APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1999, p.

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0099-2240/99/$04.000
Copyright 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 8

Culture-Independent Identification of Microorganisms That

Respond to Specified Stimuli
JAMES BORNEMAN*
Department of Plant Pathology, University of California, Riverside, California 92521
Received 5 February 1999/Accepted 17 May 1999

A new approach that permits culture-independent identification of microorganisms that respond to specified
stimuli was developed. This approach was illustrated by examination of microorganisms that grew in response
to various nutrient supplements added to soil. A thymidine nucleotide analog, bromodeoxyuridine (BrdU), and
supplements were added to soil and incubated for 3 days. DNA was extracted from the soil, and the newly
synthesized DNA was isolated by immunocapture of the BrdU-labeled DNA. The unique perspective this
approach offers was demonstrated by comparing the microbial community structures obtained from total soil
DNA and the BrdU-labeled fraction in an rRNA gene (rDNA) analysis. The traditional total DNA analysis
revealed no notable differences between the treatments, whereas the BrdU-labeled DNA showed significantly
different banding patterns between the nutrient supplement treatments and compared with total DNA banding
patterns. PCR primers were developed to specifically amplify the intergenic region of an rDNA sequence unique
to the BrdU analysis of a phosphate supplement treatment. Amplification of DNA from all treatments using
these primers showed that it was unique to the phosphate treatment and that it was present in both the total
DNA and BrdU-labeled DNA fractions. This result demonstrates the promise of this new strategy, because it
was able to permit identification of a sequence from a phosphate-responsive organism that was not discernable
in the traditional total DNA community structure analysis.
The majority of extant microorganisms have yet to be described, and fewer than one percent of these can be cultured by
standard methods (1, 7). The extent of this diversity was exemplified by a study which used genetic complexity measurements
to calculate that 4,000 completely different bacteria genomes
inhabited a Norwegian forest soil sample (19). Recently, descriptions of rRNA genes (rDNA) obtained from environmental samples have provided both a glimpse of this extensive
microbial diversity and a tool to identify uncultured organisms
(1, 14). However, progress beyond the classification stage of
microbial ecology to the physiological stage has been primarily
limited to community-level analyses such as microbial respiration, substrate utilization, N2 fixation, signature lipid biomarkers, and enzyme activities (9).
To correlate the activity of specific microorganisms with
defined environmental or physiological parameters, Stahl et al.
pioneered the use of rRNA hybridization probes to monitor
population levels and shifts (17). However, this strategy requires the prior identification and characterization of meaningful sequences. Given the immense complexity of most natural microbial communities, identification of such sequences is
a daunting task. A random analysis of rRNA or rDNA sequences from an environmental sample would likely lead to the
identification of the dominant organisms in that community
but not necessarily the ones involved in a particular physiological response. This report describes a new approach to bridge
the gap between function and phylogeny by permitting the
identification of populations that grow in response to specified
or measured stimuli.

added to sieved soil samples throughout a 3-day period and incubated in petri
dishes at room temperature. On day 1, H2O and glucose were mixed into the soil
samples (500 l of H2O was added to treatments C and D, 550 l of H2O was
added to treatment B, and 50 l of 888 mM glucose was added to treatments C
and D). On day 2, H2O and supplements were mixed into the soil samples (250
l of H2O was added to treatments B and C, 200 l of H2O was added to
treatment D, 50 l of 100 mM bromodeoxyuridine [BrdU] was added to treatments A to D, and 50 l of 89 mM KH2PO4 was added to treatment D). On day
3, H2O and supplements were mixed into the soil samples (50 l of H2O was
added to treatments B to D, and 50 l of 100 mM BrdU was added to treatments
A to D). On day 4, DNA was extracted from the soil samples with the FastDNA
Kit For Soil as described by the manufacturer (BIO 101, Vista, Calif.) (4).
BrdU immunocapture. Immunocapture of BrdU-labeled DNA was performed
by a modification of the method described by Haider et al. (8). Twenty-five
microliters of Dynabeads (M-450) coated with sheep anti-mouse immunoglobulin G was washed three times with 200 l of phosphate-buffered salinebovine
serum albumin (PBS-BSA [PBS containing 0.1% BSA]) as described by the
manufacturer (Dynal, Lake Success, N.Y.). These beads were resuspended in 84
l of PBS-BSA-herring (PBS-BSA containing 5 mg of herring sperm DNA
[Promega, Madison, Wis.] per ml) and incubated with 16 l of anti-BrdU antibody (Boehringer Mannheim, Indianapolis, Ind.) for 60 min at room temperature while being rotated. This mixture was washed three times with 200 l of
PBS-BSA and resuspended in 80 l of PBS-BSA-herring. Twenty microliters of
denatured soil DNA (95C for 5 min, 5 min on ice) was added to this suspension
and incubated at room temperature for 120 min while being rotated. This mixture was washed three times with 200 l of PBS-BSA and resuspended in 40 l
of H2O. This suspension was heated at 95C for 5 min and cooled on ice for 5
min, and the non-bead fraction was collected by magnetic capture. After this
work was completed, an alternative procedure to isolate BrdU-labeled DNA was
described (20). Comparison of the two procedures showed that the protocol
described by Urbach et al. (20) is preferable, as it isolated less unlabeled DNA
(data not shown).
Community structure analysis. Community structure analysis was performed
by resolving PCR-amplified 16S-23S rRNA intergenic fragments on a denaturing
polyacrylamide gel (5). The intergenic spacer region between the small- and
large-subunit rRNA genes was amplified in 150-l PCR mixtures with the following final concentrations or total amounts: 20 ng of soil DNA or 1 ng of
BrdU-labeled DNA, 50 mM Tris (pH 8.3), 500 g of BSA per ml, 2.5 mM MgCl2,
250 M deoxynucleoside triphosphates (dNTPs), 200 nM forward primer 1406F
TGYACACACCGCCCGT (10), 200 nM reverse primer 23R GGGTTBCCCC
ATTCRG, and 7.5 U of Taq DNA polymerase. All reagents were combined and
heated at 94C for 2 min. For the soil DNA, 25 cycles of PCR were performed;
for the BrdU-labeled DNA, 35 cycles were performed. PCRs were done with an
Air Thermo-Cycler (Idaho Technologies, Idaho Falls, Idaho) at 94C for 15 s,
52C for 15 s, and 72C for 30 s, followed by 72C for 2 min. PCR products were
purified with the QIAquick PCR Purification Kit (Qiagen, Valencia, Calif.),

MATERIALS AND METHODS

Nutrient supplementation and DNA extraction. One-gram soil samples were
amended with four different nutrient supplement treatments. Supplements were

* Mailing address: Department of Plant Pathology, University of

California, Riverside, CA 92521. Phone: 909-787-3584. Fax: 909-7873782. E-mail: borneman@ucrac1.ucr.edu.
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VOL. 65, 1999

IDENTIFICATION OF MICROBES THAT RESPOND TO STIMULI

FIG. 1. Outline of the strategy to identify microorganisms that respond to

stimuli.

eluted with 50 l of H2O, dried to a volume of 10 l, and denatured by addition

of 10 l of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol
blue, 0.05% xylene cyanol FF) and heating at 95C for 5 min. The DNA was
loaded on a prerun 5% (29:1) polyacrylamide0.6 Tris-borate-EDTA TBE gel
(0.375 mm thick) containing 8 M urea and electrophoresed at 50 W for 150 min
to maintain a temperature between 55C and 60C. Gels were silver stained with
the SILVER SEQUENCE DNA Staining Reagents (Promega) (2).
Sequence-specific PCR. Band A was excised from the polyacrylamide gel (see
Fig. 2), PCR amplified with the parameters described above, cloned into the
pGEM-T vector as described by the manufacturer (Promega), and sequenced
with an ABI PRISM Dye Terminator Cycle Sequencing Kit (Perkin Elmer,
Foster City, Calif.). Sequence-specific PCR primers were designed to hybridize
to the 16S-23S rRNA intergenic region of this sequence. Ten-microliter PCRs
were performed on total DNA and BrdU-labeled DNA from all treatments,
resolved on a 1.25% agarose gel, and stained with ethidium bromide. PCRs
contained 50 mM Tris (pH 8.3), 2.5 mM MgCl2, 500 ng of BSA per l, 250 nM
dNTPs, 400 nM forward primer (TCACTTACTGTTCGGTTT), 400 nM reverse
primer (CTTGGATAGAAGAAGCAT), 0.5 U of Taq DNA polymerase, and
either 25 ng of total DNA template or 1 ng of BrdU-captured DNA template.
PCR cycle parameters were as follows: 2 min at 94C, 40 cycles of 0 s at 94C, 20 s
at 50C, and 10 s at 72C; and 2 min at 72C.
Nucleotide sequence accession number. The 16S-23S rRNA intergenic sequence obtained from band A (see Fig. 2) has been deposited in GenBank under
the accession number AF124217.

RESULTS AND DISCUSSION

This recently designed methodology uses a modification of
the thymidine incorporation technique, which quantifies microbial growth by measuring the amount of thymidine that is
incorporated into microbial DNA (13). [3H]thymidine uptake
has been used successfully to measure in situ bacterial growth
in soil, aquatic, and other environments (6, 13, 18). By replacing [3H]thymidine with BrdU, the newly synthesized DNA can
be isolated by BrdU immunocapture. The microbes that grow
in response to a stimulant may therefore be identified by (i) the
simultaneous addition of BrdU and a stimulant to an environmental sample, (ii) sample incubation, (iii) DNA extraction,
(iv) immunocapture of the BrdU-labeled DNA, and (v) rRNA
gene analysis (Fig. 1).
To depict the unique perspective that can be obtained from
this approach, the microbial community structures of soil supplemented with four different nutrients were examined by both
the new BrdU-labeled DNA strategy and a traditional total
DNA analysis (Fig. 2). The community analyses were done by
resolving PCR-amplified 16S-23S rRNA intergenic region

3399

FIG. 2. Comparison of the microbial community structures obtained from

total DNA and BrdU-labeled DNA by analysis of the 16S-23S rRNA intergenic
region after exposure to nutrient supplements. Lanes: 1, X174/HaeIII molecular weight marker (Promega); 2 and 6, treatment A; 3 and 7, treatment B; 4 and
8, treatment C; 5 and 9, treatment D.

fragments on a denaturing polyacrylamide gel (5). The size

heterogeneity of these fragments provides a simple method to
depict bacterial community structure. When comparing the
banding patterns from the four treatments by use of total
DNA, virtually no difference can be seen. However, the BrdUlabeled DNA revealed significantly different patterns, both between the treatments and compared to that of the total DNA.
A likely explanation for the difference is that the standard total
DNA analysis examines all of the DNA extracted from soil,
including DNA from organisms that did not respond to the
treatment and naked DNA that is associated with soil particles.
Conversely, the BrdU-labeled DNA approach examines only
newly synthesized DNA, allowing the identification of the responding and actively growing organisms. In addition, the lack
of PCR products in the BrdU-only (lane 6) and no-BrdU (data
not shown) treatments shows that the BrdU did not act as a
stimulant for growth and that the immunocapture process is
specific.
To validate these different depictions of bacterial diversity,
the relative abundance of a specific sequence unique to the
phosphate treatment was assessed. This sequence was obtained
by cloning the 16S-23S rRNA intergenic region DNA contained in band A (Fig. 2, lane 9). The clone used in this analysis
has 96% sequence similarity to a 16S rRNA gene from Bacillus
subtilis (12). To verify its relative abundance, specific PCR
primers were designed to hybridize to the 16S-23S rRNA intergenic region of band A. Both total DNA and BrdU-captured DNA from all treatments were subjected to amplification
by PCR (Fig. 3). Only the phosphate treatment produced a
PCR product, suggesting that the organism represented by
band A specifically responded to the phosphate supplement.
The fact that band A was successfully amplified in both the
BrdU-captured DNA and the total DNA demonstrates the
promise of this new strategy, as it identified a sequence from a
phosphate-responsive organism that was not discernable in the
traditional total DNA analysis. The significant genetic complexity of total DNA analyses will presumably lead to depictions of microbial community structure biased towards the
most numerous gene sequences, which may have no relevance

3400

BORNEMAN

APPL. ENVIRON. MICROBIOL.

captured. It could also be used to identify microorganisms that

respond to measurable natural phenomena such as nutrient
availability and signal molecules.
ACKNOWLEDGMENTS
I thank Gary G. Judd and Donald A. Cooksey for their comments on
the manuscript.

FIG. 3. Validation of a specific rRNA gene sequence from a phosphateresponsive organism by PCR. Lanes: 9, no-template negative control; 10, 1-kb
ladder (Gibco BRL, Grand Island, N.Y.).

to a given physiological response. The BrdU capture approach

removes large pools of background DNA, enabling analysis of
only actively growing populations.
Recently, Urbach et al. (20) demonstrated the promise of
the BrdU strategy in an aquatic environment. They showed
that the BrdU approach isolated a subset of the total DNA
located in a freshwater lake, suggesting that only a fraction of
this bacterial community was actively dividing. My study expands on this concept by demonstrating the utility of this strategy to examine the effects of exogenous chemical stimuli on
bacterial community structure in soil. I also have shown the
advantage of the BrdU strategy over a total rDNA analysis in
identifying changes in community structure. The potential limitations of the BrdU strategy described in both reports include
the range of organisms capable of BrdU uptake and incorporation. Urbach et al. (20) showed that only two of four different
bacterial strains incorporated BrdU. Other reports, however,
suggest that the majority of bacteria take up and incorporate
[3H]thymidine (16). Since BrdU has been used successfully as
a thymidine analog in numerous applications, it is likely that
most organisms will also take up and incorporate BrdU. Another possible limitation of this strategy may come from the
potential toxicity of halogenated nucleotides incorporated into
DNA (11). However, the growth kinetics for specific bacterial
cultures obtained with and without BrdU were shown to be
similar (20). In addition, if used at modest concentrations,
BrdU appears to be relatively safe and is currently being administered to human cancer patients (3, 15). Despite these
potential shortcomings, both of these reports demonstrate an
approach that can assist in the investigation of uncultured
microorganisms. In future work, this strategy could be modified to include the identification of important genes, since the
genomic DNAs of the responding microorganisms are also

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