Journal

of
Methods
Microbiological
Journal of Microbiological Methods 32 (1998) 2129
Extraction of ribosomal RNA and genomic DNA from soil for
studying the diversity of the indigenous bacterial community.
a ,b a ,b a
Gabriela Frois Duarte , Alexandre Soares Rosado , Lucy Seldin , Anneke C. Keijzer-
b b,
*
Wolters , Jan Dirk van Elsas
a
Instituto de Microbiologia Paulo de Goes, Centro de Ciencias da Saude, Bloco 1, Ilha do Fundao, Rio de Janeiro, RJ 21944, Brazil
b
IPO-DLO, P.O. Box 9060, 6700GW Wageningen, The Netherlands
Received 11 December 1997; accepted 15 December 1997
Abstract
A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA
(rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA
as well as small and large ribosomal subunit RNA from four soils of different texture. DNA and rRNA yields from these
21
soils were 1530 and 0.251.0 mg g soil, respectively. Following different purication steps, the rRNA as well as
genomic DNA extracts obtained were sufciently pure for either reverse transcription and polymerase chain reaction (PCR)
amplication, or direct PCR amplication. Using a set of universal bacterial primers based on conserved regions of the 16S
rRNA sequence, both approaches yielded mixed target molecules for subsequent denaturing gradient gel electrophoresis
ngerprinting of soil microbial diversity. The amplied rRNA-based bacterial diversity assessment was compared with
diversity assessments based on amplied DNA in one selected soil. Results showed similarities as well as differences
between the proles generated on the basis of rRNA and those based on genomic DNA, which suggested that the bacterial
communities dened on the basis of their genomic DNA contained variable amounts of rRNA. 1998 Elsevier Science
B.V.
Keywords: Ribosomal RNA; Soil; Bacterial diversity; Denaturing gradient gel electrophoresis
1. Introduction cation strategies have been developed in the last
decade. Strategies for DNA extraction have been
Studies on the genetic diversity of microbial based either on direct cell lysis and DNA extraction
communities in soils have primarily been based on (pioneered by Ogram et al. [5]) or on an indirect,
analyses of total microbial community nucleic acids two-step (cell extraction followed by DNA extrac-
[14]. To obtain these nucleic acids in a form tion) approach (pioneered by Holben et al. [6]).
suitable for analysis, different extraction and puri- Since these early studies, numerous simplied proto-
cols have been developed, and the emphasis has been
increasingly placed on direct extraction as it often
provided the highest DNA yields. Whereas direct and
* indirect soil DNA extraction methods have been well
Corresponding author. Tel.: 131 317 476210; fax: 131 317
410113; e-mail: j.d.vanelsas@ipo.dlo.nl developed and are widely used [1,2,79], methods
0167-7012/ 98/ $19.00 1998 Elsevier Science B.V. All rights reserved.
PI I S0167- 7012( 98) 00004- 9
22 G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129
geared to obtain RNA from soil microbial popula- 2. Materials and methods
tions have been less well developed [1014].
The use of ribosomal RNA (rRNA) as a basis for 2.1. Soils
assessing microbial diversity offers the potential
advantage of the natural amplication of target Four soils of different texture were used in the
numbers, as one cell can contain thousands of development of the nucleic acid extraction protocol.
ribosomes. Moreover, many bacterial species are These were the Dutch soils Flevo silt loam (FSL),
known to vary their ribosome numbers in accordance Ede loamy sand (ELS) and Loss (L), and a Finnish
with their cellular activity [15], and hence rRNA- organic soil (FOS). FOS is a high-organic-matter soil
based approaches can provide a means to analyze the received from Dr. M. Salkinoja-Salonen (University
diversity of the active fractions of microbial com- of Helsinki, Finland). Characteristics of all soils have
munities in soil. Some soil rRNA extraction proto- been described before [18,22,23]. Briey, ELS is a
cols have been proposed [1114,16], and these moderately acid (pHKCl 5.5) beekeerd soil with
sometimes resulted in the simultaneous extraction of high organic matter content (3.5%), FSL is a neutral
RNA and DNA (e.g. [13]). However, these protocols (pHKCl 7.2) silt loam with about 2% organic
all isolated RNA directly from soil, which resulted in matter, and L is a moderately acid silt loam. The
drawbacks with respect to the purity of the RNA FOS soil was slightly acid (pHH O 6.0), had 30%
2
obtained. Moreover, the RNA produced might have organic carbon (% w/ w) and a water holding
originated from (partially) lysed cells [13,17] and capacity of 147% [23].
might therefore not be representative for the micro-
bial cell populations present in soil. 2.2. Glass- and plasticware and solutions
Recent molecular ecological studies in soils have
been based on both direct and indirect soil DNA All glassware used was previously treated by
extraction protocols [18], and such studies have baking at 1808C for at least 6 h. Plasticware was
permitted the assessment of the fate of both intro- soaked in a 0.1% (v/ v) diethyl pyrocarbonate
duced microorganisms [18,19] and functional genes (DEPC; Fluka, Buchs, Switzerland) solution for 2 h,
[20], and of indigenous bacterial communities ([21]). followed by autoclaving at 1258C for 30 min. All
As DNA-based studies of indigenous bacterial com- solutions were prepared with DEPC-treated water
munities in soil and rhizosphere revealed remarkable (overnight with 0.1% DEPC, 378C, followed by
stability of molecular proles over time versus autoclaving) in pretreated glassware and autoclaved
treatments ([21]), we surmised that a rRNA-based at 1218C for 20 min.
analysis might more readily reveal shifts in com-
munity structure or activity (reecting adaptational 2.3. Extraction of RNA and DNA
processes), than a DNA-based one. Hence, a novel
rapid and simple indirect rRNA extraction and A protocol based on lysis by bead beater treatment
purication protocol, based on the action of sodium of cells followed by subsequent RNA extraction and
pyrophosphate (NaPP) to disrupt cellsoil aggregate purication steps, was developed. The steps in the
bonds and the lysis and extractive power of bead procedure are described below (Fig. 1), whereas the
beating and hot acid phenol, was developed. The protocol development is described in Section 3.
protocol produced high yields of rRNA as well as Briey, 4 g of soil was taken up in 15 ml of 0.1%
DNA. It was suitable for microbial diversity mea- (w/ v) NaPP containing 2 g of gravel (24 mm
surements via reverse transcription-polymerase chain diameter), shaken for 10 min (250 rpm), and soil
reaction (RT-PCR) (rRNA) or direct PCR (DNA), particles and gravel were pelleted by centrifugation
both followed by denaturing gradient gel electro- (3 min, 121 g, room temperature). The resulting
phoresis (DGGE). We here describe this protocol and pellet was reextracted using 5 ml of sterile 0.1%
its application in soil microbial diversity measure- (w/ v) NaPP, spun (3 min, 121 g) and the supernatant
ments. separated. This process was repeated once. The
G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129 23
centrifuged (5 min; 5900 g; room temperature), and
the upper aqueous phases extracted once with acid
phenol (pH 5.0), followed by three extractions with
acid phenolchloroformisoamylalcohol (25:24:1),
pH 5.0. The nucleic acids in the resulting prepara-
tions were precipitated with 0.6 Vol of isopropanol
after addition of acid (pH 5.5) sodium acetate (0.3 M
nal concentration), washed with 70% ethanol and
resuspended in 200 ml of TCM buffer (10 mM
Tris?HCl pH 7.50.1 mM CsCl5 mM MgCl ). The
2
crude extracts thus obtained contained rRNA frac-
tions, including 16S rRNA, as well as chromosomal
DNA, and can be used for rRNA- as well as DNA-
based analyses.
DNA purication steps followed those outlined in
van Elsas et al. [18] for direct extractions, including
CsCl precipitation and Wizard (Promega, Leiden,
The Netherlands) DNA clean-up steps. Further puri-
cation of rRNA in the crude extracts was achieved
via the addition of 10 U (1 ml) RNase-free DNase-I
(Boehringer, Almere, The Netherlands) to 50 ml
crude extract and 49 ml TCM buffer, followed by
incubation at 258C for 3 h. Final purication con-
sisted of the use of RNeasy RNA clean-up columns
(Qiagen, Hilden, Germany), as described by the
manufacturer. For the (high-organic) ELS and FOS
soils, Sephadex G75 (Pharmacia, Roosendaal, The
Netherlands) spin column purication was necessary
for additional purication, in accordance with Moran
Fig. 1. Simultaneous indirect extraction of ribosomal RNA and
et al. [12]. The rRNA thus obtained was taken up in
genomic DNA from soil microbial communities.
50 ml of Milli-Q puried water and stored at 2808C.
Reverse-transcribable rRNA was obtained from all
supernatants containing the cells were pooled (total four soils.
volume about 25 ml) and centrifuged (20 min, The RNA obtained was visualized using conven-
21 000 g), after which the cell pellet was resuspend- tional agarose gel electrophoresis [25]. It was quan-
ed in 10 ml of sterile 0.1% (w/ v) NaPP and tied by using uorescent staining with the
TM
subjected to a second high-speed centrifugation step RiboGreen RNA quantitation kit (Molecular
as before. The resulting cell pellet was resuspended Probes, Leiden, The Netherlands), and reading in an
in 4 ml of 120 mM sodium phosphate buffer (pH LS50B uorometer (Perkin-Elmer, Nieuwerkerk,
5.8), and 3 g of 0.1 mm (diameter) glass beads, 500 The Netherlands). The RNA provided in the kit
ml 20% sodium dodecyl sulphate (SDS), and 3.5 ml served as the standard for calibration.
acid phenol (pH 5.0) were added. The cell suspen-
sions were subjected to two bead beating steps (23 2.4. RT-PCR and PCR amplications
60 s) in a Braun (Melsungen, Germany) cell
homogenizer, with an intermittent 10 min heat rRNA (1 ml of undiluted as well as tenfold diluted
(608C) treatment (modied after [24]). After this puried preparations) was reverse-transcribed in 50
mechanical disruption of cells, suspensions were ml mixes at various temperatures, i.e., 60, 62, 68 and
24 G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129
708C (30 min), using the commercial GeneAmp EZ lands) apparatus consisting of a waterbath main-
rTth RNA PCR kit (Perkin-Elmer). Universal bac- tained at 608C and a set-up for running denaturing
terial primer 1401R (300 nM15% dimethyl sulfox- gradient gels. Polyacrylamide (6%) gels with gra-
ide) was used [3]. The resulting cDNA product was dients between 45% and 65% denaturants (UF5
subsequently amplied via PCR using a eubacterial ureaformamide) were prepared in accordance with
universal primer set for DGGE, i.e., primers 968F Muyzer et al. [27]. The solution with 45% denaturant
and 1401R, spanning (roughly) the 9601420 region contained 3.15 M (18.9% w/ v) urea plus 18% (v/ v)
(including variable regions V6 through V8) of the formamide and the one with 65% denaturant 4.55 M
16S rRNA molecule (Escherichia coli numbering urea (27.3% w/ v) plus 26% (v/ v) formamide. The
system of Brosius et al. [26]). A GC clamp described running time and voltage were standardized at 16 h,
by Muyzer et al. [27] was xed to the 968F primer. 100 V. After electrophoresis, gels were stained for 60
These primers are very suitable for DGGE analysis min with SYBR green I nucleic acid gel stain (3 ml
4
of the diversity of whole bacterial communities, of a 10 -fold concentrated solution in 10 ml; Molec-
yielding a PCR product of about 450 base pairs (bp) ular Probes) and photographed under UV light using
[3]. As controls for the amplication of rRNA- a SYBR green gel stain photographic lter (Molecu-
derived molecules instead of any remaining DNA in lar Probes).
the preparations, mixtures containing soil rRNA that
had not been treated by RT were also subjected to
PCR amplication. These controls consistently yield-
ed negative results with highly-puried rRNA prepa- 3. Results
rations.
The PCR reaction mixes (50 ml) contained 5 ml 3.1. Nucleic acid extraction from soils
RT reaction product or 1 ml of puried soil DNA and
200 nM of both primers. They were further com- Initial attempts to obtain quantitative amounts of
posed as described [18]. AmpliTaq DNA polymer- rRNA from FSL soil by using the protocol of Felske
ase, Stoffel fragment (Perkin-Elmer) was used ac- et al. [16] failed, as rRNA yields were poor (,5 ng
21
cording to the manufacturers instructions. PCR was g soil) and no products were obtained following
performed in a Peltier thermal cycler PTC-200 (MJ RT-PCR. Moreover, this protocol was found to be
Research, Watertown, MA, USA), initially using 40 laborious as it includes several high-speed centrifu-
cycles of 948C 30 s, 558C 30 s, 728C 60 s, followed gation steps, followed by time-consuming resuspen-
by nal extension for 10 min at 728C. Alternatively, sion steps. We therefore decided to abandon the
a touch-down temperature cycling scheme was used, ribosome-rst method, in favour of the cell ex-
lowering the annealing temperature every two cycles tractionlysisrRNA extraction approach. As one of
by 28C, from 658C to 578C for 10 cycles, after which the general difculties with extractions from soil is
30 cycles at 558C were run. The latter temperature the lysis efciency of cells, highly efcient bead
cycling produced consistent results and was adopted beater lysis [18] was applied, combined with hot acid
as the standard. phenol and SDS treatment [24]. This was followed
PCR products were analyzed by electrophoresis on by extraction based on acid phenolchloroform
1.4% (w/ v) agarose gels stained (after the run) with steps. The procedure is outlined in Fig. 1. It yielded
21
crude extracts containing high amounts of both ethidium bromide (5 mg ml ); amplicons from
rRNA and DNA from four soils (Fig. 2). RNA yields high-yield PCR reactions were subjected to DGGE
ranged from 0.250.75 (FOS) to 1.0 (FSL, ELS and analysis.
21
L soils) mg g soil, whereas DNA yields were
between roughly 15 (FSL, ELS and L) and 30 (FOS)
21
2.5. DGGE mg g soil. The crude nucleic acid preparations
were subjected to parallel purication procedures to
DGGE of the mixed PCR products (2030 ml) generate pure rRNA or genomic DNA for subsequent
was performed using an Ingeny (Leiden, The Nether- PCR/ DGGE analyses.
G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129 25
were negligible. Fig. 2 shows the respective crude
and puried extracts obtained. The puried extracts
were ready for RT as described below.
To obtain pure soil microbial community DNA,
the crude nucleic acid extracts were subjected to
two-step purications, consisting of CsCl precipi-
tation of contaminating compounds and Wizard spin
column clean-up, in accordance with Refs. [18,28].
Occasionally, potassium acetate precipitation was
applied [7,28]. As expected, these steps yielded PCR
ampliable DNA devoid of rRNA for all soils.
Moreover, the losses incurred during DNA purica-
tion and recovery were low in all cases, in accord-
ance with recent data [18].
3.3. RT-PCR and PCR amplications of soil rRNA
and DNA
Fig. 2. Crude and puried rRNA extracts obtained from selected
The puried rRNA extracts obtained from the four
soils. Lanes: 1 kb5molecular size marker (Boehringer 1-kb
soils were subjected to RT and PCR. Controls
ladder, molecular sizes of three key bands in bp indicated);
performed to ascertain that rRNA instead of any
1,25FSL soil; 3,45L soil; 5,65ELS soil; 7,85FOS soil. Lanes 6
remaining DNA was reverse-transcribed and am-
and 8: inserted from parallel gel.
plied conrmed the RT activity when 60 or 628C
was used for primer annealing, but not at 68 or 708C.
3.2. Purication steps for rRNA and DNA Hence, RT with primer 1401R required a lower
temperature than published [16] for its activity.
As the crude extracts (Fig. 2) contained large Robust double-stranded DNA bands of expected
quantities of rRNA and DNA and, in addition, were sizes (about 450 bp) were obtained after the RT-PCR
sometimes brownish (ELS and FOS soils), indicating steps performed with (low) dilutions of extracts of
the presence of humic compounds, further purica- the low-organic soils FSL and L (Fig. 3), whereas
tion was applied using the steps outlined in Fig. 1. weaker bands were obtained with dilutions of ELS
To obtain pure rRNA, DNA was rst completely and FOS soils (not shown). The negative controls, in
removed using DNase treatment; the efciency of the which the RT step was omitted, showed negative
enzymatic treatment was checked via gel electro- PCR amplication in all cases, whereas a positive
phoresis. For all soils, a 3 h treatment was sufcient control, i.e., Ralstonia solanacearum rRNA subject-
to completely remove DNA. Following this step, ed to RT-PCR, revealed a band of similar size.
rRNA clean-up (RNeasy) and (optionally) Sephadex Whereas with L soil RT-PCR products were readily
columns were used, to quickly obtain rRNA suitable obtained, for FSL soil dilution was necessary to
for RT. Two soils, FSL and L, already yielded obtain optimal products, as judged by the intensities
colourless rRNA preparations (devoid of DNA) after of bands on agarose gels (Fig. 3). Apparently,
the rst RNeasy spin column purication step, undiluted rRNA extracts obtained from FSL soil still
whereas preparations from ELS and FOS soils were contained amounts of inhibitors that hindered ef-
still slightly brownish. Subsequent Sephadex G75 cient RT.
column purication removed most of the colour from The puried DNA extracts obtained with the four
the latter preparations. With the exception of the FOS soils were successfully used to generate PCR prod-
soil, the puried rRNA preparations still contained ucts for DGGE analysis using the same primers as
21
quantities of rRNA on the order of 1 mg g soil, above. Fig. 3 shows the amplicon obtained with one
indicating that any losses incurred during purication soil.
26 G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129
Fig. 3. PCR amplication products obtained from FSL and L soils after RT-PCR under different conditions. Lanes: 1 kb5molecular size
marker (Boehringer 1-kb ladder); 1 through 45FSL rRNA reverse-transcribed and PCR amplied, as follows: 15undiluted; 251:3 diluted;
351:9; 451:27; 55FSL rRNA, no RT (negative control); 6 through 95L soil rRNA, reverse-transcribed and amplied, as follows:
65undiluted; 751:3; 851:9; 951:27; 105L rRNA 1:3, no RT; 115negative (water) control; 125FSL DNA, PCR amplied; 135Ralstonia
solanacearum rRNA, reverse-transcribed and PCR amplied.
3.4. DGGE 4. Discussion
In one soil, FSL, we determined whether DGGE In this study, we developed a simple, rapid and
assessment of the diversity of the bacterial com- efcient protocol for the extraction of rRNA spe-
munities based on rRNA would reveal structures cically from bacterial cells in soil. The method was
different from or similar to those obtained via DNA- based on a combination of cell extraction from soil
based PCR/ DGGE. PCR products were generated and subsequent cell lysis via, respectively, NaPP
from undiluted as well as diluted genomic DNA and dispersal and bead beating [18], and the efciency of
rRNA extracts that had been obtained from the same hot acid phenol in extraction of RNA from cells [24].
soil samples. The results of this analysis are shown Extraction specically from cells was deemed im-
in Fig. 4. A multitude (20 up to 30) of bands were portant, as subsequent analysis of the rRNA aimed at
obtained using both approaches, and the proles an assessment of the diversity of the community of
were similar with respect to the presence of dominat- intact bacterial cells rather than free rRNA molecules
ing bands. Visual inspection of the two proles in soil. Due to their compact secondary structure,
revealed that at least 18 bands migrated to similar rRNA molecules might be rather stable in soil [13].
positions, which suggested they were similar. A few Nannipieri et al. [17] suggested both free DNA and
bands were conspicuously more intense in the DNA- RNA might become stabilized in soil following a
generated prole, whereas others were more intense release from lysing cells, and the small rRNA
in the rRNA-based patterns. A more thorough analy- molecules might be even better stabilized than the
sis of these proles, which are based on different larger genomic DNA molecules.
biomolecules representing the same bacterial com- Previously published protocols have either used
munities, encompasses cloning and sequencing stra- direct cell lysis/ nucleic acid extraction, in which
tegies that are not reported herein. total microbial cells are lysed directly in the presence
G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129 27
21
roughly between 15 and 30 mg g soil, which is
equivalent to about 2.5- to 5-fold the amounts
obtained from the same soils in a recently developed
indirect DNA extraction method [18]. The rRNA
yields were considerable as well, amounting to 0.25
21
1.0 mg g soil for all soils. These yields were in or
near the range reported by other workers. The
protocol of Hahn et al. [10] yielded 0.85 mg of rRNA
21 21
g of soil, and that of Felske et al. 0.2 mg g of
the same soil [16]. Moran et al. [12] reported rRNA
21
yields of 1.11.9 mg g forest soil. Using the
assumptions of these authors for the average ribo-
some content of bacterial cells in soil, and based on
9 21
microscopic bacterial counts of around 10 cells g
FSL soil (not shown), we estimated an rRNA ex-
traction efciency of 1030% for this soil. When
compared to yields of bacterial cells or DNA from
soil and assuming unbiased lysis and recovery, to our
view this represents a recovery level acceptable for
assessments of microbial populations in soil. We
attributed the enhanced production of nucleic acids
from the bacterial fractions obtained from FSL soil
to a combination of the high efciencies of cell
Fig. 4. Denaturing gradient gel electrophoresis analysis of micro-
bial diversity based on 16S rDNA specic PCR with total extraction, cellular lysis and nucleic acid extraction/
community DNA versus rRNA indirectly extracted from FSL soil.
recovery provided by NaPP soil dispersion, bead
Lanes: 15markers (from top to bottom, PCR products generated
beating/ SDS/ hot acid phenol lysis and nucleic acid
with genomic DNA from, respectively, Enterobacter cloacae
extraction steps.
BE1, Listeria innocua ALM105, Rhizobium leguminosarum
The current protocol will be very useful when
biovar trifolii R62, Arthrobacter sp. and (faint band) Burkhol-
deria cepacia P2); 25FSL rRNA, reverse-transcribed and PCR genomic DNA and rRNA are to be analyzed side-by-
amplied; 35FSL DNA, PCR amplied; 45markers (see lane 1).
side, like in the case of the microbial diversity
studies presented here. The advantage of parallel
nucleic acid extraction and analysis is that a com-
parison of soil microbial diversity on the basis of
of the soil matrix and RNA is subsequently obtained either the relative abundance of genomes (genetic
[1214], or direct ribosome extraction, in which diversity) or of that of ribosomes (activity diversi-
ribosomes are rst obtained from soil followed by ty) can be performed directly on one community of
rRNA extraction from the isolated ribosome mixture microbial cells. Combined DNA- and rRNA-based
[16]. Both approaches suffer from a number of diversity assessment was also reported in a recent
deciencies which relate to either the presence of study by Felske et al. [29]. However, these authors
contaminating compounds, to great losses of material based their conclusions on rRNA and DNA obtained
[16] or to uncertainty about the cellular location of by different methods. Notwithstanding their data,
the RNA obtained. For instance, the method of Hahn which suggested considerable similarity between the
et al. [10] yielded impure rRNA and posed difcul- microbial diversity on the basis of DNA- versus
ties for PCR amplication [16]. rRNA-based proles, it is likely that the use of DNA
In line with other studies [13,14], large amounts of and rRNA obtained via different strategies introduces
microbial genomic DNA were coextracted with the an experimental bias.
rRNA. The DNA obtained from all soils amounted to In this study, the different soils analyzed required
28 G.F. Duarte et al. / Journal of Microbiological Methods 32 (1998) 2129
different purication steps to obtain reverse-tran- assess these effects specically in such potentially
scribable rRNA. Whereas two silt loam soils with nutrient-responsive microbial groups, group-specic
low organic matter content, FSL and L, needed only primers are needed. It is a challenge for future
a few purication steps, extra purication steps were research to test the concept of differential activities
required for the high-organic soils ELS and FOS. of dened bacterial groups of interest by using the
This observation was in line with previous work with nucleic acid extraction method described herein in
these soils [18] and with the persistence of a brown combination with RT-PCR/ DGGE analysis using
colour in extracts of the latter soils, indicating the group-specic 16S rRNA-based primers.
presence of contaminating humic compounds. The
Sephadex G75 gel ltration step was highly efcient
in removing the colour without detectable losses of
Acknowledgements
the rRNA, which was in accordance with data
reported by Moran et al. [12] and Selenska-Pobell
G.F. Duarte and A.S. Rosado were supported by
[13].
fellowships provided by the National Research
The similarities between the DNA- and rRNA-
Council of Brazil (CNPq). We thank Dr. M. Salkino-
based bacterial diversity proles of the FSL soil may
ja-Salonen for providing the FOS soil, and Dr. K.
be ascribed to the fact that many of the organisms
Wernars for supplying the L. innocua strain.
underlying the DGGE proles (the dominating bac-
teria [3]) do not vary their rRNA content to a great
extent, resulting in similar relative abundancies of
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