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Applied and Environmental Microbiology, January 1999, p. 6-10, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Fate of Free DNA and Transformation of the Oral
Bacterium Streptococcus gordonii DL1 by Plasmid DNA in
Human Saliva
Derry K.
Mercer,1,*
Karen P.
Scott,1
Wendy A.
Bruce-Johnson,1
L. Anne
Glover,2 and
Harry J.
Flint1
Rowett Research Institute,
Bucksburn,1 and
Department of Molecular
and Cell Biology, University of Aberdeen, IMS,
Foresterhill,2 Aberdeen, Great Britain
Received 6 July 1998/Accepted 14 October 1998
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ABSTRACT |
Competitive PCR was used to monitor the survival of a 520-bp DNA
target sequence from a recombinant plasmid, pVACMC1, after admixture of
the plasmid with freshly sampled human saliva. The fraction of the
target remaining amplifiable ranged from 40 to 65% after 10 min of
exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been
exposed to degradation by fresh saliva was capable of transforming
naturally competent Streptococcus gordonii DL1 to
erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of
filter-sterilized saliva and a 1-µg/ml final concentration of pVACMC1
DNA. Addition of filter-sterilized saliva instead of heat-inactivated
horse serum to S. gordonii DL1 cells induced competence,
although with slightly lower efficiency. These findings indicate that
DNA released from bacteria or food sources within the mouth has the
potential to transform naturally competent oral bacteria. However,
further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.
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INTRODUCTION |
Interest in gene transfer in the
gastrointestinal tract has been stimulated recently by the need to
assess risks associated with the possible onward transfer of
manipulated traits from genetically modified microorganisms or
DNA-containing transgenes to gut bacteria (5, 23).
More generally, gene transfer plays an important role in the
evolution of gut bacteria and in their ability to adapt to
environmental challenges. It is widely accepted that conjugal transfer
involving plasmids and conjugative transposons, as well as
bacteriophage transduction, plays a major role in gene transfer between
bacteria in the gut (24). Conjugal transfer of recombinant
plasmids has been demonstrated between strains of Escherichia
coli and between strains of lactic acid bacteria introduced
into the digestive tract of gnotobiotic mice (4, 9). By
contrast, the possible role of transformation has received little
attention, largely because free DNA has been considered unlikely to
survive the action of gut nucleases (26). Despite this,
fragments of bacteriophage M13 DNA were recently reported to survive
passage through the mouse gastrointestinal tract and were even found in
some mouse tissues (27, 28). Many bacteria, including
representatives of the oral and gut microflora, are known to be
naturally transformable (15). Uptake of DNA by competent cells in vitro occurs rapidly, at a rate of 100 nucleotides/s in the
case of Streptococcus pneumoniae DP1601 (19);
free DNA would therefore need to survive for only short periods of time to enable competent bacteria to be transformed. Furthermore, we know
little about the effects of food components or gut microenvironments upon DNA survival in the gastrointestinal tract. Free DNA in soils is
often bound to soil minerals (7) and plant polysaccharides (31, 32), where it is more resistant to nucleolytic attack (22) while still being available for the transformation of
competent bacteria (3).
In assessing the fate of free DNA in the digestive tract, it is
important to consider all regions, including the oral cavity and
esophagus. The oral cavity is the site of first contact between incoming bacteria and free DNA in food and the resident microflora and
is one of the most complex and heterogenous microbial habitats in the
human body (8). Certain oral bacteria are important agents
of periodontal disease, dental caries, and some systemic infections
(18), and some of these species are also naturally competent
(13, 29, 34). In this study, we investigate the survival of
free DNA in human saliva and use competitive PCR to quantify DNA
degradation. Plasmid DNA that has been exposed to degradation in human
saliva is shown to be able to transform the naturally competent oral
bacterium Streptococcus gordonii DL1 in vitro.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Bacterial strains
used are described in Table 1.
Lactococcus lactis IL1403 was routinely maintained at 30°C
in M17 broth (Oxoid) supplemented with 0.5% (wt/vol) glucose. S. gordonii DL1 and Streptococcus mutans strains were
routinely maintained in brain heart infusion (BHI) broth (Difco), and
E. coli XL1-Blue cultures were maintained in Luria broth
(25), all at 37°C. All cultures were stored at 
85°C in
10% (vol/vol) glycerol after being snap-frozen in liquid
nitrogen.
DNA purification and manipulation.
All plasmids used are
described in Table 1. Plasmid DNA was purified from E. coli
cultures by the alkaline lysis method (2) and was
isolated from streptococci and lactococci by the method of O'Sullivan
and Klaenhammer (21). L. lactis IL1403
chromosomal DNA was prepared by the method of Leenhouts et al.
(14), and DNA was dissolved in sterile distilled water
(dH2O). Bacteriophage 
DNA was purchased from Boehringer
Mannheim. Restriction digestion was carried out according to the
manufacturer's instructions (Promega). The concentration of DNA in all
samples was determined by a GeneQuant II apparatus (Pharmacia Biotech).
Simulation of oral and gastrointestinal conditions.
Saliva
was taken prior to tooth brushing in the morning and was sampled from
five different healthy volunteers. Volunteers were two males and three
females aged between 25 and 35 years. To simulate stomach conditions,
saliva was acidified to pH 1 to 2 or pH 3 to 4 with concentrated
hydrochloric acid. The effect of small intestinal conditions was
stimulated with 1.5 mg (wt/vol) of pancreas acetone powder (Sigma) per
ml dissolved in 150 mM NaHCO3 (20). Colonic
lumen conditions were simulated with a 20% (wt/vol) human fecal slurry
dissolved in one-fourth strength Ringers solution.
DNA degradation experiments.
Except where otherwise stated,
0.15 µg of DNA, dissolved in 10 µl of sterile dH2O, was
added to 30 µl of saliva, acidified saliva, pancreas acetone
solution, or fecal slurry and mixed thoroughly. Samples were incubated
at 37°C, and at set time intervals degradation was stopped by the
addition of 80 µl of phenol-chloroform (1:1) followed by thorough
mixing. The aqueous layer was removed after centrifugation (Jouan A14
microcentrifuge, 10 min, 17,746 × g) and was further
purified by precipitation of DNA with 160 µl of ethanol, a wash with
70% (vol/vol) ethanol, and resuspension of DNA in 15 µl of sterile
dH2O. Samples of the aqueous phase (10 µl) were run on
agarose gels containing 0.15 µg of ethidium bromide per ml, and the
remainder was used for PCR amplification.
PCR.
PCR amplification of a 520-bp fragment of plasmid
pVACMC1 was carried out by capillary PCR with a Corbett Research
FTS-4000 thermal sequencer using primers CMCP2
(5'-GACAAGACAAAGAAGACTCC-3') and pVACrev
(5'-AGCGATCCTTGAAGCTGTC-3') (Applied Biosystems). Samples
(2 µl) containing template DNA were added to a standard PCR mixture
(11), except for the addition of extra MgCl2
(6.5 mM final concentration). Amplification was carried out with the following cycle times: one cycle of 94°C for 3 min, 58°C for
10 s, and 72°C for 15 s, followed by 31 cycles of 94°C
for 10 s, 58°C for 10 s, and 72°C for 15 s, with a
final elongation step of 72°C for 5 min and a ramp rate of 2°C/s.
Direct amplification of target DNA from transformant colonies was
carried out according to the protocol of Güssow and Clackson
(10). After PCR amplification, samples were analyzed on a
1.4% (wt/vol) ethidium bromide-stained agarose gel.
Competitive PCR.
Quantitative detection of the concentration
of the 520-bp PCR product of pVACMC1 was carried out by competitive
PCR. A competitor plasmid was constructed by the introduction of a
100-bp fragment of DNA from the 16S rRNA gene of Prevotella
albensis M384 at the end of the Ruminococcus
flavefaciens endoglucanase A (endA) gene (Fig.
1A). Upon amplification with the CMCP2
and pVACrev primers described above, the competitor plasmid
gave a 620-bp band easily distinguishable from the 520-bp band produced
by the target on a 1.4% agarose gel (Fig. 1B).
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FIG. 1.
Construction of the competitor plasmid to pVACMC1 and
its use in competitive PCR. (A) Plasmid pVACMC1 and its competitor,
pVACMCcomp, obtained by insertion of a 100-bp SalI fragment
(see text). (B) Trial competitive PCR experiment in which different
ratios of competitor plasmid and pVACMC1 were subjected to PCR
amplification with the forward PCR primer CMCP2 and the reverse primer
pVACrev. The R. flavefaciens cellulase gene,
endA, encodes CMCase activity.
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The concentration of saliva-degraded pVACMC1 was determined by adding
known concentrations of pVACMCcomp to degraded pVACMC1.
PCR
amplification was carried out with primers CMCP2 and
pVAC
rev,
and the products were separated on 1.4% (wt/vol)
agarose gels.
An initial concentration range of 0.1 to 1.0 µg of
competitor
plasmid per ml was used; if no crossover point was observed,
fivefold
dilutions of competitor were used until a crossover point was
obtained. At the point where the intensity of target and competitor
bands is identical (crossover), the concentration of competitor
plasmid
is equal to that of the degraded pVACMC1 (Fig.
1B).
Transformation of oral streptococci with pVACMC1 plasmid
DNA.
Transformation of S. gordonii DL1 with pVACMC1 was
carried out with the protocol of Macrina et al. (16). In the
experiment represented by Fig. 5, heat-inactivated horse serum was
replaced with the same volume of either horse serum, filter-sterilized saliva, or sterile dH2O as an inducer of competence
development. Competent cells (330 µl) were aliquoted into sterile
Eppendorf tubes containing a minimal volume of transforming DNA (final
concentration, 1.0 µg/ml) and incubated aerobically, with shaking,
for 2 h at 37°C. Transformants were plated on BHI agar
containing 10 µg of erythromycin per ml and incubated anaerobically
at 37°C for 48 to 72 h. Transformation, in all cases, was
confirmed by growth on selective media, analysis of plasmid DNA, PCR
amplification of colony lysates, and demonstration of
carboxymethylcellulase (CMCase) activity. For the experiment
represented by Fig. 3, a higher initial concentration, 30 µg of
pVACMC1 DNA per ml, was used in order to monitor the decrease in
concentration of transforming DNA following exposure to saliva.
Transformation of S. gordonii DL1 with pVACMC1 in the
presence of filter-sterilized saliva.
Competent cells of S. gordonii DL1 were prepared as described by Macrina et al.
(16). Competent cells (165 µl) were added to sterile
Eppendorf tubes and mixed with an equal volume of filter-sterilized saliva. pVACMC1 DNA (in a minimal volume of dH2O) was added
to the mixture to give a final concentration in the transformation mixture of 1 µg/ml. The transformation mixture was incubated at 37°C, with shaking, for 0 to 240 min. At set time points, samples of
the transformation mixture were removed and diluted appropriately, and
100 µl was plated onto selective agar (BHI agar plus 10 µg of
erythromycin per ml) and incubated anaerobically at 37°C for 48 to
72 h. Transformation, in all cases, was confirmed by growth on
selective media, analysis of plasmid DNA, PCR amplification of colony
lysates, and demonstration of CMCase activity.
Determination of CMCase activity.
Transformant cells were
plated onto BHI agar containing 0.1% (wt/vol) medium-viscosity
carboxymethylcellulose (Sigma) and stained with 1.0 mg of Congo red per
ml following overnight incubation at 37°C to detect the expression of
the endoglucanase A (endA) gene of R. flavefaciens (6).
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RESULTS |
Survival of plasmid DNA sequences in human saliva monitored by
competitive PCR and transformation of S. gordonii DL1.
The survival of DNA added to human saliva to a final concentration of
3.75 µg/ml was followed in preliminary experiments by agarose gel
electrophoresis. This DNA concentration allowed visual assessment of
degradation with L. lactis IL1403 chromosomal DNA, plasmid
DNA (pVACMC1 and pIL253), and linear bacteriophage DNA. In all
cases, DNA was visible in an incompletely degraded state for at least
3.5 min after addition to human saliva and for up to 1 min with
acidified saliva (pH 3 to 4) (data not shown). By contrast, DNA added
to acidified saliva (pH 2 or less), pancreas acetone solution, or human
fecal slurry was undetectable by this approach after 30 s (data
not shown).
In order to achieve more sensitive detection of surviving DNA sequences
present in a particular recombinant plasmid, a 520-bp
portion of
plasmid pVACMC1 was amplified by PCR following exposure
to saliva
samples. pVACMC1 comprises the
E. coli/gram-positive
shuttle
vector pVA838 carrying a cloned cellulase-encoding fragment
from the
rumen anaerobic bacterium
R. flavefaciens (
17,
35).
The two PCR primers were designed to give highly specific
detection,
since one recognizes vector sequences and the other a
sequence
in the cloned cellulase fragment (Fig.
1A). As can be seen
from
Fig.
2, a 520-bp fragment of pVACMC1
was still amplifiable after
9 min of incubation in saliva, and
amplifiable target DNA was
still detectable even after 24 h of
incubation (data not shown).
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FIG. 2.
Persistence of a 520-bp fragment of pVACMC1 DNA after
incubation with fresh, whole saliva for 9 min. PCR amplification of
degraded DNA was carried out as described in Materials and Methods, and
products were analyzed on a 1.4% (wt/vol) agarose gel. A 1-kb DNA
ladder (Gibco BRL), 0.5 to 12.2 kbp, was used.
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Quantitative detection of saliva-degraded pVACMC1 sequences was carried
out by competitive PCR. A competitor plasmid was constructed
(Fig.
1A)
and used as described in Materials and Methods. A typical
competitive
PCR gel is shown in Fig.
1B. Survival of the 520-bp
fragment was
estimated during incubation of pVACMC1 with saliva
from five different
volunteers (Table
2). After 10 min of
exposure
to saliva, between 35 and 61% of the target DNA had been
degraded,
while between 75 and 94% of the target had been degraded
after
1 h.
In the experiment represented by Fig.
3,
pVACMC1 was exposed to human saliva for varying time intervals and
plasmid DNA was
immediately extracted to inhibit further degradation.
Half of
the partially degraded DNA was run on a 0.8% (wt/vol) agarose
gel (Fig.
3A), and the other half was used to transform competent
cells
of
S. gordonii DL1 (Fig.
3B). The object of this experiment
was to determine for how long DNA fragments of a size sufficient
to
effect transformation remained, and very high initial concentrations
of
DNA (30 µg/ml) were used to allow sensitive detection. Degradation
of
pVACMC1 is evident from the disappearance of the band corresponding
to
the open circular form of the plasmid, which is visible at
time
zero, and from the initial increase and subsequent decrease
in
intensity of the band representing the linear form of the plasmid
between 4 and 10 min (Fig.
3A). Saliva-degraded pVACMC1 DNA
showed
a rapidly decreasing capacity to transform
S. gordonii DL1 (half-life,
approximately 50 s) to erythromycin
resistance, and the transformant
yield after 2 min of degradation was
some 10-fold lower than for
unexposed DNA (Fig.
3B). The authenticity
of transformants was
confirmed by demonstrating the presence of a
plasmid of the expected
size and restriction profile, by PCR
amplification with the specific
CMCP2 and pVAC
rev primer
pair and by the production of CMCase
activity. Transformation
efficiencies up to 9 min were all >1
× 10
7, which
is higher than the spontaneous mutation rate to erythromycin
resistance
(<1 × 10
8) of this strain (data not shown).
Successful transformation in
gram-positive bacteria involves
reconstitution of a functional
plasmid from different partially
degraded molecules (
15), and
this would be expected to
require fragments significantly larger
than the 520-bp PCR target
studied, since replication functions,
the endoglucanase gene, and the
selectable marker must also be
present.
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FIG. 3.
(A) Survival of pVACMC1 DNA in human saliva analyzed on
a 0.8% (wt/vol) agarose gel. A 1-kb DNA ladder (Gibco BRL), 0.5 to
12.2 kbp, was used. OC, open circular; L, linear. A faint band of
covalently closed circular DNA was visible in lane 0). (B)
Transformation of S. gordonii DL1 with pVACMC1 DNA that had
been previously exposed to fresh, whole saliva for the times
indicated.
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Transformation of S. gordonii DL1 by plasmid DNA in the
presence of human saliva.
Streptococci are normal components of
the flora of the oropharyngeal mucosa and also occur in supragingival
plaque and dental caries (12, 29, 33); a number of these
species, including strains of S. gordonii and S. mutans, are reported to be naturally competent. S. gordonii DL1 (Streptococcus sanguis Challis), a naturally transformable strain originally isolated from human serum
(13), was already known to be transformable by pVACMC1 in
vitro (35) and was chosen here as a test recipient.
Transformation was demonstrated when competent
S. gordonii
DL1 cells and plasmid DNA, at a final concentration of 1.0 µg/ml,
were simultaneously added to filter-sterilized saliva, as shown
in Fig.
4. Similar results were obtained for
saliva from a second
volunteer (results not shown). The apparent delay
in the appearance
of transformants is assumed to be due to the time
required to
build up a sufficient plasmid copy number and level of gene
expression
to achieve growth upon plating on media containing
erythromycin.
A lag phase was also observed in the absence of saliva
(Fig.
4).
Whole saliva gave a background of erythromycin-resistant
colonies
(approximately 10
6 CFU/ml from a total population
of 10
8 CFU/ml), and native bacteria present in whole saliva
would be
expected to compete with the test strain for the added DNA.
Nevertheless,
approximately 0.1% of erythromycin-resistant colonies
showed CMCase
activity in agar plate tests in similar transformation
experiments
with whole saliva. These contained amplifiable pVACMC1
plasmid
DNA and must have arisen by transformation. Degradation of
pVACMC1
in filter-sterile saliva, estimated by competitive PCR,
occurred
at a rate of approximately 60% of that in whole saliva.
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FIG. 4.
Transformation of S. gordonii DL1 with
pVACMC1 in the presence and absence of filter-sterilized saliva.
Competent cells were exposed to DNA and saliva or sterile
dH2O for the times shown before plating on selective medium
to identify transformants. Maximum transformation frequencies for the
saliva of a second volunteer were 3.7 × 105.
Experiments were carried out with different samples of S. gordonii DL1 competent cells.
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In all of the experiments reported so far,
S. gordonii DL1
was made competent according to standard procedures (
16) by
addition
of 10% (vol/vol) heat-inactivated horse serum to the growth
medium.
In the experiment represented by Fig.
5, the effect of substituting
alternative
growth medium supplements for heat-inactivated horse
serum upon
competence development and subsequent transformation
efficiency was
studied. Replacement of heat-inactivated horse
serum by untreated horse
serum decreased transformant yield, and
similar results were obtained
with both heat-inactivated and untreated
human serum (data not shown).
However, the addition of filter-sterilized
human saliva contributed to
competence development and gave transformation
efficiencies only 20%
lower than those obtained with heat-inactivated
horse serum (Fig.
5).
In the absence of any apparent inducer of
competence development, in
the case of water supplementation,
transformation efficiency was
threefold lower than with heat-inactivated
horse serum.
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FIG. 5.
Transformation of S. gordonii DL1 with
pVACMC1 after competence induction by different growth supplements. The
transformation protocol described in Materials and Methods was followed
except that heat-inactivated horse serum was replaced with the other
growth supplements.
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DISCUSSION |
This work demonstrates that free DNA can survive for a significant
time in samples of human saliva in an incompletely degraded state,
since between 40 and 65% of a 520-bp target region within plasmid
pVACMC1 was found to be amplifiable after 10 min of incubation with
saliva. Since experiments were done in vitro and involved a 4:1
admixture of saliva and added DNA solution, the precise kinetics of
degradation do not exactly reproduce those occurring in vivo. However,
over short time intervals the admixture of freshly sampled saliva with
the DNA-containing sample must be considered a reasonable simulation of
the admixture of ingested food with saliva in the mouth. High initial
concentrations of DNA were studied here in order to test the ability of
a plasmid that had been exposed to salivary nucleases to
transform naturally competent cells of S. gordonii DL1, a
species that forms part of the oral microflora. The rapid decrease
observed in transforming activity with time of exposure to saliva,
which decayed with a half-life of approximately 50 s, reflects the
decreasing population of transforming DNA molecules. Natural
transformation of gram-positive bacteria is thought to involve
reconstitution of an active plasmid from more than one partially
degraded molecule of plasmid DNA within the cell exhibiting two-hit
kinetics (1, 15). Therefore, the transformation frequency is
expected to change in proportion to the square of the DNA concentration.
We also show here that naturally competent S. gordonii DL1
can be transformed by plasmid DNA in the presence of human saliva. It
is not known what proportion of oral streptococci develop competence under in vivo conditions in the oral cavity, and we have not
attempted to address this question directly here.
However, our findings do show that heat-inactivated horse serum, which
is routinely added to S. gordonii DL1 cells to enhance
development of "natural" competence, is not an absolute requirement
for competence development. Furthermore, our data suggest that human
saliva may itself contain factors that promote competence development.
Thus, there is no reason to assume that S. gordonii and
probably other oral streptococci, such as S. mutans, that
show natural transformability under laboratory conditions (29,
34) are not capable of transformation at low frequencies in the
oral cavity in vivo. The transformation experiments reported here were
deliberately performed at high cell densities and with high initial DNA
concentrations (1.0 µg/ml) in order to maximize detection of
transformant numbers, and we make no attempt here to estimate the
frequency of such events in vivo. A plasmid concentration of 1 µg/ml
would correspond approximately to the complete plasmid content of
1 ml of a bacterial culture of 109 cells/ml, assuming a
copy number of 50 per cell for a 10-kb plasmid, and is very unlikely to
occur naturally. Assuming two-hit kinetics (1), it is
predicted that transformation frequencies would be lower by a factor of
106 for a plasmid concentration of 1 ng/ml than those
reported here for a 1-µg/ml plasmid concentration, but it is very
difficult to extrapolate from homogeneous pure cultures to
conditions in vivo, particularly for colonies or biofilms where cells
and DNA may be highly localized. The question of whether quantitatively significant rates of natural genetic transformation occur in vivo in
the oral cavity therefore remains open and requires further investigation, although it should be clear that even very infrequent transformation events can be highly significant if the transforming DNA
bestows a selective advantage on the recipient. DNA is constantly released from ingested plant, animal, or microbial cells in
different regions of the gut. Although naked DNA is predicted to
have less chance of survival under conditions prevailing lower down the digestive tract than in the oral cavity due to the effects of stomach
acid and pancreatic nucleases, survival of bacteriophage M13 DNA has
been demonstrated in the rat gut (27). In conclusion, the
possibility of microbial transformation events occurring in a variety
of gut microenvironments cannot be ruled out and deserves attention as
a potential factor in the adaptation and evolution of gut microorganisms.
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ACKNOWLEDGMENTS |
This work was supported by a grant from the United Kingdom
Ministry of Agriculture, Fisheries and Food, as part of the Novel Foods Programme.
We thank Terry Whitehead for the donation of S. gordonii DL1
and for help with streptococcal transformations. We also thank Colin
Stewart for valuable contributions during discussions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Rowett
Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB,
Great Britain. Phone: 44 (0) 1224 712751. Fax: 44 (0) 1224 716687. E-mail: d.mercer{at}rri.sari.ac.uk.
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Applied and Environmental Microbiology, January 1999, p. 6-10, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
