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Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
Institut für Agrarökologie,
Bundesforschungsanstalt für Landwirtschaft (FAL), 38116 Braunschweig, Germany
Received 26 February 2001/Accepted 15 May 2001
Genetic profiling techniques of microbial communities based on
PCR-amplified signature genes, such as denaturing gradient gel
electrophoresis or single-strand-conformation polymorphism (SSCP)
analysis, are normally done with PCR products of less than 500-bp. The
most common target for diversity analysis, the small-subunit rRNA
genes, however, are larger, and thus, only partial sequences can be
analyzed. Here, we compared the results obtained by PCR targeting
different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of
the bacterial 16S rRNA gene with primers hybridizing to evolutionarily
conserved flanking regions. SSCP analysis of single-stranded PCR
products generated from 13 different bacterial species showed fewer
bands with products containing V4-V5 (average, 1.7 bands per organism)
than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the
additional bands (>1 per organism) were caused by intraspecies operon
heterogeneities or by more than one conformation of the same sequence.
Community profiles, generated by PCR-SSCP from bacterial-cell consortia
extracted from rhizospheres of field-grown maize (Zea
mays), were analyzed by cloning and sequencing of the dominant
bands. A total of 48 sequences could be attributed to 34 different
strains from 10 taxonomical groups. Independent of the primer pairs, we
found proteobacteria ( PCR-based methods have enormously
affected our understanding of global microbial diversity because they
have contributed to both the fast differentiation and identification of
cultivated microorganisms and the access to the vast majority of
microorganisms which have not yet been cultured in the laboratory.
Studies by Woese and coworkers laid the groundwork, contributed to the
characterization of noncultivated microorganisms, and placed them into
a phylogenetic system based on the sequence analysis of their rRNAs
(41). As a main target molecule for microbial ecology
studies of diversity, the small-subunit (SSU) rRNA or the
respective genes have been used. The levels of resolution of the DNA
sequences differentiate microorganisms to approximately the species
level (34). The SSU rRNA gene is composed of
alternating evolutionarily conserved and variable regions. The
conserved regions are ideal sites for primer binding in PCRs because it
can be predicted that they will bind even to DNA of unknown organisms.
The amplified products from environmental DNA which contain the
variable regions can then be used for identification after cloning and
sequencing of the genes. By compilation of sequence data, it can be
shown that the SSU rRNA genes of all living organisms contain a total
of nine variable regions, V1 to V9, scattered in the molecule, which, for bacteria, is approximately 1,520 nucleotides long
(25).
An important objective in many ecological studies is to understand the
natural variability of microbial communities, e.g., in response to
environmental changes. In this context it is often desirable to analyze
and compare a large number of samples. A useful approach to achieve
this goal is the use of a genetic profiling technique. By this means,
microbial community structures can be compared at the level of
fingerprintlike patterns. For such purposes, techniques such as
denaturing gradient gel electrophoresis (DGGE) (24),
temperature gradient gel electrophoresis (10, 17), or
terminal restriction fragment length polymorphism analysis (21) are often applied. A useful alternative method to
separate PCR products of the same size but with different sequences is single-strand-conformation polymorphism (SSCP) (13, 20,
27). In our laboratory, we have optimized SSCP for the analysis
of complex microbial communities by removal of one complementary strand
of the double-stranded PCR product prior to SSCP on nondenaturing gels
(30).
SSCP, DGGE, and temperature gradient gel electrophoresis have a high
potential for community analysis because single bands or genetic
profiles can be isolated and identified by DNA sequencing (11,
28). However, a limitation of these methods is the fact that
only partial sequences of up to about 500 nucleotides are separated
well. Most studies of microbial community diversity so far have been
based on the analysis of only one selected region or the SSU rRNA gene
containing one to three variable regions, e.g., the V3
(24), V1-to-V3 (8), V7 and V8
(11), V3-to-V5 (37), V4 and V5 (28,
30), or V6-to-V8 (9) region. In several of these
studies, single bands were isolated from profiles and identified by DNA
sequencing. However, we assumed that in studies based on just one PCR
product the reliability of the selection of the variable region for the
results of the community analysis might not be sufficient, and
therefore this comparative study of the effects of the V regions and
primer selection was initiated.
In order to amplify different variable regions from a large diversity
of bacteria, we selected primers which were complementary to flanking
conserved regions. We applied these "universal" primers to genomic
DNA of bacterial pure cultures and to environmental DNA extracted from
rhizospheres of field-grown maize (Zea mays). The diversity
of amplified products was characterized by SSCP. For pure cultures, we
determined the number of amplified and SSCP-distinguishable operons,
and from community patterns we determined the identities of the
dominant bands by DNA sequencing and comparison to known rRNA gene sequences.
Bacteria, cultivation, and DNA extraction.
All bacterial
pure cultures used in this study were obtained from the DSMZ (Deutsche
Sammlung für Mikroorganismen und Zellkulturen, Braunschweig,
Germany) and cultured at 28°C on media containing 1.5% (wt/vol)
purified agar. Agrobacterium tumefaciens (DSM 30150), Pseudomonas putida (DSM 50906), Pseudomonas
fluorescens (DSM 50090), Bacillus subtilis (DSM 4872)
and Paenibacillus polymyxa (DSM 36) were cultivated on DSM
M1; Escherichia coli (DSM 1607) and Azotobacter beijerinckii (DSM 282) were grown on Luria-Bertani broth (Difco, Detroit, Mich.); Arthrobacter oxydans (DSM 20119) was grown
on DSM M53; Flavobacterium johnsonae (DSM 2064) was grown on
DSM M67; Rhizobium trifolii (DSM 1980) and
Acinetobacter calcoaceticus (DSM 586) were grown on R2A
(Difco); Streptomyces viridochromogenes (DSM 40736) was
grown on DSM M65. Bdellovibrio bacteriovorus (DSM 50701) was
grown on cells of Pseudomonas putida, as recommended by the DSMZ.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Primers Hybridizing to Different Evolutionarily
Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based
Microbial Community Analyses and Genetic Profiling
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
, 
, and 
subgroups) and members of the
genus Paenibacillus (low G+C gram-positive) to be the
dominant organisms. Other groups, however, were only detected with
single primer pairs. This study gives an example of how much the
selection of different variable regions combined with different
specificities of the flanking "universal" primers can affect a
PCR-based microbial community analysis.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
70°C.
PCR amplifications of rRNA gene sequences.
PCR was performed
with the thermal cycler Primus 96 (MWG-Biotech, Ebersberg, Germany).
Amplifications from cultivated pure cultures were conducted in a final
volume of 50 µl, whereas DNA fragments from environmental-DNA
solutions were processed in a final volume of 100 µl. For pure
cultures, the PCR included 1.25 U of Taq polymerase
(Amersham Pharmacia Biotech, Freiburg, Germany), 1× PCR buffer with
1.5 mM MgCl2, 0.5 µM primers, and each dNTP at
200 µM (Amersham Pharmacia Biotech). For environmental
samples, the composition was slightly different: 3.75 U of
Expand-Taq HF (Roche Diagnostics, Mannheim, Germany), 5 µg
of T4 gene 32 protein ml
1 (Roche Diagnostics)
(36), and a final concentration of 2 mM MgCl2. Three primer pairs were chosen for
amplification of partial sequences of the 16S rRNA gene. For
amplification of the V2-V3 region, the forward primer was (5'-3') ACT
GGC GGA CGG GTG AGT AA, and the reverse primer was (5'-3') CGT ATT ACC
GCG GCT GCT GG. For the amplification of the V4-V5 region, we used
primers Com1 and Com2-Ph, described previously (30), and
for the V6-V8 region, we used primers f968 and r1346 (without a GC
clamp) (26). The binding sites of these primers according
to the positions in the E. coli SSU rRNA genes
(5) are shown in Table 1.
The primers were synthesized by MWG Biotech or TIBmolbiol (Berlin, Germany). The reverse primers were phosphorylated. Environmental DNA
and pure-culture DNA were amplified under the same conditions: 95°C
for 3 min, followed by 35 cycles of 1 min at 95°C, 50°C for 1 min,
72°C for 70 s, and finally 72°C for 5 min.
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Generation of genetic profiles by SSCP. SSCP analysis was conducted according to the single-strand community approach described previously (30). After purification of the double-stranded PCR products with Qiaquick (Qiagen, Hilden, Germany), the phosphorylated strands were removed by lambda exonuclease (Amersham Pharmacia Biotech) digestions (5 U for pure-culture PCR products and 10 U for community products) at 37°C for 2 h. Proteins were removed by phenol-chloroform extraction, and the DNA was precipitated as described by Sambrook et al. (29) and resuspended in a solution consisting of 8 µl of 10 mM Tris-HCl (pH 8.0) and 8 µl of denaturing loading buffer (95% [vol/vol] formamide, 10 mM NaOH, 0.025% [wt/vol] each of bromphenol blue and xylene cyanole). Samples were incubated for 2 min at 95°C and then immediately cooled on ice.
To generate SSCP, we used the gel matrix (MDE; FMC Bioproducts, Rockland, Maine) in final concentrations of 0.7-fold for products including the V6-to-V8 region and 0.6-fold for products generated with the other two primer pairs. The stock solution was twofold. The electrophoreses were carried out in a Macrophor sequencing apparatus (Amersham Pharmacia Biotech) with gels of 21 cm under conditions described before (31), except that the PCR products with the V6-to-V8 region were run at 26 instead of 20°C. Afterward, the gels were run and DNA was stained according to the silver-staining procedure described by Bassam et al. (1).Identification of SSCP bands. The procedures to isolate single bands from SSCP profiles for DNA sequencing were carried out as described previously (28). Gel-extracted DNA was used as a template in PCR. The PCR was conducted under the same conditions as the pure-culture amplifications. The identities of the reamplified products were confirmed in another SSCP analysis using the community profiles as references.
The reamplified products were then ligated into a pGEM-T vector and transformed into E. coli JM109 (Promega, Mannheim, Germany) according to a protocol of the supplier. Transformed cells with inserts were selected by blue-white screening. Cloned DNA fragments were amplified from the vector by PCR (with conditions as recommended by the manufacturer) using primers matching the flanking regions of the vector (forward, [5'-3'] CAC GAC GTT GTA AAA CGA C, and reverse, [5'-3'] GGA TAA CAA TTT CAC ACA GG). The sizes of the PCR products were determined by agarose gel electrophoresis (1.25% [wt/vol] agarose) (29). Inserts of the expected size were then sequenced by cycle sequencing, using the SequiTherm Excel II sequencing kit (Epicenter Technologies, Madison, Wis.). The primers for sequencing were m13f, (5'-3') TGT AAA ACG ACG GCC AGT, and m13r, (5'-3') CAG GAA ACA GCT ATG AC, both infrared dye 800 labeled. Conditions for the cycle-sequencing process have been described previously (28). The sequences were automatically analyzed on a 6% (wt/vol) polyacrylamide gel (Rapid Gel XL; Amersham Pharmacia Biotech) using a LI-COR DNA 4200 GeneRead IR apparatus (LI-COR, Lincoln, Neb.). The sequences were edited and aligned with the AlignIR 1.1 program (LI-COR) and, for phylogenetic analysis and identification of related sequences, loaded into the arb program and database (http://www.arb-home.de). All sequences generated in this study were consensus sequences.Nucleotide sequence accession numbers. The sequences generated in this study can be found in GenBank (http://www.ncbi.nlm.nih.gov) (2) under accession numbers AJ311396 to AJ311442.
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Primer selection and specificities. Primer pairs were chosen which amplified products from different regions of the bacterial SSU rRNA gene by PCR. The predicted lengths of the PCR products were similar: 436 bp for primers amplifying the variable regions V2-V3, 408 bp for those amplifying V4-V5, and 378 bp for primers amplifying regions containing V6, V7, and V8. For the amplification of products containing V4-V5, we chose primers which had already been applied for PCR-SSCP analysis of microbial communities from rhizospheres (30), compost (28), and, slightly different, also for PCR-DGGE microbial community analysis of soil samples (15, 16). PCR products with V6-to-V8 regions were generated with primers broadly used for microbial community analysis by PCR-DGGE (23) but, to our knowledge, not yet tested for PCR-SSCP. The third amplified rRNA gene region was generated with primers bordering the V2-V3 region. The reverse primer had already been used in earlier studies by Muyzer et al. (24). This sequence was actually almost the complementary sequence to the forward primer used to amplify the V4-V5 region in our study. The forward primer to amplify the V2-V3 region was selected from primers used for sequencing eubacterial SSU rRNA genes (E .R. B. Moore, personal communication). A shorter partial sequence including the V3 region had been targeted with universal primers in PCR-SSCP studies of bacterial communities in bioreactors (42). In a recent DGGE study, PCR products containing V1-to-V3 regions were found useful to detect the effects of herbicides on soil microbial communities (8).
The specificities of primers selected for our study were analyzed with the arb program package (see Materials and Methods) and the most recent database release available (December 1998). In order to estimate the binding specificities for the amplification of unknown sequences from environmental DNA, we determined the proportion of organisms in the database which would hybridize to the selected primers. Due to the annealing temperature, we estimated that two mismatches would still result in product formation. Under these circumstances, we found that the forward primer used in the amplification of the V2-V3 region would match with 57% of the database sequences for the domain Eubacteria and were highly specific for that group (Table 1). The reverse primer matched with 55% of the eubacterial sequences but was less specific, since it potentially also hybridized to sequences of the domains Archaea and Eucarya. In combination with the forward primer, the PCR products, however, should be specifically generated from members of the domain Eubacteria. This was not the case for primers selected to amplify the V4-V5 region, since matches of over 75% were recorded for Eubacteria and Eucarya and more than 50% for Archaea. The primers which were chosen for amplifying the V6-to-V8 regions, in contrast, were highly specific for Eubacteria. Both primers matched with more than 70% of the sequences in the Eubacteria database but with none in the Archaea or Eucarya sequences. In addition to testing the specificity of each primer separately, we analyzed the compatibility of each primer pair shown in Table 1 and found that at least 89% of the products which would be amplified by one primer contained complementary sequences which would bind to the opposite primer (data not shown). Cases in which no match with the opposite primers were found were often caused by the presence of partial sequences or sequences with gaps of unidentified nucleotides in the databases. The result of this database analysis demonstrated that the "universal" primers in our study were not perfectly universal. It is known that at the domain level conserved regions show some degree of variability (22). It has been shown that a slight modification of a primer binding site even within one conserved region can result in big differences in 16S rRNA gene sequences amplified from environmental samples (40).PCR-SSCP analysis of bacterial pure cultures.
We selected a
total of 13 species from different phylogenetic groups. Figure
1 shows typical SSCP gels obtained with
the three different primer pairs for all selected
bacteria. The majority of bacteria showed more than one product. In
some cases, the products of a single species could have the same
intensity on the gel, e.g., as shown for S. viridochromogenes, A. tumefaciens, and P. putida (Fig. 1B, lanes 4, 8, and 10, respectively). In
other cases, one band was dominant and additional bands occurred
in lower quantities, e.g., as found for E. coli and
B. subtilis (Fig. 1A, lanes 2 and 5).
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Dissection of SSCP patterns generated from bacterial-cell consortia
extracted from rhizospheres.
PCR amplifications using community
DNA as a template generated patterns of similar complexity for all
primer pairs tested (Fig. 1, lanes 15). In order to understand what had
been amplified from the community with the different primer sets, we
tried to isolate all dominant single bands from each profile. The
results of this analysis are shown in Table
3. Identifications of the products
amplified with the first primer set (V2-V3) showed the dominance of
P. polymyxa sequences. P. polymyxa is known to be a colonizer of maize rhizospheres (32, 39). The sequences detected in these profiles were all different from each other except
for the two sequences with 100% similarity to P. polymyxa X60623. Thus, the majority of products were caused by different operons
and not by metastable conformers. This is in accordance with the
pure-culture analyses with this organism shown in Table 2. The
"species" richness detected with the other PCR products (V4-V5 and
V6-V8) was higher than that detected with the products containing
V2-V3. The V6-V8-targeted PCR generated more different sequences
belonging to the group of gram-positive bacteria with a low-G+C DNA
content and in the subgroup of Proteobacteria. Members
of the Cytophaga-Flavobacterium-Bacteroides (CFB)
group were only detected with PCR products containing V4-V5. This could be explained by different primer specificities: in databases, a higher
number of homologous sequences was found for the CFB group (85%,
including two mismatches) with the V4-V5-targeting primers than with
the other two primer pairs (42% for V2-V3 and 56% for V6-V8 [data
not shown]). Even a sequence of a member of the kingdom
Crenarchaeota was found within the V4-V5 profile. This also
could be explained by looking at the primer specificities (Table 1).
Crenarchaeota-related sequences have also been found in
other studies based on cultivation-independent PCR-based methods with
soil DNA (3, 6, 18, 38).
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and subgroups) and low-G+C
gram-positive bacteria as the dominant eubacteria. Additionally, all
three primer sets amplified one sequence of proteobacteria from the subgroup. The PCR-amplified sequences within each of these phylogenetic
groups led to different identifications of closest relatives, except
for P. polymyxa. It may be argued that the number of bands
which were sequenced in our study from each community profile was too
low to expect an overlap between the identifications. This would be
true if the number of bands were much higher than those which were
actually sequenced. However, in our study we identified most bands
which were detectable in the profiles. A serious limitation for
identifications at the species level, however, is the use of partial
sequences. It is known that the reliability of relating an unknown
sequence to known sequences and, thus, of identifying it increases with
the length and the number of variable regions of the PCR-amplified product (35). The diversity of closest relatives detected
in our study may thus not only be a result of sequences amplified from
different organisms but also of the use of partial sequences for
comparison to database sequences.
It is interesting that all bands which were isolated from community
SSCP profiles exhibited different DNA sequences except for one case
(P. polymyxa with V2-V3). Thus, in contrast to our pure-culture results reported above, the formation of metastable conformers was of only minor importance and did not contribute to the
pattern complexity of the community profiles. These results are
corroborated by earlier PCR-SSCP studies which we conducted of the
diversity of eubacterial, actinomycetes, and fungal communities in
composts (28). Our analysis underlines the high potential of the community PCR-SSCP approach (30) as an alternative
to more commonly used profiling techniques for microbial community analyses.
Conclusions. Our study shows that intraspecies operon heterogeneities can contribute significantly to complex genetic profiles in microbial community analysis. In studies based only on profile comparisons and not on sequencing, this effect may be misinterpreted as a high microbial diversity, and dramatic pattern changes may be an effect of the reduction of only one or two organisms. The effect of operon heterogeneities can be reduced by choosing appropriate variable regions with less intraspecies diversity, e.g., V4 and V5 in our study. We also point out that even primers which bind to evolutionarily conserved regions of the SSU rRNA gene are never 100% universal at the domain level. Therefore, biases with different universal primers are inevitable and will contribute, in addition to the choice of variable regions, to the diversity found in genetic profiles in microbial community analyses.
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ACKNOWLEDGMENTS |
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We thank Karin Trescher for her excellent technical assistance. We also thank Sabine Peters, Ingo Fritz, and Erko Stackebrandt for discussions.
This work was supported by a grant from the German Ministry for Education and Research (BMBF; grant no. 0311740).
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FOOTNOTES |
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* Corresponding author. Mailing address: FAL-Institut für Agrarökologie, Bundesallee 50, 38116 Braunschweig, Germany. Phone: 49 (531) 596 2553. Fax: 49 (531) 596 2599. E-mail: christoph.tebbe{at}fal.de.
Present address: AMODIA Bioservice GmbH, 38124 Braunschweig, Germany.
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Abstract
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References
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Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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