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Applied and Environmental Microbiology, April 1999, p. 1786-1788, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Quantitative Selective PCR of 16S Ribosomal DNA Correlates Well
with Selective Agar Plating in Describing Population Dynamics of
Indigenous Pseudomonas spp. in Soil Hot Spots
Kaare
Johnsen,1,2,*
Øivind
Enger,2
Carsten S.
Jacobsen,1
Laila
Thirup,1 and
Vigdis
Torsvik2
Geological Survey of Denmark and Greenland,
Copenhagen, Denmark,1 and Department
of Microbiology, University of Bergen, Bergen, Norway2
Received 24 August 1998/Accepted 7 February 1999
 |
ABSTRACT |
We used a quantitative PCR method targeting 16S ribosomal DNA using
competitive PCR for specific detection of indigenous
Pseudomonas DNA in soil hot spots. The amount of
Pseudomonas DNA corresponded to the number of culturable
Pseudomonas bacteria on Gould's S1 agar. This represents
the first use of PCR for quantification of indigenous bacteria in
more than one sample of soil.
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TEXT |
It is a well-established fact
that only a small fraction of environmental bacterial communities
can be cultivated by current techniques. As a consequence, doubt has
been raised that results obtained with traditional agar plating are
representative of the actual processes in nature. Among the methods
that have been suggested for studying the unculturable fraction of
indigenous bacterial communities, some of the most prominent are based
on nucleic acids. Extraction and purification of DNA from soil have
come in focus (7, 18), and now commercial kits based on
recent techniques are successfully marketed (1).
As a means of quantifying unculturable bacterial populations,
competitive PCR (cPCR) is promising. cPCR employs the highly sensitive
PCR and bypasses quantification problems caused by differences in the
exponential PCR amplification of DNA by using an internal standard
(4). The internal standard is a competitive DNA
template which shares two primers and thus is coamplified in
competition with specific DNA sequences in the sample. Since the
lengths of the fragments differ, amplification products from the
internal standard and sample DNAs are readily separated on a gel.
Competitive PCR has been used in, e.g., marine environments
(14), but soil poses problems, as it is heterogeneous and
consists of large amounts of inhibitory compounds, so reproducible
results are more difficult to obtain. Lee et al. (13)
added 16S ribosomal DNA from strain EA25 to soil and could
thereafter quantify it by cPCR with good correlation. Lechner and
Conrad (12) compared cPCR with traditional cultivation
techniques for estimation of hydrogenase-containing bacteria in one
rice rhizosphere sample and obtained approximately the same enumeration
result by cPCR and cultivation in a single sample. Hallier-Soulier et
al. (6) monitored introduced toluene degraders by CFU
counting and cPCR with xylE in sterilized soil. They found
no clear correlation between CFU counting and DNA. Rosado et al.
(15) likewise found varying correlation between a DNA assay
(most-probable-number PCR) and CFU counting of introduced bacteria in
soil. Hence, only one study (12) has applied it for
detection of indigenous bacteria in one soil sample, and no study
has provided a comparison between quantitative PCR and traditional
cultivation techniques for detection and quantification of
indigenous bacterial population dynamics in soil.
Our purpose was to assess the reproducibility and appropriateness of
competitive PCR in soil ecosystems for quantification of an
indigenous Pseudomonas population in a soil hot spot.
The hot spots were excised bits of young barley roots submerged in soil and monitored during degradation. This report presents, for the
first time, a tight association between the number of
culturable bacteria and the DNA of indigenous bacteria in soil.
PCR mixtures.
Each PCR tube contained a total volume of 46 µl, with 33.4 µl of twice-distilled water, 4.5 µl of 10× Ampli
Taq PCR buffer (Perkin-Elmer Cetus, Norwalk, Conn.), 0.2 µl of each primer (in a 0.1 mM solution), 4.5 µl of 1% DNase-free
bovine serum albumin (Pharmacia, Uppsala, Sweden), 2 µl
of Gene Amp 10 mM deoxynucleoside triphosphate mixture
(Perkin-Elmer), 0.2 µl of Ampli Taq polymerase (Perkin-Elmer), and 1 µl of template DNA. cPCRs contained 1 µl of internal standard and 1 µl of sample as templates. All
primers (Table 1) were Gibco BRL custom
primers of desalted purity purchased from Life Technologies, Roskilde,
Denmark.
Specific PCR amplification.
To ensure PCR amplification
specific for Pseudomonas DNA, a
Pseudomonas-specific primer set should be found. Probe
PSMG (2) was reported to be
Pseudomonas specific, and we checked the specificity of
the Pseudomonas primer again on 1 August 1998 (11). The second primer was 9-27 (16). This
sequence is one of several specific for eubacterial DNA
conveniently placed 445 bp upstream of PSMG, hence giving
Pseudomonas-specific amplification. In order to test if
amplification using these two primers was specific, a panel of
Fast Soil DNA (Bio 101, Vista, Calif.)-purified DNAs from seven strains
were used for PCR (6 min at 94°C; 35 cycles of 30 s at 92°C,
30 s at 52.5°C, and 1 min at 68°C; 6 min at 68°C; and final cooling at 4°C). The PCR tubes were placed in the PCR machine after
the temperature had reached 94°C. The organisms used were Pseudomonas aeruginosa DSM50071, P. putida
DSM50208, P. fluorescens biovar I DSM50090,
P. fluorescens biovar V DSM50148, Vibrio
vulnificus DSM10143, Aeromonas hydrophila DSM30016, and
Corynebacterium glutamicum DSM20300. The V. vulnificus strain has 16S sequences closest to PSMG
(differing in 2 of 15 bases). The amplification proved specific for the
strains tested (Fig. 1).
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FIG. 1.
Test for specific PCR amplification with
PSMG as a Pseudomonas-specific primer.
Lanes: 1, P. aeruginosa; 2, P. putida;
3, P. fluorescens I; 4, P. fluorescens V; 5, V. vulnificus; 6, A. hydrophila; 7, C. glutamicum; 8, no template DNA; 9, pGEM ladder (fragment sizes [from the top], 2,645, 1,605, 1,198, 676, 517, 460, 396, 350, 222, 179, 126, and 75 bp).
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Construction of an internal standard.
In order to get an
internal standard, a shorter fragment with the same primers in the end
was constructed as described by Hallier-Soulier et al.
(6). Essentially, two primers within the fragment
spanning 9-27 and PSMG in P. aeruginosa
DSM50071 were constructed (Intern1R and Intern2) (Table 1). The
primers also had a 15-bp overlapping region, where the sequences
were complementary. The two outer fragments were amplified (9-27 plus Intern1R and Intern2 plus PSMG), and the products
were mixed and PCR amplified with 9-27 and PSMG as
primers. Gel electrophoresis confirmed the presence of one band
of the correct size, and the remaining PCR product was purified by the
use of a QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany)
as described by the manufacturer. Tenfold dilutions of the internal
standard were prepared and amplified with DNA from P. aeruginosa DSM50071. Products of the correct sizes were made,
indicating that the cPCR system worked. DNA from a soil
Pseudomonas strain, JAJ137 (8), was purified by
Fast Soil DNA and mixed with the internal standard in different
mixtures as described by Suzuki and Giovannoni (16). The
ratio of JAJ137 DNA to internal-standard DNA was the same in the PCR
products as in the template (data not shown). This suggests that the
relative efficiencies of amplification of the template and the internal
standard were the same (16). DNA was quantified
(A260) on a Shimadzu UV-240 Graphicord
UV-visible-light recording spectrophotometer.
DNA purification from soil samples.
The Fast Soil DNA
purification kit was used in accordance with the manufacturer's
instructions. However, cell lysis by the Fast Prep machine was replaced
by three freeze-thaw cycles with 15 s of vortexing after each
thawing. Samples likely to represent high and low levels of
Pseudomonas DNA in the sample set were then subjected to PCR
with a 10-fold dilution of the internal standard to find the
appropriate DNA level range. Twofold dilutions of the internal standard
were made and used as competitive template DNA in the PCR tests. An
example is shown in Fig. 2. Results for four samples per sampling day were assessed, and average values for
each day are shown in Fig. 3 (assuming
100% efficiency of DNA extraction from the soil). The numbers of
culturable Pseudomonas CFU on Gould's S1 agar, which is
known to be Pseudomonas specific (5, 9, 10), are
also depicted in Fig. 3 (17). The two methods give
strikingly similar results. Considering all of the soil samples tested
during the 56 days point by point, there is a coefficient of
correlation (r2) between CFU numbers and DNA
amounts of 0.60, corresponding to a level of significance of
P > 0.001 in a product moment correlation coefficient
analysis (3). Thus, our results demonstrate the ability of a
competitive PCR to quantify DNA in soil reproducibly, even though both
the DNA content and the number of culturable bacteria only varied
within approximately 1 order of magnitude. Furthermore, the results
show that culturability dependent methods, in some cases, are as
suitable as molecular methods for describing soil
microbial ecology.
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FIG. 2.
Example of a gel with products of a cPCR. Lanes: 1, pGEM
ladder (fragment sizes, 2,645, 1,605, 1,198, 676, 517, 460, 396, 350, 222, 179, 126, and 75 bp); 2 to 5, twofold dilution series with sample
+B, day 3; 6 to 9, twofold dilution series with sample +B, day 6.
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FIG. 3.
Comparison of numbers of CFU on Gould's S1 agar
specific for Pseudomonas and copies of
PSMG-amplified DNA (assuming 100% extraction efficiency
from the soil) specific for Pseudomonas. Error bars
represent standard deviations. Samples are excised bits of young barley
roots submerged in soil and monitored during degradation. Soil was
incubated at 10°C for 56 days, and agar plates were incubated at
20°C for 3 days (17). rDNA, ribosomal DNA.
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ACKNOWLEDGMENTS |
This study was supported by Danish Biotechnological Research and
Development Program grant 9502015. Kaare Johnsen's stay in Bergen was
partly funded by NorFa (Nordisk Forskerutdanningsakademi).
We thank laboratory technician Frida Lise Daae for skillful help in the laboratory.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Geological
Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV,
Denmark. Phone: 45 38 14 20 00. Fax: 45 38 14 20 50. E-mail:
kj{at}geus.dk.
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Applied and Environmental Microbiology, April 1999, p. 1786-1788, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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