization

Arch Microbiol (1997) 168 : 185192 Springer-Verlag 1997

Received: 29 March 1997 / Accepted: 28 May 1997

ORIGINAL PAPER

B. Zarda D. Hahn () A. Chatzinotas J. ZeyerSwiss Federal Institute of Technology (ETH Zrich),Institute of Terrestrial Ecology, Soil Biology, Grabenstrasse 3,CH-8952 Schlieren, SwitzerlandTel. +41-1-633-6039; Fax +41-1-633-1122e-mail: [email protected]

W. Schnhuber A. Neef R. I. AmannTechnische Universitt Mnchen, Lehrstuhl fr Mikrobiologie,D-80290 Mnchen, Germany


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hybridization with rRNA-targeted probes depends on thecontent of ribosomes, a low cellular rRNA content can beone reason for the low detection yield in bulk soil. Detec-tion may also be prevented by impermeability of the fixedcells (Boenisch 1989) for probes due to alterations in thecell wall structure of dormant cells (e.g., spores) that in-crease their resistance to adverse environmental condi-tions (Roszak and Colwell 1987).

The permeability of cells is also influenced by method-ological factors such as the fixation protocol. Fixationusually increases the permeability of the cell membranes.Depending on the fixation protocol, however, a reductionof the permeability of cells for macromolecules such asprobes can be obtained. A further problem is backgroundfluorescence, which may severely hinder the detection ofweak hybridization signals especially. Some of these dif-ficulties may partially be overcome by the use of ad-vanced detection technology, e.g., by confocal laser scan-ning microscopy (Assmus et al. 1995; Manz et al. 1995).An alternative approach depends on the availability ofhigh-quality images. It requires an optimization of thesample preparation, ensuring an even dispersion of thetarget cells in thin layers and the concomitant use ofprobes labeled with highly contrasting dyes.

The aim of our study was to evaluate Cy3-labeledprobes for the detection of bacterial populations in bulksoil. Cy3 is a very photostable carbocyanine dye and pro-duces bright signals due to a high molar extinction coeffi-cient (150,000 M1 cm1) and a high quantum yield (Mu-jumdar et al. 1989). Initial studies focused on the opti-mization of the sample preparation with the aim of ob-taining an even dispersion of target cells in thin layers ofsoil slurries. Afterwards, Cy3-labeled oligonucleotide

probes were used to evaluate the effect of fixation proto-cols and pretreatments on the permeability and, thus, on

the detectability of bacterial cells. The optimized protocolwas finally used to estimate the abundance of major phy-logenetic groups of Bacteria, i.e. the -, -, -, and -sub-divisions of Proteobacteria, gram-positive bacteria with ahigh DNA G+C content, bacteria of the Cytophaga-Flavo-bacterium cluster of the CFB phylum, and the plancto-mycetes in addition to Archaea and Eukarya in bulk soil.

Materials and methods

Characteristics, fixation, and dispersion of soil samples

Surface samples down to a depth of 10 cm from the pristine forestsoil Hau, an Aquic Eutrochrept (Birmensdorf, Switzerland; asilty clay with 5.6% organic material; vegetation: Aro-Fagetum;Richard et al. 1978) were collected at the end of August 1995. Soilsamples were either directly fixed and stored in 96% ethanol orfixed in 4% paraformaldehyde/phosphate-buffered saline (com-posed of 0.13 M NaCl, 7 mM Na2HPO4 and 3 mM NaH2PO4; pH7.2 in water) at 0C for 16 h (Hahn et al. 1992). Paraformaldehyde-

fixed samples were subsequently washed in phosphate-bufferedsaline and stored in 96% ethanol at 20C at a concentration of 50mg of soil (dry wt.) per ml. Before application to filters or slides,20 l of the samples (containing the equivalent of 1 mg of soil, drywt.) was dispersed in 980 l of 0.1% sodium pyrophosphate in dis-tilled water by mild sonication with a probe (diameter 2 mm) at asetting of 20 for 1 min (Branson Sonifier B-12; Danbury, Conn.,USA).

Probes and stains

Oligonucleotide probes (Table 1) were synthesized with a primaryamino group at the 5-end (MWG Biotech, Ebersberg, Germany).Cy3 Reactive Dye (Cy3; Amersham, Zurich, Switzerland) was co-valently bound to the amino group of the oligonucleotide probe.

The dye-oligonucleotide conjugate (1:1) was purified from unre-acted components and stored at 20C in distilled water at a con-centration of 25 ng l1 (Amann et al. 1990a).

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Table 1 Oligonucleotide probes

Probe Target Sequence Reference

Eub338 Bacteria 5-GCTGCCTCCCGTAGGAGT Amann et al. (1990b)16S rRNA, position 338355

Euk516 Eukarya 5-ACCAGACTTGCCCTCC Amann et al. (1990b)16S rRNA, position 502516

Arch915 Archaea 5-GTGCTCCCCCGCCAATTCCT Stahl and Amann (1991)16S rRNA, position 915934

Alflb -Subdivision of Proteobacteria 5-CGTTCGYTCTGAGCCAG Manz et al. (1992)16S rRNA, position 1935

Bet42a -Subdivision of Proteobacteria 5-GCCTTCCCACTTCGTTT Manz et al. (1992)23S rRNA, position 10271043

Gam42a -Subdivision of Proteobacteria 5-GCCTTCCCACATCGTTT Manz et al. (1992)23S rRNA, position 10271043

SRB385 -Subdivision of Proteobacteria 5-CGGCGTCGCTGCGTCAGG Amann et al. (1990a)16S rRNA, position 385402

HGC69a Gram-positive bacteria with high DNA G+C content 5-TATAGTTACCACCGCCGT Roller et al. (1994)23S rRNA. position 19011918

CF319a Cytophaga-Flavobacterium cluster of the CFB phylum 5-TGGTCCGTGTCTCAGTAC Manz et al. (1996)16S rRNA, position 319336

Pla5a Planctomycetes 5-GACTTGCATGCCTAATCC A. Neef and R. I. Amann16S rRNA, position 4562 (unpublished results)


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The DNA intercalating dye 4,6-diamidino-2-phenylindole(DAPI; Sigma, Buchs, Switzerland) was stored as a solution of 1mg ml1 at 20C (Porter and Feig 1980). A dilution of 200 ngml1 was stored at 4 C and used to stain bacterial cells nonspecif-ically (final concentration, 20 ng l1).

Total cell counts

For the determination of total cell counts, 1 ml of the dispersed soilsolution was supplemented with 4 l of DAPI solution (200 ngml1) and incubated for 1 h in the dark. Afterwards, the solutionwas transferred into the filter tower of a filtration device (Milli-pore, Volketswil, Switzerland) along with 5 ml of distilled waterand was filtered under vacuum through prewetted membrane fil-ters (Millipore GVWP 02500: diameter, 25 mm, pore size, 0.22m), and the filter was washed with 10 ml distilled water. Excessstain was finally removed from the filter by incubation in 96%ethanol for 2 min. Filters were transferred onto slides, mountedwith Citifluor solution (Citifluor, Canterbury, UK), and the prepa-rations were examined with a Zeiss Axiophot microscope (Zeiss,Oberkochen, Germany) fitted for epifluorescence with a high-pres-sure mercury bulb (50 W) and filter set 02 (Zeiss; G 365, FT 395,LP 420).

In situ hybridization

For in situ hybridization, 10 l from each fixed and dispersed sam-ple was spotted onto gelatin-coated slides [0.1% gelatin, 0.01%KCr(SO4)2] and dried at room temperature for at least 4 h. Follow-ing dehydration in 50, 80, and 96% ethanol for 3 min each, thepreparations were subjected to different pretreatments in order toincrease the permeability of microbial cells (Table 2). Afterwards,the slides were rinsed with distilled water and dehydrated in 50,80, and 96% ethanol for 3 min each.

Hybridizations with oligonucleotide probes were performed in9 l of hybridization buffer [0.9 M NaCl, 20 mM Tris-HCl, 5 mMEDTA, and 0.01% SDS (pH 7.2)] in the presence of 1035% for-mamide (Alf1b = 10%; HGC69a, SRB385, Arch915, and Euk516= 20%; Eub338, Pla5a, Bet42a, and Gam42a = 30%; CF319a =

35%), 1 l of the probe (25 ng l1), and 1 l of the DAPI solution(200 ng l1) at 42C for 2 h. After hybridization, the slides were

washed in buffer containing 20 mM Tris-HCl (pH 7.2), 10 mMEDTA, 0.01% SDS, and either 440, 308, 102, or 80 mM NaCl de-pending on the formamide concentration during hybridization (10,20, 30, and 35%, respectively) for 15 min at 48C, subsequentlyrinsed with distilled water, and air-dried.

Slides hybridized with fluorescent probes were mounted withCitifluor solution, and the preparations were examined with aZeiss Axiophot microscope fitted for epifluorescence with a high-

pressure mercury bulb (50 W) and filter sets 02 and HQ-Cy3 (AHFAnalysentechnik, Tbingen, Germany; G 535/50, FT 565, BP610/75).

Statistical analysis

Bacteria were counted at 1,000 magnification. Forty fields se-lected at random and covering an area of 0.01 mm2 each were ex-amined from a sample distributed over eight circular areas of 53mm2 each. Log-transformed data of these counts were assessed bya one-way analysis of variance and afterwards by multiple pair-wise comparisons with Tukeys HSD test (SYSTAT). The signifi-cance level was set at = 0.05.

Results

Optimization of the sample preparation

A mild ultrasonic treatment of soil samples resuspendedin 0.1% pyrophosphate resulted in an even dispersion oftarget cells in thin layers of soil slurries on both filters andslides that allowed a reliable detection after DAPI stain-ing. On filters, the numbers of bacteria were 420 66 108 for ethanol-fixed samples and 479 71 108 forparaformaldehyde-fixed samples after the ultrasonic treat-ment. These numbers were not significantly differentfrom those obtained on slides in which ultrasonic treat-

ment enabled detection of larger numbers of cells than insamples only resuspended in pyrophosphate. The increase

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Table 2 Pretreatments to in-crease the permeability of bac-terial cells for oligonucleotideprobes

Treatment Protocol Reference

0.1% Lysozyme Incubation with lysozyme (Fluka, Buchs, Hnerlage et al.Switzerland; 1 mg corresponding to 37,320 U (1995)dissolved in 1 ml of 100 mM Tris-HCl, pH7.5, 5 mM EDTA) at 37C for 15 min

0.1 N HCl Incubation with 0.1 N HCl at room temper- Macnaughton et al.ature for 1 h (1994)

0.1 N HCl Subsequent treatment with lysozyme as This study+ 0.1% lysozyme described above

SDS/DTT Incubation with 10 mg SDS ml1, 50 mM Nicholson anddithiothreitol (DTT, Fluka) in water, freshly Setlow (1990)prepared, at 65C for 30 min

SDS/DTT Subsequent treatment with lysozyme as Fischer et al.+ 0.1% lysozyme described above (1995b)

10% Peracetic acid Incubation with acetic anhydride/30% H2O2, El-Gammal and1:1 (v/v) in water for 1 h at room temperature Sadek (1988)

10% Peracetic acid Subsequent treatment with lysozyme as Hahn et al. (1993)+ 0.1% lysozyme described above

SDS/DTT Subsequent treatment with peracetic acid as This study+ 10% peracetic acid described above

SDS/DTT Subsequent treatment with lysozyme as This study+ 10% peracetic acid described above+ 0.1%lysozyme


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in detectability was more pronounced in ethanol-fixed soilsamples, where cell numbers increased significantly from170 54 108 to 404 15 108 cells (g soil, dry wt.)1,while the number of cells detected in paraformaldehyde-fixed soil samples increased only slightly from 394 154 108 to 450 11 108 cells (g soil, dry wt.)1.

Effects of fixation and pretreatmentson the detectability of bacteria

The choice of the fixation protocol influenced the numberof cells detected after DAPI staining. In ethanol-fixed soilsamples, lower numbers of cells were detected after DAPIstaining than in paraformaldehyde-fixed samples (Table3). In ethanol-fixed samples, cell numbers were 226 26 108 cells (g soil, dry wt.)1, while in paraformaldehyde-fixed samples, 330 26 108 cells (g soil, dry wt.)1 weredetected. The percentage of cells detected by in situ hy-bridization with the bacterial probe Eub338 was not sig-nificantly different in ethanol- or paraformaldehyde-fixedsoil samples that were not subjected to pretreatments(Table 3). In ethanol-fixed samples 37 7%, and in para-formaldehyde 41 8% of the DAPI-stained cells were de-

tected.Similar results were obtained when pretreatments were

applied to increase the permeability of bacterial cells be-fore in situ hybridization with probe Eub338. While inparaformaldehyde-fixed soil samples none of the pretreat-ments had a significant influence on the percentage ofcells detected, in ethanol-fixed samples the percentage ofcells detected decreased after several pretreatments (Table3). Pretreatments with SDS/dithiotreitol (DTT), a combi-nation of this treatment and peracetic acid, and these treat-ments combined with subsequent lysozyme treatments re-duced the number of cells detected from 37 7% down to19 6% (Table 3).

In contrast to these findings, the detectability of bacte-ria increased in both ethanol- and paraformaldehyde-fixedsamples during long-term storage in ethanol. The largerdetection yield was mainly caused by an increase in thedetectability of members of the -subdivision of Pro-teobacteria from 2 1% after 2 months to 10 3% ofDAPI-stained cells after 7 months of storage, while thenumbers of DAPI-stained cells remained unchanged. Inparaformaldehyde-fixed samples, for example, cell num-bers detected by in situ hybridization with probe Eub338then increased from 41 8 to 47 8% of DAPI-stainedcells.

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Table 3 Effect of pretreat-ments prior to hybridization onnumbers of bacteria in ethanol-or paraformaldehyde-fixed soilsamples. Total numbers of bac-teria were determined afterDAPI staining and are ex-pressed as numbers of cells

[ 108 (g soil, dry wt.)1],while cells detectable by in situhybridization with the Cy3-la-beled oligonucleotide probeEub338 are expressed as % ofDAPI-stained cells of the samesample (n = 40; X SD)

Pretreatment Ethanol fixation Paraformaldehyde fixation

DAPI Eub338 DAPI Eub338( 108) (%) ( 108) (%)

Untreated 226 26 37 7 330 26 41 8

0.1% Lysozyme 196 18 37 7 350 42 41 8

0.1 N HCl 220 34 31 8 350 36 39 50.1 N HCl 210 20 33 10 320 26 39 5

+ 0.1% lysozyme

SDS/DTT 176 16 19 6 320 22 36 6

SDS/DTT 156 26 21 6 306 38 37 7+ 0.1% lysozyme

10% Peracetic acid 176 18 34 6 306 20 44 6

10% Peracetic acid 220 16 36 6 350 26 45 9+ 0.1% lysozyme

SDS/DTT 120 14 22 7 306 16 36 6+ 10% peracetic acid

SDS/DTT 126 16 24 7 320 34 36 8+ 10% peracetic acid+ 0.1%lysozyme

Table 4 Counts [ 108 (g soil, dry wt.)1] in ethanol- or para-formaldehyde-fixed bulk soil with and without previous pretreat-ment (peracetic acid and subsequent 0.1% lysozyme treatment) ob-tained after DAPI staining (n = 600; X SD) or by in situ hy-bridization with group-specific probes (n = 40; X SD)

Probes Ethanol fixation Paraformaldehyde fixation

Untreated Peracetic acid Untreated Peracetic acidand lyzozyme and lyzozyme

DAPI 404 15 402 16 450 11 467 17

Eub338 142 32 130 37 178 44 198 36

Arch915 4 4 2 2 4 5 1 3

Alflb 39 12a 32 12b 44 16a 31 12a

Bet42a 1 3 2 4 3 3 2 4

Gam42a 2 4 1 3 3 5 4 5

SRB385 14 11 13 6 17 9 15 7

HGC69a 1 3 8 6 1 3 2 4

CF319a 1 2 1 1 1 2 1 2

Pla5a 22 12 20 10 32 12 31 11

a Determined after storage in ethanol for 56 monthsb Determined after storage in ethanol for 7 months


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persal of soil aggregates and the dissociation of microor-ganisms from soil particles. However, in both ethanol- andparaformaldehyde-fixed samples, the ultrasonic treatmentwas necessary to ensure an even distribution of target cellsin thin layers on microscopic slides.

Effects of fixation and pretreatmentson the detectability of bacteria

The choice of the fixation protocol influenced the numberof cells detected after DAPI staining. The lower cell num-bers in ethanol-fixed samples, however, may be due to areduced signal-to-noise ratio caused by an increase inbackground signals that consequently resulted in a re-duced detectability of DAPI-stained cells. A second rea-son may be a decreased stabilization of bacterial cells bycoagulating fixatives such as ethanol as compared tocrosslinking fixatives such as paraformaldehyde. Thisassumption was supported by a significant decrease ofethanol-fixed cells subjected to pretreatments with SDS/DTT, peracetic acid, a combination of both treatments,and these treatments combined with subsequent lysozymetreatments (Table 3). Cell numbers in paraformaldehyde-fixed samples remained unchanged after these treatments.

In contrast to these findings, the number of cells de-tected by in situ hybridization was not significantly differ-ent in ethanol- and paraformaldehyde-fixed samples.Compared to earlier studies that reported detection yieldsin bulk soil of approximately 1% (Hahn et al. 1992), theoptimized protocol with Cy3-labeled probes raised the de-tection yield with probe Eub338 to approximately 40% ofthe DAPI-stained cells. This is a large increase in cells de-

tectable by in situ hybridization, exceeding by far those0.10.3% of bacteria that can be isolated from soil (Faegriet al. 1977; Torsvik et al. 1990) and potentially identified.The increase in detectability was mainly due to the appli-cation of the fluorescent cyanine dye Cy3 as label insteadof tetramethylrhodamine isothiocyanate (Hahn et al. 1992).With the concomitant use of optimized filter systems suchas the Filter System 20 (Zeiss; data not shown) or HQ-Cy3, Cy3 allowed a sensitive detection of probe-con-ferred signals and a reliable differentiation between auto-fluorescent and probe-conferred signals (Fig.1).

A detection yield of approximately 40% of the DAPI-stained cells also means that 60% of the community re-

mained undetected. This could be due to a restricted per-meability of dormant cells for probes (Fischer et al.1995b; Zepp et al. 1997). Various pretreatments that hadbeen shown to enhance the permeability of bacteria,failed, however, to enhance the detectability of bacterialcells. This failure may be due to the inadequacy of per-meabilization protocols for dormant cells but may also re-flect sequence differences between probe Eub338 andrRNA of the cells remaining undetected. Furthermore,low levels of rRNA per cell may also account for the fail-ure to detect significant numbers of cells by whole-cellhybridization because the morphological accomodationswhereby bacteria survive conditions, for example, of ex-

treme nutrient limitation may be accompanied by thedegradation of macromolecules such as rRNA (Kaplanand Apirion 1975; Hood et al. 1986). Although many rest-ing cells or spores still contain amounts of rRNA between30 and 50% of the amount in vegetative cells (Quiros etal. 1989; Setlow 1994), which is sufficient for their detec-tion by whole-cell hybridization (Fischer et al. 1995b;Zepp et al. 1997), detectability may be reduced when theamount of rRNA declines to lower levels.

Analysis of bacterial community structure in bulk soil

To date, studies on bacterial populations in soil have usu-ally been based on viable counting methods such as the de-termination of colony-forming units or the MPN technique.One of the main problems of viable counting methods isthe difficulty in defining conditions that are suitable for theactivation and outgrowth of all cells present (Sorheim et al.1989; Both et al. 1990; Richaume et al. 1993). They can,

therefore, be extremely selective and usually underestimate(1) numbers of total bacteria when nonselective media areused (Daley 1979; Sorheim et al. 1989; Richaume et al.1993) and (2) numbers of specific bacteria when selectivemedia are used (Both et al. 1990; Wagner et al. 1993; Wag-ner et al. 1994a). Direct microscopic counts after stainingwith fluorochromes usually exceed plate counts by severalorders of magnitude (Fischer et al. 1995a, b).

In soil, total numbers of bacteria determined by viablecounting methods usually range between 106 and 108 cells(g soil, dry wt.)1 with wide differences in the relative pro-portion of the individual bacterial genera or groups foundin particular soils [for reviews, see Clark (1967), Alexan-

der (1991), and Atlas and Bartha (1993)]. General state-ments on the abundance of bacterial populations in soilare, therefore, often contradictory. Some studies report thatgram-negative bacteria predominate in soil (Atlas andBartha 1993), although gram-positive bacteria with a lowDNA G+C content such as bacilli (767%, Alexander1991) and those with a high DNA G+C content (1033%,Atlas and Bartha 1993) may comprise a significant pro-portion of the bacterial population. Commonly found gen-era of the gram-negative bacteria include members of the-subdivision of Proteobacteria, e.g., members of thegenus Agrobacterium (120% relative proportion of theaerobic/facultatively anaerobic bacterial genera), the -

subdivision of Proteobacteria, e.g., members of the genusAlcaligenes (120%), the -subdivision of Proteobacteria,e.g., members of the genus Pseudomonas (315%), andthe Cytophaga-Flavobacterium cluster of the CFB phy-lum, e.g., members of the genus Flavobacterium (120%).In contrast to these findings, other studies have shown thepredominance of gram-positive bacteria with a low or highDNA G+C content in soil, whereas gram-negative bacteriaare present only in low numbers (7%) with pseudomonadsas the only significant group (5.5%) (Clark 1967).

Our findings using the in situ analysis of bacterial com-munity structure with Cy3-labeled oligonucleotide probesrevealed that bacteria of the - and -subdivisions of Pro-

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teobacteria, of the Cytophaga-Flavobacterium cluster, andof the gram-positive bacteria with a high DNA G+C con-tent were present only in relatively low numbers (approxi-mately 18 108 cells (g soil, dry wt.)1]. The numbers ofthese bacteria, however, were still much higher than the to-tal number of bacteria determined by viable counting meth-ods, i.e., as colony-forming units [5.6 0.4 107 cells (gsoil, dry wt.)1], determined by serial dilutions incubatedon 5% PTYG plates (Balkwill and Ghiorse 1985) supple-mented with 0.05% cycloheximide at 28C for 3 days].

The most abundant groups we could detect in soilHau are likely members of the - and -subdivisions ofProteobacteria and of the planctomycetes. These groupsare usually detected in soil in low numbers by viablecounting methods or (as in the case of the plancto-mycetes) have never been detected. Especially the detec-tion of planctomycetes demonstrates the potential of thein situ hybridization technique for the analysis of bacterialcommunity structure because it confirms earlier observa-tions on their occurrence in soil by PCR-based methods(Liesack and Stackebrandt 1992; Lee et al. 1996). Theplanctomycetes are an unusual group of budding bacteriathat do not synthesize the universal bacterial cell wallpeptidoglycan. Planctomycetes were initially discoveredand isolated as aquatic freshwater microorganisms be-longing to four genera: Planctomyces, Pirellula, Gem-mata, andIsosphaera (Staley et al. 1991; Schlesner 1994;Fuerst 1995). To date, however, they have never been iso-lated from terrestrial environments. Because of their highabundance, planctomycetes may have a significant func-tion in soil although their role in chemical transformationsis as yet unknown. According to the nutritional require-ments of aquatic isolates (which include the use of N-

acetyl-glucosamine as C and N sources; Schlesner 1994),a role of planctomycetes in the degradation of chitin maybe assumed in both aquatic and terrestrial environments.

The results of this study show that in situ hybridizationcan be a valuable tool in analyzing bacterial communitystructure in bulk soil and encourage further studies on bac-terial community structure and specific bacterial popula-tions (e.g., the planctomycetes) in this environment. How-ever, future studies will also have to address questions onthe still unidentified bacteria that are detectable only by insitu hybridization with probe Eub338 and on those that arenot detected at all by in situ hybridization. As shown forthe planctomycetes, the failure to detect bacteria with

probe Eub338 may reflect sequence differences betweenprobe Eub338 and the rRNA of the cells remaining unde-tected. Therefore, future studies should be based on se-quence analysis of universal clone libraries of genes en-coding for 16S rRNA (Borneman et al. 1996) and shouldcover topics such as the design of probes that target spe-cific sequences of this library or bacterial groups such asgram-positive bacteria with a low DNA G+C content.

Acknowledgements This work was supported by grants of theSwiss National Science Foundation (Priority Program Biotechnol-ogy), the Swiss Federal Office of Environment, Forests, and Land-scape (BUWAL), and the EU (contract no. BIO2-CT94-3098).

References

Alexander M (1991) Introduction to soil microbiology. Krieger,Malabar, Florida, USA

Alfreider A, Pernthaler J, Amann R, Sattler B, Glckner F-O,Wille A, Psenner R (1996) Community analysis of the bacter-ial assemblages in the winter cover and pelagic layers of a high

mountain lake by in situ hybridization. Appl Environ Microbiol62:21382144

Amann RI, Binder BJ, Olsen RJ, Chisholm SW, Devereux R, StahlDA (1990a) Combination of 16S rRNA-targeted oligonu-cleotide probes with flow cytometry for analyzing mixed mi-crobial populations. Appl Environ Microbiol 56:19191925

Amann RI, Krumholz L, Stahl DA (1990b) Fluorescent-oligonu-cleotide probing of whole cells for determinative, phylogenetic,and environmental studies in microbiology. J Bacteriol 172:762770

Amann RI, Springer N, Ludwig W, Grtz H-D, Schleifer K-H(1991) Identification in situ and phylogeny of uncultured bac-terial endosymbionts. Nature 351:161164

Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identi-fication and in situ detection of individual microbial cells with-out cultivation. Microbiol Rev 59:143169

Assmus B, Hutzler P, Kirchhof G, Amann R, Lawrence JR, Hart-mann A (1995) In situ localization of Azospirillum brasilensein the rhizosphere of wheat with fluorescently labeled, rRNA-targeted oligonucleotide probes and scanning confocal lasermicroscopy. Appl Environ Microbiol 61:10131019

Atlas RM, Bartha R (1993) Microbial ecology: fundamentals andapplications. Benjamin/Cummings, Redwood City, CA, USA

Bakken LR (1985) Separation and purification of bacteria fromsoil. Appl Environ Microbiol 49:14821487

Balkwill DL, Ghiorse WC (1985) Characterization of subsurfacebacteria associated with two shallow aquifers in Oklahoma.Appl Environ Microbiol 50:580588

Boenisch T (1989) Background. In: Naish SJ (ed) Handbook ofimmunochemical staining methods. Dako, Carpinteria, CA,USA, pp 2123

Borneman J, Skroch PW, OSullivan KM, Palus JA, Rumjanek

NG, Jansen JL, Nienhuis J, Triplett EW (1996) Molecular mi-crobial diversity of an agricultural soil in Wisconsin. Appl En-viron Microbiol 62:19351943

Both GJ, Gerards S, Laanbroek HJ (1990) Enumeration of nitriteoxidizing bacteria in grassland soil using most probable num-ber technique: effect of nitrite concentration and sampling pro-cedure. FEMS Microbiol Ecol 74:277286

Clark FE (1967) Bacteria in soil. In: Burges A, Raw F (eds) Soilbiology. Academic Press, London, pp 1549

Daley RJ (1979) Direct epifluorescence enumeration of nativeaquatic bacteria: uses, limitations, and comparative accuracy.In: Costerton JW, Colwell RR (eds) Native aquatic bacteria:enumeration, activity, and ecology. American Society for Test-ing and Materials, Philadelphia, pp 2945

El-Gammal SMA, Sadek MA (1988) Enzymatic saccharificationof some pretreated agricultural wastes. Zentralbl Mikrobiol

143:5562Faegri A, Torsvik VL, Goksoyr J (1977) Bacterial and fungal ac-tivities in soil: separation of bacteria and fungi by rapid frac-tionated centrifugation techniques. Soil Biol Biochem 9:105112

Fischer K, Hahn D, Amann RI, Daniel O, Zeyer J (1995a) In situanalysis of the bacterial community in the gut of the earthwormLumbricus terrestris L. by whole-cell hybridization. Can J Mi-crobiol 41:666673

Fischer K, Hahn D, Hnerlage W, Schnholzer F, Zeyer J (1995b)In situ detection of spores and vegetative cells of Bacillusmegaterium in soil by whole-cell hybridization. Syst Appl Mi-crobiol 18:265273

Fuerst JA (1995) The planctomycetes: emerging models for mi-crobial ecology, evolution and cell biology. Microbiology 141:14931506

191


8/8

Hahn D, Amann RI, Ludwig W, Akkermans ADL, Schleifer K-H(1992) Detection of microorganisms in soil after in situ hy-bridization with rRNA-targeted, fluorescently labelled oligonu-cleotides. J Gen Microbiol 138:879887

Hahn D, Amann RI, Zeyer J (1993) Whole-cell hybridization ofFrankia strains with fluorescence- or digoxigenin-labeled, 16SrRNA-targeted oligonucleotide probes. Appl Environ Micro-biol 59:17091716

Harmsen HJM, Kengen HMP, Akkermans ADL, Stams AJM, DeVos WM (1996) Detection and localization of syntrophic pro-pionate-oxidizing bacteria in granular sludge by in situ hy-bridization using 16S rRNA-based oligonucleotide probes.Appl Environ Microbiol 62:16561663

Hnerlage W, Hahn D, Zeyer J (1995) Detection of mRNA ofnprM in Bacillus megaterium ATCC 14581 grown in soil bywhole-cell hybridization. Arch Microbiol 163:235241

Hood MA, Guckert JB, White DC, Deck F (1986) Effect of nutri-ent deprivation on lipid, carbohydrate, RNA, DNA, and proteinlevels in Vibrio cholerae. Appl Environ Microbiol 52:788793

Hopkins DW, Macnaughton SJ, ODonnell AG (1991) A disper-sion and differential centrifugation technique for representa-tively sampling microorganisms from soil. Soil Biol Biochem23:217225

Kaplan R, Apirion D (1975) Decay of ribosomal ribonucleic acid

in Escherichia coli cells starved for various nutrients. J BiolChem 250:31743178

Kingsley MT, Bohlool BB (1981) Release ofRhizobium spp. fromtropical soils and recovery for immunofluorescence enumera-tion. Appl Environ Microbiol 42:241248

Lee S-Y, Bollinger J, Bezdicek D, Ogram A (1996) Estimation ofthe abundance of an uncultured soil bacterial strain by a com-petitive quantitative PCR method. Appl Environ Microbiol62:37873793

Liesack W, Stackebrandt E (1992) Occurrence of novel groups ofthe domain Bacteria as revealed by analysis of genetic materialisolated from an Australian terrestrial environment. J Bacteriol174:50725078

Lindahl V, Bakken LR (1995) Evaluation of methods for extrac-tion of bacteria from soil. FEMS Microbiol Ecol 16:135142

Macnaughton SJ, ODonell AG, Embley TM (1994) Permeabiliza-

tion of mycolic-acid-containing actinomycetes for in situ hy-bridization with fluorescently labelled oligonucleotide probes.Microbiology 140:28592865

Manz W, Amann R, Ludwig W, Wagner M, Schleifer K-H (1992)Phylogenetic oligodeoxynucleotide probes for the major sub-classes of proteobacteria: problems and solutions. Syst ApplMicrobiol 15:593600

Manz W, Amann R, Szewzyk R, Szewzyk U, Stenstrm T-A, Hut-zler P, Schleifer K-H (1995) In situ identification ofLegionel-laceae using 16S rRNA-targeted oligonucleotide probes andconfocal laser scanning microscopy. Microbiology 141:2939

Manz W, Amann R, Ludwig W, Vancanneyt M, Schleifer K-H(1996) Application of a suite of 16S rRNA-specific oligonu-cleotide probes designed to investigate bacteria of the phylumcytophaga-flavobacter-bacteroides in the natural environment.Microbiology 142:10971106

Mujumdar RB, Ernst LA, Mujumdar SR, Waggoner AS (1989)Cyanine dye labeling reagents containing isothiocyanate groups.Cytometry 10:1119

Nicholson WL, Setlow P (1990) Sporulation, germination and out-growth. In: Harwood CR, Cutting SM (eds) Molecular biologi-cal methods forBacillus. Wiley, Chichester, pp 391450

Porter KG, Feig YS (1980) The use of DAPI for identifying andcounting aquatic microflora. Limnol Oceanogr 25:943948

Quiros LM, Parra F, Hardisson C, Salas JA (1989) Structural andfunctional analysis of ribosomal subunits from vegetativemycelium and spores of Streptomyces antibioticus. J Gen Mi-crobiol 135:16611670

Ramsay AJ (1984) Extraction of bacteria from soil: efficiency ofshaking or ultrasonication as indicated by direct counts and au-toradiography. Soil Biol Biochem 16:475481

Ramsing NB, Khl M, Jorgensen BB (1993) Distribution of sul-fate-reducing bacteria, O2, and H2S in photosynthetic biofilmsdetermined by oligonucleotide probes and microelectrodes.Appl Environ Microbiol 59:38403849

Reyes VG, Schmidt EL (1979) Population densities ofRhizobiumjaponicum strain 123 estimated directly in soil and rhizos-pheres. Appl Environ Microbiol 37:854858

Richard F, Lscher P, Strobel T (1978) Physikalische Eigen-

schaften von Bden der Schweiz. Eidgenssische Anstalt frdas forstliche Versuchswesen, Birmensdorf, Switzerland

Richaume A, Steinberg C, Jocteur-Monrozier L, Faurie G (1993)Differences between direct and indirect enumeration of soilbacteria: the influence of soil structure and cell location. SoilBiol Biochem 25:641643

Roller C, Wagner M, Amann R, Ludwig W, Schleifer K-H (1994)In situ probing of gram-positive bacteria with a high DNAG+C content using 23S rRNA-targeted oligonucleotides. Mi-crobiology 140:28492858

Roszak DB, Colwell RR (1987) Survival strategies of bacteria inthe natural environment. Microbiol Rev 51:365379

Schlesner H (1994) The development of media suitable for the mi-croorganisms morphologically resembling Planctomyces spp.,Pirellula spp., and other Planctomycetales from variousaquatic habitats using dilute media. Syst Appl Microbiol 17:

135145Setlow P (1994) Mechanisms which contribute to the long-term

survival of spores of bacillus species. J Appl Bacteriol 76:S49S60

Sorheim R, Torsvik VL, Goksoyr J (1989) Phenotypical diver-gences between populations of soil bacteria isolated on differ-ent media. Microb Ecol 17:181192

Spring S, Amann R, Ludwig W, Schleifer K-H, Van Gemerden H,Petersen N (1993) Dominating role of an unusual magnetotac-tic bacterium in the microaerobic zone of a freshwater sedi-ment. Appl Environ Microbiol 59:23972403

Stahl DA, Amann RI (1991) Development and application of nu-cleic acid probes. In: Stackebrandt E, Goodfellow M (eds) Nu-cleic acid techniques in bacterial systematics. Wiley, NewYork, pp 205248

Staley JT, Fuerst JA, Giovannoni S, Schlesner H (1991) The order

Planctomycetales and the genera Planctomyces, Pirellula,Gemmata, andIsosphaera. In: Balows A, Trper HG, DworkinM, Harder W, Schleifer K-H (eds) The prokaryotes. Springer,New York, pp 37103731

Torsvik V, Goksoyr J, Daae FL (1990) High diversity in DNA ofsoil bacteria. Appl Environ Microbiol 56:782787

Wagner M, Amann R, Lemmer H, Schleifer K-H (1993) Probingactivated sludge with oligonucleotides specific for proteobacte-ria: inadequacy of culture-dependent methods for describingmicrobial community structure. Appl Environ Microbiol 59:15201525

Wagner M, Amann R, Lemmer H, Manz W, Schleifer K-H(1994a) Probing activated sludge with fluorescently labeledrRNA targeted oligonucleotides. Water Sci Technol 29:1523

Wagner M, Erhart R, Manz W, Amann R, Lemmer H, Wedi D,Schleifer K-H (1994b) Development of an rRNA-targeted

oligonucleotide probe specific for the genusAcinetobacterandits application for in situ monitoring in activated sludge. ApplEnviron Microbiol 60:792800

Weiss P, Schweitzer B, Amann R, Simon M (1996) Identificationin situ and dynamics of bacteria on limnetic organic aggregates(lake snow). Appl Environ Microbiol 62:19982005

Zepp K, Hahn D, Zeyer J (1997) Evaluation of a 23S rRNA inser-tion as target for the analysis of uncultured Frankia popula-tions in root nodules of alders by whole-cell hybridization. SystAppl Microbiol 20:124132

Zvyagintsev DG, Galinka GM (1967) Ultrasonic treatment as amethod for preparation of soils for microbiological analysis.Microbiologiia 36:910916

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Analysis of Bacterial Community Structure in Bulk Soil by in Situ Hybridization

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