Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

Environmental Microbiology (2001) 3(7), 431±439

Quantification of bacterial subgroups in soil:comparison of DNA extracted directly from soil or fromcells previously released by density gradientcentrifugation

Sophie Courtois,1,2 AÊ sa FrostegaÊrd,3,4 Pernilla

GoÈ ransson,3 Geraldine Depret,1 Pascale Jeannin2 and

Pascal Simonet1*1Laboratoire d'Ecologie Microbienne, UMR CNRS 5557,

UniversiteÂ Claude Bernard Lyon 1, 43 bd du 11 novembre

1918, 69622 Villeurbanne Cedex, France.2Aventis Pharma, Centre de Recherche de Vitry-

Alfortville, 13, quai Jules Guesde BP 14, 94403 Vitry sur

Seine, Cedex, France.3Department of Microbial Ecology, Lund University,

Ecology Building, SE-223 62 Lund, Sweden.4Department of Chemistry and Biotechnology, Agricultural

University of Norway, PO Box 5040, N-1432 Aas, Norway.

Summary

All molecular analyses of soil bacterial diversity are

based on the extraction of a representative fraction of

cellular DNA. Methods of DNA extraction for this

purpose are divided into two categories: those in

which cells are lysed within the soil (direct extraction)

and those in which cells are first removed from soil

(cell extraction) and then lysed. The purpose of this

study was to compare a method of direct extraction

with a method in which cells were first separated from

the soil matrix by Nycodenz gradient centrifugation in

order to evaluate the effect of these different

approaches on the analysis of the spectrum of

diversity in a microbial community. We used a

method based on polymerase chain reaction (PCR)

amplification of a 16S rRNA gene fragment, followed

by hybridization of the amplified fragments to a set of

specific probes to assess the phylogenetic diversity

of our samples. Control parameters, such as the

relationship between amount of DNA template and

amount of PCR product and the influence of compet-

ing DNA on PCR amplification, were first examined.

Comparison between extraction methods showed

that less DNA was extracted when cells were first

separated from the soil matrix (0.4 mg g21 dry weight

soil versus 38±93 mg g21 obtained by in situ lysis

methods). However, with the exception of the g-

subclass of Proteobacteria, there was no significant

difference in the spectrum of diversity resulting from

the two extraction strategies.

Introduction

Nucleic acid-based techniques have led to a revised

classification of previously isolated bacteria (Pace et al.,

1986; Woese, 1987; Stackebrandt et al., 1988) and have

also provided an extensive range of new tools to detect

these bacteria in their natural environment (for reviews,

see Amann et al., 1995; Head et al., 1998). However, the

sensitivity and specificity of these detection techniques

remain hampered by problems encountered when extract-

ing and purifying template DNA molecules from complex

environments, such as soils or sediments (Tebbe and

Vahjen, 1993; Zhou et al., 1996; FrostegaÊrd et al., 1999).

In one DNA extraction strategy, bacteria are lysed directly

in the soil matrix before DNA purification (Ogram et al.,

1987; Tsai and Olson, 1991; Picard et al., 1992; Smalla

et al., 1993; MoreÂ et al., 1994; Van Elsas et al., 1997).

Alternatively, DNA can be extracted from bacteria

previously separated from the soil (Faegri et al., 1977;

Torsvik, 1980; Jacobsen and Rasmussen, 1992; Holben

and Harris, 1995). In a recent paper (FrostegaÊrd et al.,

1999), we demonstrated that each step of the direct

extraction technique, including cell lysis, recovery and

purification of the DNA, showed considerable variability

depending on soil type, indicating that data obtained from

such analyses have to be considered very cautiously.

Possible reasons for potential biases generated by direct

extraction methods include incomplete extraction of DNA

owing to DNA adsorption to soil colloids, localization of

bacteria in inner or outer soil compartments and inefficient

lysis in situ for some types of bacteria (More et al., 1994;

Kuske et al., 1998). Moreover, extracellular DNA from

dead plant cells or bacteria can persist in soils for long

periods of time (Widmer et al., 1996; Paget et al., 1998),

and DNA molecules released from cells during the lysis

treatment cannot easily be distinguished from extracel-

lular DNA that was already present in the soil before the

treatment. Finally, the mean size of the DNA extracted by

this method is relatively small, and the purity of the DNA is

Q 2001 Blackwell Science Ltd

*For correspondence. E-mail: [email protected]; Tel.(133) 04 72 44 82 89; Fax (133) 04 72 43 12 23.

often unsatisfactory, particularly in the case of soils rich in

humic compounds.

Studies by Leff et al. (1995) and Steffan et al. (1988)

indicated that problems such as DNA smearing and

purification efficiency could be circumvented using proto-

cols in which bacteria are isolated from the soil matrix

before lysis. However, their techniques included several

successive washing and differential centrifugation cycles,

which are time consuming and thus less suitable for

routine analyses that require the handling of numerous

samples. Moreover, the number of extracted cells and the

quantities of extracted DNA were low. Recently, an

improved method for separating cells from the soil matrix

was accomplished using centrifugation in a Nycodenz

buoyant density gradient (Lindahl and Bakken, 1995). The

number of extracted cells, as estimated by acridine

orange direct counts (AODC), rose from 1% of the total

counts using washing/centrifugation techniques to up to

50%, depending on soil type, using the Nycodenz

gradient. This improvement made possible the use of

the indirect DNA extraction approach (which provides

several advantages including a higher degree of purity

and larger fragment size of the extracted DNA compared

with the more commonly used direct in situ lysis method)

for analyses of soil microbial communities.

Although the development of DNA-based methods for

analysing soil microbial communities is critical because of

the inability to culture most soil microorganisms, the

extent to which these techniques introduce bias by

selecting certain organisms within the soil microbial

community is still poorly understood. The aim of the

present study was to assess the extent of such biases by

comparing whether soil DNA extracted by the two

different methods ( i.e. direct extraction from cells lysed

in situ versus cell extraction in which bacteria are first

isolated from the soil) revealed significant differences in

the 16S rRNA gene analysis of the soil microbial

community. This was carried out by amplifying a 700 bp

region within the 16S rRNA gene, derived either from total

soil DNA after in situ lysis according to the protocol

described by FrostegaÊrd et al. (1999) or after lysis of a

bacterial fraction previously recovered on Nycodenz

gradient. Polymerase chain reaction (PCR) products

were subsequently hybridized with taxa-specific oligonu-

cleotide probes and quantified by phosphorimaging to

determine the proportions of these different groups in

relation to the total soil bacterial community.

Results and discussion

PCR amplification and oligonucleotide probe hybridization

Two PCR primers (FGPS612 and FGPS669), located at

positions 506 and 1194 of the Escherichia coli 16S rDNA

sequence, were designed to be complementary to

conserved regions of the gene that flank variable regions

in order to design and use group-specific oligonucleotide

probes that could hybridize to the inside of the amplified

fragments. We chose to amplify the shortest fragment

possible in order to minimize the chance of chimeric

sequence formation (Liesack et al., 1991). After PCR

amplification, we hybridized the resulting PCR products

with probes (Table 1) that targeted various bacterial

groups, including Eubacteria, a-, b- and g-subgroups of

the Proteobacteria, Gram-positive bacteria with a low

G1C content and actinomycetes. We also included two

probes of higher taxonomic specificity within the actino-

mycete group, targeting the genera Streptomyces and

Streptosporangium.

Validation of PCR primers and oligonucleotide probes We

designed primers and probes targeting the 16S rRNA

gene according to available sequence information (Gen-

Bank database). Probe specificity was checked using the

PROBE MATCH program available through the Ribosomal

Database Project (Maidak et al., 2000) and tested

experimentally with DNA isolated from about 30 bacterial

species covering the phylogenetic groups targeted in this

study. PCR amplifications gave rise to products of the

expected size (<700 bp) for each of the DNA templates,

and subsequent dot-blot hybridization experiments con-

firmed a high degree of specificity for the various probes.

In no case was a signal obtained from isolates not

belonging to the target group. The probe specific for

Eubacteria produced a positive signal when hybridized to

all the amplified DNA templates, except those of

Staphylococcus sp. and Streptoverticillium abikoensis.

The probes targeting the subgroups of Proteobacteria

produced the expected hybridization signal, as did the

probes specific for actinomycetes and the genera

Streptomyces and Streptosporangium. The probe specific

for low G1C Gram-positive bacteria also showed speci-

ficity, except that it did not detect the Streptococcus

subgroup. This result could be explained by the presence

of a mismatch in the sequence alignment between

Streptococcus 16S rDNA and the probe. Staphylococcus

DNA was not detected by the probes specific to low G1C

Gram-positive bacteria and for Eubacteria, suggesting the

possibility that inhibitory compounds in the DNA extract

may have interfered with hybridization.

Validation of PCR amplification and oligonucleotide probe

hybridization To address potential biases in the quantita-

tive analysis of microbial diversity based on PCR

amplification and probe hybridization (Reysenbach et al.,

1992; Wagner et al., 1994; Polz and Cavanaugh, 1998),

initial experiments were carried out to check the relation-

ship between amount of DNA template and amount of

432 S. Courtois et al.

Q 2001 Blackwell Science Ltd, Environmental Microbiology, 3, 431±439

PCR product and the influence of competing DNA on PCR

amplification.

In a first experiment, a < 700 bp region of the 16S

rRNA gene of Bacillus subtilis was PCR amplified using

primers targeting Eubacteria (Table 1), and the products

were quantified using dot-blot hybridization. Different

amounts of template (256 fg2500 ng of B. subtilis DNA)

were amplified. To recalculate hybridization signals into a

number of 16S rDNA copies, a standard curve was

prepared consisting of known amounts of purified 16S

rDNA amplicons (derived from B. subtilis as described

above); which were applied to the hybridization mem-

branes. The linearity of the hybridization was checked

preliminarily in order to ensure that the measurements

were performed within the linear range of the method.

Five portions (2±16 ml) of PCR products were taken from

each PCR tube and applied to membranes. A linear

relationship (r2 � 0.99±1.0), based on logarithmic values,

was found between hybridization signals and the amounts

of PCR products applied to the membrane, except for

products obtained from the lowest quantity of template

DNA used for PCR (256 fg), for which the r2-values were

lower (r2 � 0.91; results not shown). The hybridization

signals were plotted against the number of 16S rDNA

copies present in the template solution before amplifica-

tion (not shown). A linear response (r2 � 0.99), based on

logarithmic values, was found for template concentrations

up to 4 ng, corresponding to about 107 16S rDNA

templates, in the 50 ml PCR mixture. Similar curves

were obtained using DNA from other species (Agrobac-

terium tumefaciens, Burkholderia cepacia, Ralstonia

solanacearum, Escherichia coli, Bacillus anthracis, Strep-

tomyces lividans, Frankia sp.), with r2-values between

0.95 and 0.98 in the same interval of template DNA. In

addition, various amounts of S. lividans DNA were

amplified (three replicates) simultaneously in the same

or different PCR runs. Linearity was maintained, and all

the slopes were included in confidence intervals of 95%

(data not shown), demonstrating that tube-to-tube and

day-to day-variations were not significant.

To support the assumption that DNA amplified from a

mixture of bacterial templates is representative of the

microbial community from which the templates are

derived, it is important to analyse whether bias may be

introduced by differences in amplification efficiency

among template DNAs. We investigated how the ampli-

fication of DNA from one species could be affected by the

presence of DNA from another species by amplifying

mixtures of DNA derived from B. subtilis (low G1C

content) and S. lividans (high G1C content). These

species were chosen because templates with low G1C

content, which denature more readily, have been reported

to be favoured in the PCR amplification (Reysenbach

et al., 1992). From both species a < 700 bp region in the

16S rRNA gene was amplified using primers targeting

Eubacteria (Table 1), meaning that the PCR products

consisted of amplicons from both species. The number of

16S rDNA copies derived from the respective species was

then determined by dot- blot hybridization/phosphorima-

ging using group-specific probes (FGPS621 or FGPS

617; Table 1). Hybridization signals were recalculated into

number of 16S rDNA copies using standard curves

consisting of known amounts of purified 16S rDNA

amplicons derived from B. subtilis or S. lividans respec-

tively (for details, see Experimental procedures).

Increasing amounts of S. lividans DNA added to 25 pg

of B. subtilis DNA before amplification resulted in a

gradual decrease in B. subtilis PCR product. Similarly,

amplification of 6 ng of S. lividans DNA in the presence of

increasing amounts of B. subtilis DNA resulted in

decreasing amounts of S. lividans PCR product (higher

amounts of S. lividans DNA were used because previous

hybridizations had shown that the hybridization signals

from this species were weaker than those from B.

subtilis). When the ratio of S. lividans to B. subtilis DNA

was 1:10 (based on the weight of template DNA), the S.

lividans product was reduced to 19% of that obtained from

the pure sample, whereas at a 1:10 ratio of B. subtilis to S.

lividans DNA, the product was reduced to 43% of that

from the pure B. subtilis DNA sample (not shown). Thus,

Table 1. Target and bacterial groups and sequences of primers and probes.

Target (primer or probe) Sequence (5' to 3') Position

FGPS612 Eubacteria (forward primer) C(C/T)A ACT (T/C/A)CG TGC CAG CAG CC 506±525FGPS669 Eubacteria (reverse primer) GAC GTC (A/G)TC CCC (A/C)CC TTC CTC 1174±1194FGPS618 Eubacteria (probe) ATG G(T/C)T GTC GTC AGC TCG 1056±1073FGPS614 a-Proteobacteria (probe) GTG TAG AGG TGA AAT TCG TAG 683±703FGPS615 b-Proteobacteria (probe) CGG TGG ATG ATG TGG ATT 939±956FGPS616 g-Proteobacteria (probe) AGG TTA AAA CTC AAA TGA 900±917FGPS621 Low G1C %, Gram1 (probe) ATA CGT AGG TGG CAA GCG 532±549FGPS617 Actinomycetes (probe) GCC GGG GTC AAC TCG GAG G 1159±1177FGPS680 Streptomyces (probe) TGA GTC CCC A(A/C/T)C (T/A)CC CCG 1132±1149FGPS619 Streptosporangium (probe) GCT TGG GGC TTA ACT CCA GG 609±628

a. Position on E. coli 16S rRNA gene.

Quantification of bacterial subgroups in soil 433


the amplification of S. lividans DNA appeared to be more

negatively affected by the presence of B. subtilis DNA

than vice versa. However, the genome of B. subtilis

contains 10 copies of 16S rDNA (Kunst et al., 1997),

whereas the genome of S. lividans only contains six

(Suzuki et al., 1988). Moreover, the B. subtilis genome is

smaller (4215 kb; Kunst et al., 1997) than the genome of

S. lividans (7901 kb; Leblond et al., 1993), so that an

equivalent mass of B. subtilis genomic DNA contains

three times as many copies of the 16S rDNA region as S.

lividans genomic DNA. When adjusting for this (Fig. 1),

we found that amplification of DNA mixtures containing

the same template proportions with respect to copy

number resulted in the same percentage reduction in

the product derived from the DNA present in lower

concentrations, regardless of species, i.e. for both

species, a 1:3 ratio (based on number of 16S rDNA

copies) to the other species resulted in a 43% decrease in

product. Our results thus indicate that, at least in this

case, the G1C contents of the two DNAs were not a

significant biasing factor. Instead, the amplification effi-

ciency appeared to be affected by template amounts,

regardless of which types of templates they were. The

relationship is shown in Fig. 2, in which the number of

products obtained per template followed a straight line

when plotted against the number of templates, based on

logarithmic values (r2 � 0.91). The regression function's

intercept with the y-axis estimates the number of 16S

rDNA copies that would be obtained if only one template

copy was present in the PCR solution. The value was

10.52, which is very close to the theoretical value 10.54

because 35 cycles were used, and log(1*235) � 10.5. If

the relationship shown in Fig. 2 is also true for other

mixtures of species, this would mean that the detection of

a species present in low numbers within a complex

community might not necessarily improve by increasing

the concentration of DNA in the PCR amplification.

Considering the potential PCR biasing issues described

above, it seemed more appropriate to use the PCR

hybridization method for relative rather than absolute

quantification. Therefore, we used this methodology to

compare the spectrum of bacterial subgroups obtained

from soil extracted after direct in situ cell lysis with the

method in which the bacteria are separated from the soil

matrix before lysis and DNA extraction.

Extraction of bacteria from soil

Preliminary work was carried out to optimize the release of

indigenous bacterial cells trapped inside soil microaggre-

gates. Based on data from Lindahl and Bakken (1995) and

Lindahl (1996), demonstrating enhanced homogenization

efficiency using a Waring blender compared with sonica-

tion or chemical treatments, we examined the effects of

length of homogenization and choice of buffer on bacterial

extraction from soil. Direct bacterial counts (AODC),

carried out on soil extracts and on whole soil samples,

showed that the percentage of extracted bacteria was

similar after 3 � 1 min homogenization compared with a

Fig. 1. The influence of competing DNA on amplification efficiency.The ratio indicates the number of 16S rDNA copies of competingDNA/number of 16S rDNA copies of template DNA.W DNA from B. subtilis was mixed with DNA from S. lividans indifferent proportions and PCR amplified. Values indicate thenumber of 16S rDNA copies obtained per template from B. subtilis.D DNA from S. lividans was mixed with DNA from B. subtilis indifferent proportions and PCR amplified. Values indicste thenumber of 16S rDNA copies obtained per template from S. lividans.In all cases a ,700 bp region in the 16S rDNA gene was amplifiedusing primers for Eubacteria (Table 1). Quantification of the PCRproducts was done by dot-blot hybridization/phosphorimaging. Theprobe FGPS621 (Table 1), specific for bacteria with a low GCcontent, was used to enumerate the number of 16S rDNA copiesderived from B. subtilis and the probe FGPS617 (Table 1), specificfor actinomycetes, was used to enumerate the number of 16SrDNA copies derived from S. lividans. n�1 for all values.

Fig. 2. Number of PCR amplification products obtained pertemplate when different amounts of template were present in 50 mlof PCR solution. A , 700 bp region in the 16S rRNA gene wasamplified using primers for Eubacteria (Table 1).V DNA from only B. subtilis was present in the PCR solution.W DNA from B. subtilis was mixed with DNA from S. lividans indifferent proportions and PCR amplified. Values indicate thenumber of 16S rDNA copies obtained per template from B. subtilis.D DNA from S. lividans was mixed with DNA from B. subtilis indifferent proportions and PCR amplified. Values indicate thenumber of 16S rDNA copies obtained per template from S. lividans.Quantification of the PCR products was carried out using dot-blothybridization/phosphorimaging (see Fig. 1). n�1 for all values.



single treatment of 1 min 30 s (12.6% and 14.5%

respectively). However, the amount of DNA extracted

was significantly higher (P � 0.05) after 3 � 1 min homo-

genization [428 ^ 9 ng g21 dry weight (dw) of soil

compared with 185 ^ 5 ng after 1 min 30 s]. Less than

2% of the recovered bacteria were able to form colonies

(Table 2), in agreement with previous studies of the

culturability of indigenous soil bacteria (Bakken, 1985;

Amann et al., 1995).

The percentage of bacteria extracted after 3 � 1 min

was not affected by the buffer (0.05 M pyrophosphate,

pH 8.0, 0.9% NaCl, or pure water), confirming previous

results (Lindahl, 1996). However, because 0.05 M pyro-

phosphate buffer resulted in a brownish solution, sub-

sequent extractions were carried out using Waring

blender homogenization for 3 � 1 min in the presence

of 0.9% NaCl, as recommended by Lindahl and Bakken

(1995). Bacteria were then separated from soil in a

Nycodenz density gradient (see Experimental proce-

dures). A second extraction of the soil pellet, suggested

by Lindahl (1996), was attempted in order to increase the

bacterial yield. The soil pellet was resuspended in 0.9%

NaCl by vortexing, with or without subsequent sonication.

Sonication conditions were optimized to obtain a disper-

sion efficiency similar to that obtained using Waring

blender treatment (determined by plate counts; data not

shown). The second extraction resulted in only a 10%

increase in the bacterial yield, as determined by AODC

counts, viable counts and amount of DNA extracted

(Table 2). Therefore, we eliminated the second extraction

in order to expedite the processing of a large number of

samples.

Comparison of DNA yield and quality using direct

extraction and extraction from cells previously separated

from the soil

We previously described in situ lysis treatments for

releasing DNA directly from soil without previous extrac-

tion of bacteria (FrostegaÊrd et al., 1999), using the same

sandy loam soil as used in the current study. We found

that the amount of extracellular DNA, as determined by

vortex homogenization of the soil suspension, was 36 mg

DNAg21 dw soil, whereas lysis treatments, including

grinding, grinding plus sonication and grinding, sonication

plus enzymatic/chemical treatments (lysozyme±achromo-

peptidase±SDS) resulted in 59, 93 and 38 mg DNAg21 dw

soil respectively. In the current study, we found that the

amount of crude DNA isolated from bacteria extracted

previously from the soil matrix, but not yet purified on CsCl

gradient, was only 428 ng g21 dw soil. This value is

consistent with the number of extracted cells (1.9 � 108

bacteria), assuming a mean DNA content of 1.6±2.4 fg

per cell (Bakken and Olsen, 1989).

Using direct extraction protocols, particularly those

including a sonication step, the extracted DNA ranged in

size between 2 and 23 kb, resulting in smears on agarose

gels. However, DNA isolated from extracted bacteria and

purified on a CsCl gradient contained mainly 23 kb

molecules (Fig. 3), with few smaller fragments.

Analysis of soil bacterial diversity

Using our optimized PCR conditions and 1 ng of template

DNA, we amplified the various soil-derived DNA samples

Table 2. Direct counts and colony counts of bacteria extracted from soil, using single or repeated extraction proceduresa

Treatments AODCb

cells g21 dwTotal culturablebacteriacfu g21 dw

Total culturableA ctinomycetescfu g21 dw

DNA extractng g21 dw

Soil suspension 1.3 � 109 (^ 0.1) 6.9 � 106 (^ 0.4) 8.6 � 106 (^ 1.2)First Nycodenz extraction 1.9 � 108 (^ 0.2) 4.1 � 106 (^ 1.5) 2.5 � 106 (^ 0.7) 333 (^ 35)Second Nycodenz extraction

Vortex homogenization 3.9 � 107 (^ 0.2) 2.0 � 105 (^ 0.2) 2.7 � 105 (^ 0.2) 28 (^ 9)Vortex 1 sonication 4.1 � 107 (^ 0.6) 4.5 � 104 (^ 1.5) 5.2 � 104 (^ 0.4) c

a. All values are means ^ standard deviation (n � 3).b. Acridine Orange Direct Count.c. Recovered DNA below detection limit.

Fig. 3. Gel electrophoresis (0.8% agarose in Tris borate±EDTAbuffer) of DNA extracted from the Nycodenz gradient-purified cellfraction after 1 min 30s (1) or 3 � 1 min (2) Waring blenderdispersion time. Various concentrations of calf thymus DNA wereblotted (A±D: 260, 130, 65 and 32.5 ng respectively) to allow DNAquantification. M, l DNA size marker.



obtained by either direct extraction or cell extraction. We

used prokaryote-specific primers FGPS612 and

FGPS669, which gave rise to PCR products that formed

a thick band in agarose gels, indicating a preponderance

of heterogeneous fragments of < 700 bp. This result

confirmed the sufficient size and purity of the template

DNA and its origin from diverse bacteria that differed in

the size of the amplified region.

After hybridization with the various oligonucleotide

probes, the level of hybridization was quantified by

phosphorimaging, based on calibration curves estab-

lished using amplified DNA from several representatives

of each phylogenetic group. The ratio of prokaryotic to

eukaryotic DNA was not determined, but some experi-

ments using PCR amplification with Eukarya-specific

primers (designed by Amann et al., 1995) on the small

subunit rRNA gene showed the presence of eukaryotic

DNA in all in situ lysis extracts, whereas eukaryotic DNA

was not detected in DNA derived from the Nycodenz

procedure.

When hybridization signals with each probe were

calculated as a percentage of the total signal obtained

with the probe specific for Eubacteria, more than half the

DNA amplified from lysed soil bacteria escaped hybridiza-

tion with the group-specific probes (data not shown). This

might reflect that not all the bacterial groups were

represented by the probes (for example, the cyanobac-

teria, as well as the d-subclass of Proteobacteria, were

not targeted in this study). A more probable hypothesis is

the presence of DNA from unknown and/or uncultivated

soil bacteria. New probes targeting new phyla of

uncultured bacteria, such as Acidobacterium or Verruco-

microbia phyla, have now been designed and could be

used to obtain a more reliable picture of the soil bacterial

diversity (Lee et al., 1996; Ludwig et al., 1997).

Table 3 shows the percentages of the different bacterial

groups, based on the sum of the signals obtained from the

different groups. Except for the genus Streptosporangium,

a positive signal was detected for each of the probes,

indicating the presence of the corresponding microorgan-

isms in the soil sample. According to the results based on

the relative ratio of the different taxa detected after direct

DNA extraction, the pattern representing the fraction

obtained after washing of the soil was similar to those

obtained after the different direct lysis treatments. Inter-

estingly, the positive effect of additional lysis on detection

of the actinomycete population confirmed previous obser-

vations (FrostegaÊrd et al., 1999), based on in vitro culture

data, that additional lysis steps are required to release

DNA from Streptomyces. This also demonstrated the

sensitivity of the PCR hybridization method in detecting

variations in the composition of DNA extracts of different

population levels.

A comparison of the DNA distribution patterns obtained

using direct extraction and cell extraction procedures

shows that the results are generally similar except for (i)

the actinomycetes, which dropped from 28% to 42% using

direct extraction to less than 17% using the cell extraction

method (Table 3); and (ii) the g-Proteobacteria, which

showed more than a twofold percentage increase using

the cell extraction method. With regard to the actinomy-

cetes, an additional step in the indirect procedure

(incubation in 6% yeast extract solution for 1 h at 408C

to induce bacterial spore germination before the Waring

blender dispersion) resulted in an increase in detection of

actinomycetes and Streptomyces DNA to levels observed

using the direct DNA extraction procedure (data not

shown). This suggests that spores may escape the

Nycodenz extraction or the lysis treatment with detergent

and lysozyme.

Assuming that the DNA extracted by the two methodol-

ogies is a reflection of the microbial spectrum in the soil,

Table 3. Percentages of a2, b2, g ±subclasses of Proteobacteria, low GC% G1 bacteria, Actinomycetes and Streptomyces DNA extracted bydirect extraction or cells previously separated from the soil, as determied by PCR hybridization (the sum of signals obtained with each probe,except the Streptomyces probe, was taken as 100%).

Target probe

a-Proteobacteria% (^ S.D)a

b-Proteobacteria% (^ S.D) a

g-Proteobacteria% (^ S.D) a

low GC Gram1% (^ S.D) a

Actinomycetes% (^ S.D.) a

Streptomyces% (^ S.D) a

Direct extractionSoil washing with buffer 25.3 (^ 3.1) 18.6 (^ 3.1) 12.5 (^ 0.2) 15.4 (^ 1.2) 28.2 (^ 2.3) b

Grinding 22.6 (^ 1.4) 15.5 (^ 1.5) 9.7 (^ 2.6) 9.1 (^ 4.9) 43.1 (^ 1.7) 2.3 (^ 0.3)Grinding 1 sonication 28.5 (^ 5.2) 18.1 (^ 1.4) 13.3 (^ 1.4) 9.7 (^ 0.4) 30.4 (^ 0.2) 5.1 (^ 1.0)Grinding 1 sonication 1enzymatical lysis

20.3 (^ 0.4) 18.1 (^ 1.6) 11.9 (^ 2.2) 10.2 (^ 0.6) 39.6 (^ 4.3) 6.3 (^ 0.8)

Indirect extractionCell fraction lysis 1 CsClgradient

22.7 (^ 2.5) 13.3 (^ 1.0) 29.8 (^ 1.0) 16.5 (4.6) 17.7 (5.8) 6.2 (^ 2.7)

a. All values are means ^ standard deviation (n � 3).b. Values below the detection limit.



our results show that the targeted taxonomic groups are

represented fairly evenly. Using group-specific hybridiza-

tion of soil rRNA, Felske et al. (1998) quantified the most

active bacteria in a grassland soil and found a dominance

of Bacillus species. a-Proteobacteria, b-Proteobacteria, d-

Proteobacteria, low GC% Gram-positive and high GC%

Gram-positive represented 22% (^ 5%), 1.5%, 0%, 49%

(^ 11%) and 19% (^ 6%), respectively, of the eubacterial

rRNA present. As suggested by in vitro culture data, the

spectrum of bacteria present in a given soil depends on

biological conditions as well as chemical and physical

properties of the soil.

In conclusion, results of microbial diversity analyses,

using both the direct DNA extraction and cell extraction

strategies described here, gave rise to a similar spectrum

of microbial diversity. These results suggest that the

Nycodenz procedure may be a useful alternative to the

direct extraction approach. Desired quantity and purity of

DNA are critical points for the method choice. Cell

extraction procedure provides lower DNA yield, but the

relatively high number of cells obtained by centrifugation

in the Nycodenz gradient and the high purity of the DNA

extracted from these cells underscores the potential

usefulness of this technique for studies of soil microbial

communities based on molecular methods. The cell

extraction technique provides a major advantage in

overcoming the bias of cultivation and, in addition, it

avoids problems of extracting microbial DNA directly from

soil, including the co-extraction of large amounts of

extracellular DNA as well as contaminating impurities

such as humic acids.

Experimental procedures

Strains

Proteobacteria strains (Agrobacterium tumefaciens, Azospir-illum brasilense, Nitrobacter, Rhizobium leguminosarum,Alcaligenes eutrophus, Burkholderia cepacia, Ralstoniasolanacearum, Acinetobacter calcoaceticus, Azotobactervinelandii, Escherichia coli, Pseudomonas putida, Pseudo-monas fluorescens) and low G1C content Gram-positivebacteria strains (Bacillus subtilis, Bacillus anthracis, Lacto-coccus lactis, Rhodococcus sp., Staphylococcus sp., Strep-tococcus sp.) were grown in Luria±Bertani (LB) broth.Actinomycetes strains (Streptomyces alboniger, Strepto-myces ambofaciens, Streptomyces lividans, Streptomycesviolascens, Streptosporangium fragile, Streptosporangiumvulgare, Streptoverticillium abikoensis, Actinoplanes sp.,Frankia) were cultured using the method of Hickey andTresner (1952).

Soil

Soil samples were collected from the upper 5±10 cm of anarable field at La CoÃte Saint AndreÂ, IseÁre, France. The soil

was a sandy loam (pH 5.6) with an organic matter content of40.6 g kg21 dw. All visible roots were removed, and thesamples were was stored at 48C.

Direct extraction of total DNA from soil

Soil samples were dried for 24 h at room temperature andsieved (2 mm mesh). Several protocols were used to comparedifferent lysis treatments: (i) Extraction of extracellular DNA ±no lysis treatment. Soil samples (2 g dw) were suspended in5 ml of TENP buffer [50 mM Tris, 20 mM EDTA, 100 mMNaCl, 1% (w/v) polyvinylpolypirrolidone, pH 9.0], vortexed for10 min and centrifuged (4000 g for 5 min). The supernatantwas precipitated with isopropanol and resuspended in 100 ml ofsterile TE (10 mM Tris, 1 mM EDTA, pH 8.0). After purificationthrough two successive columns, Sephacryl S400 HR column(Pharmacia Biotech) and Elutip d (Schleicher and Schuell), theDNA was precipitated with ethanol and resuspended in TEbuffer. (ii) Soil grinding. Dry soil samples were ground at fullspeed for 10 min in a Reitsch centrifuge grinding mill (Polylabo)equipped with tungsten beads and then suspended in TENPbuffer (2 g 5 ml21), vortexed and centrifuged (see above). TheDNA in the supernatants (corresponding to 2 g of soil) wasextracted and purified as described above. (iii) Soil grinding andsonication. After grinding and suspension in TENP buffer, thesamples were sonicated using a 600 W Cup Horn Vibracellultrasonicator (Bioblock) at 15 W for 10 min with 50% activecycles. The remainder of the protocol was as per protocol (ii).(iv) Soil grinding, sonication and enzymatic/chemical treatment.After sonication, the soil suspensions were incubated for 1 h at378C in the presence of 0.3 mg ml21 lysozyme±achromopep-tidase, followed by the addition of lauryl sarcosyl (finalconcentration 1%) and incubation for 30 min at 608C. Thesamples were then centrifuged, and the DNA was precipitatedand purified as described above.

Extraction of bacteria from the soil matrix and subsequent

lysis

Soil was sieved through a 2-mm mesh. Soil samples (5 g)were suspended in 50 ml of 0.9% NaCl and homogenized ina Waring blender for 1.5 min or for 3 � 1 min at full speedwith intermittent cooling on ice after each minute. Cells andsoil particles were separated using high-speed centrifugationon a Nycodenz density gradient (Nycomed Pharma). A 7 mlNycodenz cushion with a 1.3 g ml21 density (8 g of Nyco-denz to 10 ml of ultrapure water) was placed below 20 ml ofsoil suspension in a centrifuge tube, followed by centrifuga-tion at 10 000 g in a Beckman SW 28 swing out rotor for40 min at 48C (Bakken and Lindahl, 1995). The bacterial cellfraction, floating at the top of the density gradient, wascarefully recovered with a pipette. The Nycodenz solutionwas removed from the bacteria by diluting the suspension insterile water before centrifugation for 20 min at 10 000 g. Thecell pellet was resuspended in 10 mM Tris2500 mM EDTA,pH 8.0. The number of bacteria recovered was estimated bycounting cells stained with acridine orange (Hobbie et al.,1977) and viewed through a microscope. Approximately 30randomly selected areas were counted per filter, each areacontaining not more than 20 cells. Total culturable aerobic



bacteria were plated on TS agar (Balestra and Misaghi,1997), and colonies were counted, whereas culturableactinomycete populations were monitored specifically onHV medium, as described previously (FrostegaÊrd et al.,1999). One additional extraction was carried out on the soilpellet obtained after the first Nycodenz density gradientcentrifugation. The soil pellet was resuspended in sterile0.9% NaCl solution by vortexing or sonication (CupHorn600 W ultrasonicator, 20 s, power 1, 10% active cycle). Cellsextracted from soil were lysed in a lysozyme±achromopepti-dase solution (5 mg ml21 and 0.5 mg ml21 respectively) for1 h at 378C. Lauryl sarcosyl (1% final concentration) wasadded, and the solution was incubated at 608C for 30 min.The DNA solution was then purified on a caesium chloride±ethidium bromide density gradient (35000 r.p.m. in a 65.13Kontron rotor for 36 h at 158C). DNA yields were estimatedon agarose gels (0.8% w/v) in Tris-borate±EDTA buffer bycomparing sample fluorescence intensity against a standardcurve of calf thymus DNA.

PCR conditions and dot-blot hybridization

PCR amplifications were carried out in a Perkin-Elmer 2400thermal cycler with Gibco PCR SuperMix (Gibco BRL LifeTechnologies), using primers FGPS612 and FGPS669(1 mM; Table 1), complementary to areas of highly conservedregions in the 16S rRNA gene. After an initial denaturation at948C for 3 min, 35 amplification cycles (948C for 30 s, 608Cfor 30 s, 728C for 60 s) were followed by a terminalelongation at 728C for 3 min. To eliminate amplifications ofprevious PCR products, we consistently used the carry-overprevention kit (Perkin-Elmer). Genomic DNA, extracted frombacterial strains using standard protocols (Hintermann et al.,1981; Sambrook et al., 1989), and soil DNA (1 ng) wereamplified under the same conditions to establish a calibrationrange. For soil DNA, we added T4 gene 32 protein (2.5 mg in50 ml of PCR mix; Boehringer Mannheim) to increaseamplification efficiency in case of contamination by humicsubstances (Kreader, 1996; Schwieger and Tebbe, 1997). Toassess amplification efficiency, 1/10th of the total volume ofPCR products was visualized after electrophoresis on 2%agarose gels. After denaturation, the PCR products wereblotted on a nylon membrane (GeneScreenPlus; Life ScienceProducts) and hybridized according to the method ofFrostegaÊrd et al. (1999). Hybridization signals were mea-sured with a radioanalytical imaging system (MOLECULAR

ANALYST software; Bio-Rad). Soil DNA concentrations wereestimated by interpolation from a calibration curve, obtainedby amplification of various amounts of appropriate templateDNA (DNA from various species were used to establishcalibration curves) and subsequent blotting of the products.

To quantify the influence of competing DNA on PCRamplification, DNA from two Gram-positive bacteria, B.subtilis (low G1C content) and S. lividans (high G1Ccontent), were mixed in various proportions, amplified andthe resulting amplified DNA quantified using dot-blot hybridi-zation. In one experiment, 25 pg of B. subtilis DNA wasmixed with increasing amounts of S. lividans DNA so that theproportions of Bacillus DNA to Streptomyces DNA, based onthe weight of the DNA, were 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10.Controls containing 25 pg of Bacillus DNA alone were

amplified in parallel. A similar experiment was carried outwith a constant amount of Streptomyces DNA (6 ng) andvarying the amount of Bacillus DNA. The ratios (w/w) oftemplate DNA to contaminating DNA were the same for bothexperiments. After amplification, the PCR products wereblotted on membranes and hybridized with probe FGPS621(B. subtilis) or FGPS617 (S. lividans) (Table 1). Thehybridization intensity of each dot was first recalculated intong of 16S rDNA using the standard curves derived for B.subtilis and S. lividans. The number of 16S rDNA fragmentswas then calculated based on the length of the amplified 16SrDNA region of B. subtilis (689 bp, which is 6.98 � 1024 fgbased on an average molar weight for DNA basepairs of610 Da), and S. lividans (694 bp, which is 7.03 � 1024 fg).

Acknowledgements

The work was supported by RhoÃne Poulenc Rorer and theSwedish Council for Forestry and Agricultural Research (AÊ .F.).

S.C. received a grant from the Agence Nationale pour la

Recherche Technique. We are grateful to Dominique Bernillon

and AgneÁs Combe for technical assistance, and Marcia Osburnefor comments on the manuscript.

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Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation