Previous Article | Next Article 
Applied and Environmental Microbiology, March 1999, p. 1045-1049, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Polynucleotide Probes That Target a Hypervariable Region of 16S
rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of
Community Fingerprints
Holger
Heuer,1
Kathrin
Hartung,1
Gabriele
Wieland,1
Ina
Kramer,2 and
Kornelia
Smalla1,*
Biologische Bundesanstalt für Land- und
Forstwirtschaft (BBA), D-38104
Braunschweig,1 and DSMZ-German
Collection of Microorganisms and Cell Cultures, D-38124
Braunschweig,2 Germany
Received 5 October 1998/Accepted 8 December 1998
 |
ABSTRACT |
Temperature gradient gel electrophoresis (TGGE) is well suited for
fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S
rDNA from community fingerprints to pure culture isolates from
the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE
community fingerprints as a template, and applied in hybridizations
with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S
rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is
a subset of the region used for TGGE (positions 968 to 1401),
best met the criteria of high phylogenetic variability, required for
sufficient probe specificity, and closely flanking conserved priming
sites for amplification. Removal of flanking conserved bases was
necessary to enable the differentiation of closely related species.
This was achieved by 5' exonuclease digestion, terminated by
phosphorothioate bonds which were synthesized into the primers. The
remaining complementary strand was removed by single-strand-specific
digestion. Standard hybridization with truncated probes allowed
differentiation of bacteria which differed by only two bases within the
probe target site and 1.2% within the complete 16S rDNA. However, a
truncated probe, derived from an excised TGGE band of a rhizosphere
community, hybridized with three phylogenetically related isolates with
identical V6 sequences. Only one of the isolates comigrated with the
excised band in TGGE, which was shown to be due to identical
sequences, demonstrating the utility of a combined TGGE and V6
probe approach.
| |
INTRODUCTION |
Temperature gradient gel
electrophoresis (TGGE) and the related technique, denaturing gradient
gel electrophoresis, are now frequently applied in microbial ecology to
compare the structures of complex microbial communities and to study
their dynamics (18). The steps in the procedure are
extraction of genomic DNA from environmental samples,
amplification of a segment of the 16S rRNA genes (16S ribosomal DNA
[rDNA]) in PCR, and electrophoretical separation of PCR
products with differing sequences in a denaturing gradient. This allows
analysis of many samples, which is essential for studying spatial and
temporal variations of microbial community structures in relation to
environmental factors, and shifts due to perturbation or experimental
treatment. The diversity of complex communities can be explored in a
"top-to-bottom" analysis with primer sets of varying phylogenetic
specificity (12, 13, 15, 21). Individual bands in the TGGE
fingerprints can be assigned to taxa by hybridization with
oligonucleotide probes (17, 31), or the sequence can be
determined and phylogenetically analyzed (19). However, the
ecological role of an organism often cannot be inferred from a
comparison of its 16S rRNA sequence to those of known bacteria. The
limited sequence database may lack a well-studied closely related
reference strain, or the strain may differ in the trait of interest
even if the sequences of the 16S rDNA regions used for TGGE are
identical. Several groups of organisms have been identified which share
almost identical 16S rRNA sequences but in which DNA hybridization is
lower than 70% (30).
Thus, a tool is needed to link the bands from community fingerprints to
the strains which are present in the environmental sample analyzed. In
order to study their properties and autecology, either corresponding
cultivated bacterial isolates from the sample must be identified or
corresponding unique DNA sequences cloned from DNA of the sample have
to be found, which allows the detection and study of the population
members in situ (1) or helps in selecting the proper medium
for their cultivation (26, 33).
In this study we investigated whether the hypervariable region V6
(20) of the small-subunit rRNA gene could be utilized as a
probe target to detect bacterial isolates corresponding to bands of
TGGE community fingerprints. A generally applicable method was
developed for generating highly specific digoxigenin
(DIG)-labeled probes targeting the V6 region (V6 probes) without prior
DNA sequence knowledge.
| |
MATERIALS AND METHODS |
Rhizosphere samples.
Rhizosphere communities from transgenic
and control potato plants were compared. The transgenic plant lines DL4
and DL5 constitutively expressed and secreted T4 lysozyme, which
mediates improved resistance to the bacterial soft rot disease (4,
5, 7). A transgenic control line contained the same construct,
including the nptII gene but without the T4 lysozyme gene.
All transgenic cultivars were derived from variety Désirée.
Tubers were provided by K. Düring (BAZ, Quedlinburg, Germany).
The potato cultivars were planted in a field located near Quedlinburg
in a randomized block design (four cultivars each in eight replicate
plots). The soil type was a silt loam. Roots with adhering soil
particles were sampled 16 weeks after the plants sprouted (shortly
before the potatoes were harvested). Independent samples from eight
replicate plots of each cultivar were collected (32 samples). DNA was
extracted from bacterial pellets (28) derived by repeated
Stomacher blending and differential centrifugation (29).
Bacteria.
The extracted bacterial pellets were serially
diluted and plated on R2A agar (Difco, Detroit, Mich.). Randomly picked
colonies were characterized by their fatty acid profiles, using the
Microbial Identification System (MIS) (MIDI Inc., Newark, Del.). Cells
of 192 strains, which were selected to represent the diversity of cultivated species, were lysed by freeze-boiling and directly applied to PCR mixtures. Amplification of 16S rDNA fragments, using the
primer pair F984GC-R1378 (Table 1), was
performed as described previously (12) to compare the
migrations of bands from pure cultures to those of the community
fingerprints in TGGE. The PCR products were used as targets for
Southern blot hybridizations.
TGGE.
Community fingerprinting of rhizosphere samples by
TGGE was carried out as described previously (12). The 16S
rDNA fragments (positions 968 to 1401 [Escherichia coli
rDNA sequence]) were amplified by PCR from rhizosphere DNA extracts
with the primer pair F984GC-R1378. Acid silver staining was used for
the routine detection of DNA bands in TGGE gels (24). When
DNA was to be recovered from excised bands, staining was performed with
SYBR Green I (FMC, Vallensbaek Strand, Denmark).
V6 probes.
Gel pieces containing bands from the TGGE gel
were washed with 0.5 ml of Tris-EDTA buffer, frozen at 
70°C, and
broken into smaller pieces. After centrifugation at 13,000 × g for 30 min at 4°C, 1 µl of the solution extracted from
the gel pieces was used in a PCR to reamplify the 16S rDNA fragments
with the primer pair F984GC-R1378. The enrichment of the excised band
was confirmed by TGGE. The PCR products were ligated into the pGEM-T
vector (Promega, Madison, Wis.). Competent cells of E. coli
JM109 were transformed as described in the manual of the supplier.
Plasmids with the correct insert, as determined by TGGE, were
isolated with a plasmid extraction kit (Qiagen, Hilden, Germany). The
purified plasmid DNAs were used as templates for preparation of V6
probes by PCR. Two types of V6 probes were prepared. One was amplified with primers F971 and R1057 (Table 1), as described by Smalla et al.
(29). The PCR mixture contained DIG-dUTP to label the probes. The other type of V6 probe, intended for removal of flanking conserved bases, was amplified with primers F985PTO and R1046PTO (Table
1), using the same PCR mixture but with the following cycles: one cycle
of 5 min at 94°C and 35 cycles of 30 s at 94°C, 30 s at
50°C, and 30 s at 72°C. These primers had a phosphorothioate bond between bases of the 3' terminus, which was intended to resist cleavage by T7 gene 6 exonuclease, a double-strand-specific 5'-3' exonuclease (14). The PCR products were purified, using the QIAEX II kit (Qiagen), and digested for 15 min at 37°C with 1 U of T7
gene 6 exonuclease/µl in the supplied buffer (U. S. Biochemicals, Cleveland, Ohio) to remove the primer nucleotides from
the PCR product up to the phosphorothioate bond. The products were
again purified by QIAEX II for buffer exchange. The remaining single strands (complementary to the primers) were digested by the
single-strand-specific mung bean nuclease (16), as
recommended by Amersham International (Little Chalfont, England). The
size reduction of individual V6 probes compared to untreated aliquots
was checked by electrophoresis in a 15% polyacrylamide gel.
Southern hybridizations.
Equal amounts of PCR products
(primer pair F984GC-R1378) from cloned 16S rDNA and from a selection of
bacterial isolates were blotted from an agarose gel onto an uncharged
nylon membrane (25) or with a dot blot device onto Hybond N+
nylon membranes (Amersham) (3). High-stringency
hybridization at 62°C, washing, and detection of DNA hybrids was
carried out as recommended by Boehringer (Mannheim, Germany), but the
hybridization solution contained 5% (wt/vol) blocking powder, 1× SSC
(0.15 M NaCl plus 0.015 M sodium citrate), 0.1% (vol/vol)
N-laurylsarkosinate, 0.02% (wt/vol) sodium dodecyl sulfate,
and 50% (vol/vol) formamide.
DNA sequence analysis.
Selected cloned TGGE bands were
sequenced with standard primers SP6 and T7 (IIT GmbH, Bielefeld,
Germany). PCR products from 16S rDNA of bacterial rhizosphere isolates
were sequenced with primers 536r, 357r, FoxF, and 1385r (DSMZ,
Braunschweig, Germany). Construction of consensus sequences, alignments
to most-similar database sequences, and matrix calculations of sequence
similarities to closely related database sequences were done with ARB
software (32) and the supplied database, 6pubmrz97, which
was supplemented by 16S rDNA sequences from the GenBank database.
Nucleotide sequence accession numbers.
GenBank accession
numbers of the sequences from this study are given in Table
2, except that of the 5' partial 16S rDNA
sequence of strain B3a, which is AF060537.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Analysis of partial 16S rRNA gene sequences of excised
bands from potato rhizosphere TGGE fingerprints (E. coli
positions 985 to 1377)
|
|
| |
RESULTS AND Discussion |
Fingerprinting of bacterial communities by separation of
amplified 16S rDNA fragments with TGGE provides the
opportunity to compare the community structure features of
multiple environmental samples. Although sequencing of bands
for analysis of TGGE fingerprints provides insight into the
community structure through the phylogenetic affiliations of
community members (19), the information about their
physiological and ecological traits derived from the partial sequences is often rather limited (11, 22). Thus, a
link between bands of TGGE rhizosphere fingerprints and corresponding
bacterial isolates from the same habitat was developed, which is based
on probes that target a region of the 16S rDNA including the
hypervariable region V6. A method was developed to improve the
specificity of the V6 probes by removal of phylogenetically
conserved nucleotides. The approach is illustrated in Fig.
1.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 1.
Scheme of the V6 probe approach. Polynucleotide probes
that target the hypervariable region V6 were used to screen for
bacterial isolates corresponding to bands of community fingerprints
derived by electrophoretic separation of PCR-amplified 16S rDNA
fragments in a temperature gradient (TGGE).
|
|
Development of V6 probes.
In a first attempt, V6 probes were
synthesized and DIG labeled, using PCR with the primer pair F971-R1057
(Table 1), which amplified a fragment between E. coli 16S
rDNA sequence positions 971 and 1057. The specificities of probes
derived from strains of Agrobacterium tumefaciens and
Sinorhizobium meliloti were tested with dot blotted 16S
rDNAs of six phylogenetically closely related species of the
Rhizobiaceae and a selection of species from more distantly
related groups. Both probes cross-hybridized only with the 16S rDNA of
the closest relative, Agrobacterium rubi or
Sinorhizobium fredii, respectively (data not shown), which
have identical sequences of the V6 regions (37). A probe
from Ralstonia solanacearum slightly cross-hybridized only
with the 16S rDNA of Ralstonia eutropha (data not shown).
The V6 probe approach was recently applied successfully to prove the
identities of rhizobacterial isolates (29) or cloned 16S
rDNA from soil (9, 10) with prominent populations of the
natural community which had equally migrating bands in denaturing
gradients. However, for the dense cluster of
Enterobacteriaceae isolates, specificities at the genus level were not achieved (Fig. 2a).
Attempts to increase the specificity of the probe by hybridization at a
higher temperature (65°C) or by adding an excess of primers to block
conserved binding sites resulted in only minor improvements.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 2.
Hybridization specificity of the V6 probes derived from
the 16S rDNA of band B1 in the TGGE fingerprint of the bacterial
rhizosphere community from T4 lysozyme-producing potato line DL4. The
blot was hybridized with the nontruncated V6 probe (a) or with the
truncated V6 probe (b). Targets on the Southern blot were 16S rRNA gene
fragments from rhizobacteria and from bands B1 to B7 of rhizosphere
community fingerprints (Rtr, Rathayibacter tritici; Agl,
Arthrobacter globiformis; Fre, Flavobacterium
resinovorum; Atu, A. tumefaciens; Ppu,
Pseudomonas putida; Vpa, Variovorax paradoxus;
Kkr, Kokuria kristinae; Aes, Aureobacterium
esteroaromaticum; Pfl, Pseudomonas fluorescens; Cac,
Comamonas acidovorans; Eca, E. carotovora; Fjo,
Flavobacterium johnsoniae; Pci,
Pseudomonas cichorii; Aru, A. rubi; Pag,
P. agglomerans; Psy, Pseudomonas syringae. See
Table 2 for sequence similarities of bands B1 to B7.)
|
|
Truncated V6 probes with increased specificities.
One-third of
the length of the V6 probes consists of phylogenetically conserved
nucleotides which are required for priming the PCR but counteract probe
specificity. Thus, a method was developed and applied to remove the
conserved parts of the probe, i.e., the incorporated primers and their
complements. To achieve this, the primers F985PTO and R1046PTO were
synthesized, with a phosphorothioate replacing a phosphate in the bond
of the last 2 nucleotides at the 3' end. After PCR amplification of the
fragment from 968 to 1062, the nucleotides of the incorporated primers
were removed 5' to 3' by exonuclease digestion, but hydrolysis was
stopped at the phosphorothioate bond (Fig.
3, lane 2). The remaining complementary single strand could subsequently be eliminated by the
single-strand-specific mung bean nuclease (Fig. 3, lane 3).
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 3.
Preparation of a truncated V6 probe. Lane 1 contains the
95-bp PCR product amplified with phosphorothioate primers F985PTO and
R1046PTO. On average 11 nucleotides of DIG-dUTP were introduced during
PCR (maximum, 33). Lane 2 contains the partially single-stranded probe
after hydrolysis of the incorporated primer nucleotides by T7 gene 6 exonuclease. Lane 3 contains the truncated 64-bp V6 probe after removal
of the single-stranded primer complements by mung bean nuclease. The
probe and intermediate products were separated by polyacrylamide gel
electrophoresis, electroblotted, and detected by exposure of the film
to chemiluminescence.
|
|
The specificities of the V6 probes increased dramatically after
truncation. The nontruncated V6 probe from TGGE band B1 hybridized
with
all 16S rDNA sequences related to the
Enterobacteriaceae (Fig.
2a), i.e., 16S rDNA of isolates of
Erwinia carotovora
and
Pantoea agglomerans and cloned TGGE bands B1, B2, B3,
and B6 (sequence
affiliations are shown in Table
2). In contrast, the
truncated
V6 probe was observed to be specific for
E. carotovora 16S rDNA
(Fig.
2b). Weak cross-hybridization was
detected with fragments
B2 and B3, which had sequence similarities of
96.2 or 95.4% with
B1 and 10- or 8-bp differences from the probe
derived from B1,
respectively. The truncated V6 probe of fragment B3
cross-hybridized
with neither fragment B2, which had six mismatches
with the probe,
nor the other sequences tested (data not shown). The
sequences
of B2 and B3 were 97.2% similar. The results of the sequence
analyses
of the excised TGGE bands are given in Table
2.
Cloned 16S rDNAs of 15 phylogenetically closely related members of the
subdivision of the class
Proteobacteria were hybridized
to a truncated V6 probe derived from one of the clones to determine
the
number of nucleotide mismatches, which prevent hybridization
of
truncated V6 probes. The sequences differed from that of the
probe
within the target region by 2, 3, 6, 10, or 17 nucleotides.
The only
clone which produced a hybridization signal was the one
from which the
probe originated (data not shown). The most similar
target sequence had
A or T at
E. coli 16S rDNA sequence positions
1001 and 1039 instead of G or C in the probe. Thus, the truncated
V6 probe
hybridization stringency was sufficient to distinguish
targets
differing by only 2 nucleotides. The similarity between
the two
complete 16S rDNAs was 98.8%, which indicates that differentiation
on
the species level may well be possible (
30). The theoretical
increase of specificity due to the truncation of the V6 probe
was
predicted by the equation of Baldino et al. (
2), which
accounts for the percentage of mismatches. It estimated a
1.5-fold-stronger
destabilizing effect of each probe-target mismatch
for the truncated
V6 probe compared to the nontruncated probe.
Moreover, nucleotide
mismatches are relatively less clustered in
hybrids with truncated
V6 probes, which increases their destabilizing
effects (
8).
Rhizosphere.
We investigated whether truncated V6 probes
derived from bands in TGGE fingerprints of bacterial rhizosphere
communities could be used to screen for the corresponding bacterial
strains isolated from the same habitat. Community fingerprinting by
TGGE separation of amplified 16S rDNA fragments was applied to compare
bacterial rhizosphere communities of transgenic T4 lysozyme-producing
potato plants, a transgenic control and the wild type. In Fig.
4, TGGE patterns from five of the eight
rhizosphere samples of each plant line are shown. In most of the
patterns from one of the T4 lysozyme-producing plant lines, DL4, band
B1 was much stronger than in the other patterns. In only one of the
eight samples of DL4 was band B3 stronger than B1 (not shown). The
bands B1 and B3 were excised from the TGGE gel, reamplified, subcloned,
and used to generate truncated V6 probes. The probes were hybridized to
the dot blotted 16S rDNAs of 192 strains, representing the diversity of
cultivated bacteria from the potato rhizosphere.
View larger version (99K):
[in this window]
[in a new window]
|
FIG. 4.
Fingerprints of bacterial rhizosphere communities
by TGGE separation of amplified 16S rDNA fragments. Dési,
wild-type potato variety Désirée; DL4 and DL5, T4
lysozyme-expressing potato lines; DC1, transgenic control with
nptII gene but no T4 lysozyme gene. Truncated V6 probes from
bands B1 and B3 were used to screen for corresponding bacterial
isolates from the potato rhizosphere.
|
|
The truncated V6 probe derived from TGGE band B1 specifically
hybridized with the 16S rDNA of four isolates. All were identified
by
fatty acid methyl ester-MIS analysis as
E. carotovora
subsp.
carotovora. This was in agreement with the
identification by sequence
analysis of band B1. Three of the isolates
had 16S rDNA fragments
which migrated as band B1 in TGGE. Evidence
from band intensities
in TGGE community fingerprints showed that these
bacteria represented
the most abundant population associated with the
roots of old
DL4-type potato plants and were much less abundant in the
rhizosphere
of the wild-type plants. The affiliation of this isolate
with
those
E. carotovora strains that might cause soft
rot (
23) may
be of agronomic interest. It is unclear whether
the growth of
E. carotovora is an indirect effect of
the T4 lysozyme, which
had a 20- to 70%-higher expression level in DL4
than in DL5 (
6),
or whether, e.g., somaclonal variation
caused a weakening of the
cultivar DL4, which had a significantly
reduced stem length, smaller
leaves, and a lower root mass compared to
those of the other cultivars.
The other T4 lysozyme-producing potato
line, DL5, did not show
the prominent
E. carotovora
band and thus might be a better candidate
for further field tests. The
fragment of one
E. carotovora isolate
migrated slightly
differently from band B1, demonstrating that
this isolate was not
identical to the dominant population in the
rhizosphere of potato type
DL4. Also, the type strain of
E. carotovora subsp.
carotovora, DSM 30168, could be differentiated by TGGE
from
the four
isolates.
Although the specificity of the truncated V6 probe from TGGE band B1
was sufficient to identify
E. carotovora sequences, the
truncated V6 probe from band B3 hybridized with 16S rDNA of three
bacterial isolates which were affiliated with different species
as
determined by fatty acid profiling and partial 16S rDNA sequence
analysis (Table
3). All were closely
related to members of the
genus
Enterobacter (Table
3). To
verify whether the correct targets
were detected by the truncated V6
probe, the 16S rDNAs of the
three isolates were partially sequenced.
The V6 regions of all
three isolates were identical to the V6 probe
sequence, showing
the high stringency of the hybridization. However,
the strains
could be differentiated by subsequent TGGE analysis based
on differences
in 16S rDNA regions V7 and V8. Only the 16S rDNA
fragment of strain
B3a was identical in electrophoretic mobility to
band B3, and
DNA sequencing, in fact, revealed sequence identity. The
different
migrations of the 16S rDNA fragments of strains B3b and B3c
could
be explained by five or seven base substitutions, respectively,
of A to G or C to T in positions 1244 to 1367. Sequencing
demonstrated
that phylogenetically very similar strains were detected
by the
truncated V6 probe. The strains could be differentiated by
TGGE
analysis because the separation in TGGE is mainly caused by
sequence
differences in melting domains distant from the nonmelting
GC-clamped
end of the TGGE fragment. The V6 region is located next to
the
GC clamp. Thus, TGGE analysis complements the V6 probe approach.
As
little as one base difference could be detected by TGGE
(
27).
However, screening of many strains by TGGE is not
practical. In
addition, fragments with largely different DNA sequences
may have
similar migration distances in TGGE (
15,
35). These
could
easily be differentiated with probes.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Identification of potato rhizosphere isolates with 16S
rRNA genes that hybridized with the truncated V6 probe derived from
band B3 of the rhizosphere TGGE fingerprint
|
|
Polynucleotide probes targeting rRNA genes were also applied by
Trebesius et al. (
34). They used a variable region of the
23S rRNA as a target site, but as not all parts of the ca. 220
nucleotides were hypervariable, specificity at the species level
could
be achieved only by tedious optimization of the hybridization
stringency for a
Pseudomonas stutzeri-specific probe. The
hypervariable
region V6 is probably the optimal target within the 16S
rDNA to
generate polynucleotide probes which are specific at standard
hybridization conditions. This was evident from a profile of the
positional phylogenetic conservation of the 16S rDNA. It was
constructed
from the recently published quantitative map of nucleotide
substitution
rates (
36), using calculated 45-mer average
substitution rates.
The V6 region had the highest average phylogenetic
variability
and, in contrast to other variable regions, it was closely
flanked
by conserved sites which are suitable for the annealing of
eubacterial
primers. Amplification of other highly variable regions
(i.e.,
V2 and V3) by PCR with eubacterial primers leads to products
which
also contain moderately variable nucleotide stretches,
counteracting
probe
specificity.
A possible aid for the analysis of bacteria, when they cannot be
cultivated, is to use the V6 probe approach to identify a
clone
containing a complete 16S rRNA gene (or larger fragments)
which
corresponds to the TGGE band of interest. In our work,
E. coli JM109 hosts, containing multicopy pGEM-T plasmids with
complete
16S rDNA inserts, could be grown and lysed in microtiter
plates
and directly applied to dot blots for hybridization analysis.
The additional sequence information can be useful for designing
specific oligonucleotide probes to be used for in situ detection
(
1). The direct applicability of truncated V6 probes for
identification
of individual cells remains to be
tested.
| |
ACKNOWLEDGMENTS |
We are grateful to Bert Engelen and the team of workgroup Smalla
for helpful discussions and cooperation, Henrike Westphal for excellent
technical assistance, and Ed Moore (GBF, Braunschweig) for reading the
manuscript. We thank Stefan Weidner (University of Bielefeld) for
providing some of the clones and their sequences.
This work was supported by BMBF grant 0311295.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: BBA, Institut
für Pflanzenvirologie, Mikrobiologie und biologische
Sicherheit, Messeweg 11-12, D-38104 Braunschweig, Germany. Phone and
fax: 49531299-3814/3013. E-mail: k.smalla{at}bba.de.
| |
REFERENCES |
| 1.
|
Amann, R. I.,
W. Ludwig, and K. H. Schleifer.
1995.
Phylogenetic identification and in situ detection of individual microbial cells without cultivation.
Microbiol. Rev.
59:143-169[Abstract/Free Full Text].
|
| 2.
|
Baldino, F., Jr.,
M.-F. Chesselet, and M. E. Lewis.
1989.
High-resolution in situ hybridization histochemistry.
Methods Enzymol.
168:761-777[Medline].
|
| 3.
|
Brown, T.
1993.
Dot and slot blotting of DNA, p. 2.9.15-2.9.20.
In
F. M. Ausubel, R. Brent, R. E. Kingston, et al. (ed.), Current protocols in molecular biology. John Wiley & Sons, New York, N.Y.
|
| 4.
|
Düring, K.
1994.
A plant transformation vector with a minimal T-DNA.
Transgen. Res.
3:138-140[Medline].
|
| 5.
|
Düring, K.
1996.
Genetic engineering for resistance to bacteria in transgenic plants by introduction of foreign genes.
Mol. Breeding
2:297-305.
|
| 6.
| Düring, K., and A. Mahn. Personal
communication.
|
| 7.
|
Düring, K.,
P. Porsch,
M. Fladung, and H. Lörz.
1993.
Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora.
Plant J.
3:587-598.
|
| 8.
|
Dyson, N. J.
1991.
Immobilization of nucleic acids and hybridization analysis, p. 111-156.
In
T. A. Brown (ed.), Essential molecular biology. Oxford University Press, New York, N.Y.
|
| 9.
|
Felske, A., and A. D. L. Akkermans.
1998.
Prominent occurrence of ribosomes from an uncultured bacterium of the Verrucomicrobiales cluster in grassland soils.
Lett. Appl. Microbiol.
26:219-223[Medline].
|
| 10.
|
Felske, A.,
H. Rheims,
A. Wolterink,
E. Stackebrandt, and A. D. L. Akkermans.
1997.
Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils.
Microbiology
143:2983-2989[Abstract/Free Full Text].
|
| 11.
|
Fox, G. E.,
J. D. Wisotzkey, and P. Jurtshuk, Jr.
1992.
How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.
Int. J. Syst. Bacteriol.
42:166-170[Abstract/Free Full Text].
|
| 12.
|
Heuer, H.,
M. Krsek,
P. Baker,
K. Smalla, and E. M. H. Wellington.
1997.
Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.
Appl. Environ. Microbiol.
63:3233-3241[Abstract].
|
| 13.
|
Heuer, H., and K. Smalla.
1997.
Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) for studying soil microbial communities, p. 353-373.
In
J. D. van Elsas, E. M. H. Wellington, and J. T. Trevors (ed.), Modern soil microbiology. Marcel Dekker, New York, N.Y.
|
| 14.
|
Kerr, C., and P. D. Sandowski.
1972.
Gene 6 exonuclease of bacteriophage T7. I. Purification and properties of the enzyme.
J. Biol. Chem.
247:305-310[Abstract/Free Full Text].
|
| 15.
|
Kowalchuk, G. A.,
J. R. Stephen,
W. De Boer,
J. I. Prosser,
T. M. Embley, and J. W. Woldendorp.
1997.
Analysis of ammonia-oxidizing bacteria of the subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments.
Appl. Environ. Microbiol.
63:1489-1497[Abstract].
|
| 16.
|
Kroeker, W. D.,
D. Kowalski, and M. Laskowski.
1976.
Mung bean nuclease. I. Terminally directed hydrolysis of native DNA.
Biochemistry
15:4463-4467[Medline].
|
| 17.
|
Manz, W.,
R. Amann,
W. Ludwig,
M. Wagner, and K. H. Schleifer.
1992.
Phylogenetic oligonucleotide probes for the major subclasses of proteobacteria: problems and solutions.
Adv. Microb. Ecol.
15:593-600.
|
| 18.
|
Muyzer, G., and K. Smalla.
1998.
Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology.
Antonie Leeuwenhoek
73:127-141[Medline].
|
| 19.
|
Muyzer, G.,
A. Teske,
C. O. Wirsen, and H. W. Jannasch.
1995.
Phylogenetic relationship of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments.
Arch. Microbiol.
164:165-172[Medline].
|
| 20.
|
Neefs, J.-M.,
Y. Van de Peer,
L. Hendriks, and R. De Wachter.
1990.
Compilation of small ribosomal subunit RNA sequences.
Nucleic Acids Res.
18:2237-2242.
|
| 21.
|
Nübel, U.,
F. Garcia-Pichel, and G. Muyzer.
1997.
PCR primers to amplify 16S rRNA genes from Cyanobacteria.
Appl. Environ. Microbiol.
63:3327-3332[Abstract].
|
| 22.
|
Pace, N. R.
1997.
A molecular view of microbial diversity and the biosphere.
Science
276:734-740[Abstract/Free Full Text].
|
| 23.
|
Pishchik, V. N.,
I. I. Chernyaeva,
N. I. Vorob'ev, and A. M. Lazarev.
1996.
Characteristic features of virulent and avirulent strains of Erwinia carotovora.
Microbiology
65:262-268.
|
| 24.
|
Riesner, D.,
G. Steger,
R. Zimmat,
R. A. Owens,
M. Wagenhöfer,
W. Hillen,
S. Vollbach, and K. Henco.
1989.
Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.
Electrophoresis
10:377-389[Medline].
|
| 25.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 26.
|
Santegoeds, C. M.,
S. C. Nold, and D. M. Ward.
1996.
Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat.
Appl. Environ. Microbiol.
62:3922-3928[Abstract].
|
| 27.
|
Sheffield, V. C.,
R. D. Cox,
L. S. Lerman, and R. M. Myers.
1989.
Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.
Proc. Natl. Acad. Sci. USA
86:232-236[Abstract/Free Full Text].
|
| 28.
|
Smalla, K.,
N. Cresswell,
L. C. Mendoca-Hagler,
A. Wolters, and J. D. van Elsas.
1993.
Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification.
J. Appl. Bacteriol.
74:78-85.
|
| 29.
|
Smalla, K.,
U. Wachtendorf,
H. Heuer,
W.-T. Liu, and L. Forney.
1998.
Analysis of BIOLOG GN substrate utilization patterns by microbial communities.
Appl. Environ. Microbiol.
64:1220-1225[Abstract/Free Full Text].
|
| 30.
|
Stackebrandt, E., and B. M. Goebel.
1994.
Taxonomic note: a place for DNA-DNA reassociation and 16S RNA sequence analysis in the present species definition in bacteriology.
Int. J. Syst. Bacteriol.
44:846-849[Abstract/Free Full Text].
|
| 31.
|
Stahl, D. A.,
B. Flesher,
H. R. Mansfield, and L. Montgomery.
1988.
Use of phylogenically based hybridization probes for studies of ruminal microbial ecology.
Appl. Environ. Microbiol.
54:1079-1084[Abstract/Free Full Text].
|
| 32.
| Strunk, O., W. Ludwig, O. Gross, B. Reichel, N. Stuckmann, M. May, B. Nonhoff, M. Lenke, T. Ginhart, A. Vilbig,
and R. Westram. 22 October 1998, revision date. [Online.] ARB a
software environment for sequence data. Department of Microbiology,
Technical University of Munich, Munich, Germany.
http://www.biol.chemie.tu-muenchen.de. [19 January 1999, last date
accessed.]
|
| 33.
|
Teske, A.,
P. Sigalevich,
Y. Cohen, and G. Muyzer.
1996.
Molecular identification of bacteria from coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures.
Appl. Environ. Microbiol.
62:4210-4215[Abstract].
|
| 34.
|
Trebesius, K.,
R. Amann,
W. Ludwig,
K. Mühlegger, and K. H. Schleifer.
1994.
Identification of whole fixed bacterial cells with nonradioactive 23S rRNA-targeted polynucleotide probes.
Appl. Environ. Microbiol.
60:3228-3235[Abstract/Free Full Text].
|
| 35.
|
Vallaeys, T.,
E. Topp,
G. Muyzer,
V. Macheret,
G. Laguerre,
A. Rigaud, and G. Soulas.
1997.
Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs.
FEMS Microbiol. Ecol.
24:279-285.
|
| 36.
|
Van de Peer, Y.,
S. Chapelle, and R. De Wachter.
1996.
A quantitative map of nucleotide substitution rates in bacterial rRNA.
Nucleic Acids Res.
24:3381-3391[Abstract/Free Full Text].
|
| 37.
|
Yanagi, M., and K. Yamasoto.
1993.
Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer.
FEMS Microbiol. Lett.
107:115-120[Medline].
|
Applied and Environmental Microbiology, March 1999, p. 1045-1049, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Youssef, N., Sheik, C. S., Krumholz, L. R., Najar, F. Z., Roe, B. A., Elshahed, M. S.
(2009). Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys. Appl. Environ. Microbiol.
75: 5227-5236
[Abstract]
[Full Text]
-
Andreote, F. D., de Araujo, W. L., de Azevedo, J. L., van Elsas, J. D., da Rocha, U. N., van Overbeek, L. S.
(2009). Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte, Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities. Appl. Environ. Microbiol.
75: 3396-3406
[Abstract]
[Full Text]
-
Ott, S. J., El Mokhtari, N. E., Musfeldt, M., Hellmig, S., Freitag, S., Rehman, A., Kuhbacher, T., Nikolaus, S., Namsolleck, P., Blaut, M., Hampe, J., Sahly, H., Reinecke, A., Haake, N., Gunther, R., Kruger, D., Lins, M., Herrmann, G., Folsch, U. R., Simon, R., Schreiber, S.
(2006). Detection of Diverse Bacterial Signatures in Atherosclerotic Lesions of Patients With Coronary Heart Disease. Circulation
113: 929-937
[Abstract]
[Full Text]
-
Corgie, S. C., Beguiristain, T., Leyval, C.
(2004). Spatial Distribution of Bacterial Communities and Phenanthrene Degradation in the Rhizosphere of Lolium perenne L.. Appl. Environ. Microbiol.
70: 3552-3557
[Abstract]
[Full Text]
-
Van Hamme, J. D., Singh, A., Ward, O. P.
(2003). Recent Advances in Petroleum Microbiology. Microbiol. Mol. Biol. Rev.
67: 503-549
[Abstract]
[Full Text]
-
Gomes, N. C. M., Fagbola, O., Costa, R., Rumjanek, N. G., Buchner, A., Mendona-Hagler, L., Smalla, K.
(2003). Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics. Appl. Environ. Microbiol.
69: 3758-3766
[Abstract]
[Full Text]
-
Bertilsson, S., Cavanaugh, C. M., Polz, M. F.
(2002). Sequencing-Independent Method To Generate Oligonucleotide Probes Targeting a Variable Region in Bacterial 16S rRNA by PCR with Detachable Primers. Appl. Environ. Microbiol.
68: 6077-6086
[Abstract]
[Full Text]
-
van Dillewijn, P., Villadas, P. J., Toro, N.
(2002). Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa. Appl. Environ. Microbiol.
68: 4201-4208
[Abstract]
[Full Text]
-
Heuer, H., Kroppenstedt, R. M., Lottmann, J., Berg, G., Smalla, K.
(2002). Effects of T4 Lysozyme Release from Transgenic Potato Roots on Bacterial Rhizosphere Communities Are Negligible Relative to Natural Factors. Appl. Environ. Microbiol.
68: 1325-1335
[Abstract]
[Full Text]
-
Hughes, J. B., Hellmann, J. J., Ricketts, T. H., Bohannan, B. J. M.
(2001). Counting the Uncountable: Statistical Approaches to Estimating Microbial Diversity. Appl. Environ. Microbiol.
67: 4399-4406
[Full Text]
-
Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H., Berg, G.
(2001). Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed. Appl. Environ. Microbiol.
67: 4742-4751
[Abstract]
[Full Text]
-
Ranjard, L., Brothier, E., Nazaret, S.
(2000). Sequencing Bands of Ribosomal Intergenic Spacer Analysis Fingerprints for Characterization and Microscale Distribution of Soil Bacterium Populations Responding to Mercury Spiking. Appl. Environ. Microbiol.
66: 5334-5339
[Abstract]
[Full Text]
-
Smalla, K., Heuer, H., Götz, A., Niemeyer, D., Krögerrecklenfort, E., Tietze, E.
(2000). Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids. Appl. Environ. Microbiol.
66: 4854-4862
[Abstract]
[Full Text]
-
Berg, G., Roskot, N., Smalla, K.
(1999). Genotypic and Phenotypic Relationships between Clinical and Environmental Isolates of Stenotrophomonas maltophilia. J. Clin. Microbiol.
37: 3594-3600
[Abstract]
[Full Text]
Top
Abstract
Introduction
