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Applied and Environmental Microbiology, September 1999, p. 3800-3804, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
An Effective Strategy, Applicable to
Streptococcus salivarius and Related Bacteria, To Enhance or
Confer Electroporation Competence
Nicole D.
Buckley,1
Christian
Vadeboncoeur,1
Donald J.
LeBlanc,2,
Linda N.
Lee,3 and
Michel
Frenette1,*
Groupe de Recherche en Écologie
Buccale, Département de Biochimie et de Microbiologie,
Faculté de Sciences et de Génie, et Faculté de
Médecine Dentaire, Université Laval, Québec, Canada,
G1K 1P41; Department of Oral Biology,
Indiana University School of Dentistry, Indianapolis, Indiana
462022; and Department of Medicine,
University of Texas Health Science Center at San Antonio, San
Antonio, Texas 78284-77583
Received 2 March 1999/Accepted 10 June 1999
 |
ABSTRACT |
Despite the large number of techniques available for transformation
of bacteria, certain species and strains are still resistant to
introduction of foreign DNA. Some oral streptococci are among the
organisms that can be particularly difficult to transform. We performed
a series of experiments that involved manipulation of growth and
recovery media and cell wall weakening, in which the electroporation
conditions, cell concentration, and type and concentration of the
transforming plasmid were varied. The variables were optimized such
that a previously untransformable Streptococcus salivarius
strain, ATCC 25975, could be transformed reproducibly at a level of
105 transformants per µg of DNA. The technique was used
to introduce a plasmid into other strains of S. salivarius,
including a fresh isolate. Moreover, the same technique was applied
successfully to a wide range of oral streptococci and other
gram-positive bacteria.
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INTRODUCTION |
Genetic manipulation of bacteria has
become a powerful tool for elucidating fundamental biological
mechanisms. While there is a plethora of techniques available to
introduce foreign DNA into several bacterial species (for reviews see
references 15 and 22), some
bacteria have proved to be refractory to most or all of the protocols
that have been described. Occasionally, there is an entire species
whose members are difficult to transform, such as Streptococcus
salivarius or Streptococcus sobrinus, while in other
species (e.g., Streptococcus mutans) there are large differences in transformability among strains. Characterization of such
species is difficult, as it relies on often painstaking, alternative
approaches. Moreover, in some cases, information accumulated with one
bacterial strain cannot be built upon, and the strain is abandoned in
favor of a related, more amenable strain.
Certain streptococci can become naturally competent; i.e., they can
take up free DNA from the surrounding medium (6). Natural competence is expressed by these bacteria during growth under defined
conditions, usually only at certain stages of growth (22). The streptococci have been divided into the following six major phylogenetic clusters based on their 16S RNA sequences: the pyogenic group, the anginosus group, the mitis group, the mutans group, the
bovis group, and the salivarius group (9). Natural
competence of streptococci has been associated with the products of a
few genes, and in a survey, Håverstein et al. (6) found
that these genes were present in strains belonging to the mitis and
anginosus groups and more rarely in members of the mutans group.
Strains belonging to the other groups lacked these genes, as well as
the associated natural competence phenotype. In the absence of natural competence, what Saunders et al. (22) called chemical
competence would have to be induced. Thus, transformation techniques
for members of nonnaturally competent streptococci have been developed. Transformation of Streptococcus bovis involves generation of
protoplasts, followed by transformation (13). Conjugation
(or mobilization by a conjugative plasmid) is a reproducible, efficient
method for introducing DNA into S. sobrinus (1a),
and electroporation of whole cells has been used with several different
Streptococcus species and strains (2, 4, 10, 12, 13,
19, 23, 24, 27). The different techniques are needed because what works for one strain or species does not necessarily work for another.
In our laboratory, numerous attempts were made over several years to
transform S. salivarius ATCC 25975. All were unsuccessful. In this paper we describe a straightforward, reproducible method for
producing high-efficiency, electroporation-competent S. salivarius. Our protocol involves glycine treatment of cells
growing in rich medium, followed by electroporation. The technique was
used successfully with other streptococci and related bacteria.
(Some of the results were presented at the 75th General Session and
Exhibition of the International Association for Dental Research,
Orlando, Fla., 1997.)
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
The electroporation
technique described in this paper was developed and optimized for
S. salivarius ATCC 25975 (kindly provided by I. R. Hamilton, University of Manitoba, Winnipeg, Manitoba, Canada). The
following other bacteria were also subjected to the electroporation
technique described here: Lactococcus lactis LM 0230 (16), Pediococcus acidilactici SMQ-250,
Streptococcus thermophilus DT1 (now designated S. thermophilus SMQ-310), all of which were generously provided by S. Moineau, Laval University, Quebec, Canada; S. salivarius
ATCC 7073 and ATCC 13419 and a fresh S. salivarius isolate,
strain 30.1 (18); S. sobrinus 6715 (26); Streptococcus vestibularis ATCC 49124;
S. thermophilus ATCC 19258; Streptococcus sanguis
ATCC 10556; and S. mutans XS123 (30). An
Escherichia coli-Streptococcus shuttle vector, pDL278
(11), and a bridge vector, pNZ123 (3), were used
in this study. These vectors were maintained in E. coli XL1
Blue (Stratagene, La Jolla, Calif.) grown at 37°C with agitation in
Luria-Bertani medium (21) supplemented with 100 µg of
spectinomycin per ml and 10 µg of chloramphenicol per ml,
respectively. These antibiotics were used at the same concentrations
when we selected for the plasmids in streptococci and related bacteria.
Tryptone-yeast extract-glucose broth (TYE) (18), Hogg-Jago
glucose broth (HJG) (12), and M17 broth (28) were
used for cultivation of streptococci. Medium designations to which an S
has been added indicate the addition of sorbitol to a final
concentration of 0.4 M. Streptococci and related bacteria were
incubated aerobically at 37°C without agitation. Agar was added to a
final concentration of 1.5% when agar petri plates were prepared.
Chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.),
while Difco Laboratories (Detroit, Mich.) supplied other medium
reagents. Culture purity was verified by microscopic examination of
cells, by determining the homogeneity of isolated colonies grown on
nonselective media, and by performing carbohydrate fermentation tests.
Cell preparation and electroporation.
Culture growth was
monitored by measuring the optical density at 660 nm
(OD660) with a Spectronic 20 spectrophotometer (Milton Roy
Co., Rochester, N.Y.). Glycine was added to cultures at the concentrations and times indicated below, and cells were collected by
centrifugation, washed twice in electroporation buffer (EPB) (5 mM
potassium phosphate with or without MgCl2 and 10%
glycerol), frozen in an ethanol dry ice bath, and stored at 
80°C.
For electroporation, cells were thawed on ice, combined with 200 ng to
1 µg of pNZ123 or 1 µg of pDL278, and transferred to prechilled
1-mm electroporation cuvettes (Bio-Rad Laboratories, Richmond, Calif.).
Electroporation was performed with a Bio-Rad Gene Pulser apparatus set
at 25 µF with an attached Bio-Rad pulse controller that allowed us to
adjust the resistance.
Transformation analysis.
To optimize the transformation
process, multiple variables were examined in single experiments by
using the multifactorial experimental designs and analyses described by
Marciset and Mollet (12). Factors were tested at maximal,
intermediate, and minimal levels (indicated in designations by +, 0, and , respectively). Trial runs with all of the test factors at
intermediate levels were performed in triplicate or quadruplicate, and
the variation coefficient [VC(0)] was determined as follows:
VC(0) = 
n 
1/
× 100,
where is 
ni/n (the average
number of transformants obtained under intermediate conditions) and
n1 is the standard variation,
{[(ni )2])/(n 1)}. Test runs in which factors were at the maximal or minimal levels were analyzed as follows. The variation coefficient (VC)
for an individual test factor (F1) was determined as follows: VC(F1) = 100/
× [(F1 + runs) (F1 runs)]/8, where 
is
ai/8 (the average number of transformants
obtained for eight tests runs).
The interaction between two factors (F1 and F2) or the variation
coefficient [VC(F1,F2)] was determined as follows: VC(F1,F2) = 100/ × [(F1,F2 +,+ runs and ,) (F1,F2 ,+ runs and +,)]/8. The absolute values of VC(F1) were
compared with the absolute value of VC(0), and differences greater than
zero were considered possibly significant and differences more than
twice VC(0) were considered very significant. Marciset and Mollet
(12) stated that the optimal level of a factor was when its
variation coefficient changed sign after its maximal value increased or
its minimal value decreased from one experiment to the next.
Plasmid DNA isolation.
Plasmid DNA was isolated from
E. coli by the alkaline lysis method described by Sambrook
et al. (21), and large-batch preparations were obtained with
a Qiagen Plasmid Maxi kit (Qiagen Inc., Chatsworth, Calif.). Plasmid
DNA were isolated from streptococci and related bacteria by using the
method of O'Sullivan and Klaenhammer (17), with the
following modifications: ethidium bromide was omitted from the
preparations, mutanolysin (12 U/ml) was added to the lysozyme solution
used for oral streptococci, and the incubation time in the presence of
lysozyme-mutanolysin was increased from 15 min to 1 h. Plasmid DNA
was quantified by agarose gel electrophoresis followed by staining with
ethidium bromide and comparison to an EcoRI-HindIII-digested 
standard
(Pharmacia Biotech Inc., Baie d'Urfe, Canada).
Southern blot hybridization.
Restriction
endonuclease-digested plasmid DNA was separated by agarose gel
electrophoresis, and the DNA fragments were transferred from the gels
to Hybond-N nylon membranes (Amersham Life Science, Buckinghamshire,
England) by using a Posiblot pressure blotter (Stratagene). The blots
were then treated and hybridized with labelled plasmid DNA probes by
using a nonradioactive DNA labeling and detection kit (Roche
Diagnostics, Laval, Canada).
EcoRI-HindIII-digested (Pharmacia Biotech
Inc.) was included in all hybridization experiments as a molecular
weight marker.
Plasmid stability.
The stability of pDL278 and pNZ123 in
S. salivarius grown in HJG was tested as previously
described (1a). Briefly, cultures were grown overnight and
then serially diluted with fresh HJG containing 0.4 M (final
concentration) sorbitol (HJGS) with or without antibiotic and incubated
at 37°C. The cultures were incubated until they reached an
OD660 of 0.5. Dilutions of the cultures were spread onto
HJG agar plates without antibiotic, which then were incubated for 24 to
48 h. Two hundred isolated colonies were then picked onto fresh
plates with and without antibiotic and incubated for 48 h. The
isolates were scored for the presence of plasmids on the basis of their
resistance to the appropriate antibiotics.
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RESULTS AND DISCUSSION |
Optimization of S. salivarius transformation.
It
has not been possible to transform S. salivarius ATCC 25975 by previously described protocols that are effective with other oral
streptococci and related bacteria. These protocols include electroporation of cells grown in the presence of 40 mM
DL-threonine, as described by Suvorov et al.
(27), and a wide variety of other electroporation protocols
(4, 14, 19, 23, 24). In addition, mobilization, as described
by Buckley et al. (1a), did not yield transconjugants of
S. salivarius ATCC 25975. The flow chart for "devising a
plasmid transformation protocol in a `new' bacterial species," as
described by Saunders et al. (22), had been followed to the
box labelled "give up."
Glycine is commonly used as a cell wall-weakening agent prior to
electroporation (
2-4,
7,
8,
25,
27). The cell
wall of
S. salivarius should be susceptible to glycine because
its
interpeptide bridges contain both
L- and
D-alanine (
20),
which are replaced by glycine,
which, in turn, impedes synthesis
and assembly of the cell wall
(
5,
29). Thus, we attempted
to produce
electroporation-competent
S. salivarius by growing
cells
with glycine. A single transformant of
S. salivarius ATCC
25975 was obtained after electroporation of cells grown in TYE
containing 1.5% glycine. However, this successful transformation
was
not reproducible when we used pNZ123 isolated from either
S. salivarius or
E. coli. One major problem was the
inconsistency
of growth in the presence of glycine. Dunny et al.
(
4) previously
observed that the glycine concentration that
allowed growth of
cells (albeit slowly) in one experiment resulted in
complete growth
inhibition in the next
experiment.
We predicted that addition of glycine to an exponentially growing
culture would maximize the deleterious effects of this compound
on cell
walls, whose synthesis must occur rapidly, and at the
same time ensure
that there is a sufficient number of cells for
preparation of competent
cells. The only other study that we are
aware of in which glycine was
added to late-stage cultures is
a study in which cryotransformable
Bacillus anthracis was prepared
(
25). Stepanov et
al. (
25) grew cells to the mid-log phase
in the presence of
5% glycine prior to freezing in the presence
of a plasmid and then
thawed the cells and screened for
transformants.
Thus, to overcome glycine toxicity, glycine was added to cultures
during early exponential growth. In addition, rich medium
was
substituted for TYE, and 0.4 M sorbitol, a sugar not metabolized
by
S. salivarius, was added to growth medium and EPB as an
osmoprotectant.
A comparison of the efficiencies of transformation of
pNZ123 into
cells grown in three different media indicated that more
transformants
(1,351 versus 0 to 5 transformants/µg) were obtained
with cells
grown in HJGS than with cells grown in either MRSS or M17S.
Thus,
HJGS was the medium used for all subsequent
experiments.
The multifactorial experimental design and statistical analyses
described by Marciset and Mollet (
12) were used to optimize
transformation efficiency. A strong point of the method of Marciset
and
Mollet is that it allows interactions between factors to be
identified.
Intuitively, a researcher may or may not appreciate
interactions, but
Marciset and Mollet (
12) describe a technique
for rapidly
and easily quantitating these relationships. The results
of an
experiment in which glycine concentration, the optical density
when
glycine was added, the concentration factor of the cells
(initial cell
volume/final cell volume), MgCl
2 content, and the
pH of EPB
were examined are shown in Table
1. The
sevenfold difference
in the numbers of transformants obtained in the
trial and test
runs (
= 3.9 × 10
4
transformants [Tf]/µg versus
= 5.6 × 10
3 Tf/µg) indicates that there were strong
interactions between
factors. The VC(0) value was 22%, while in the
test runs VC(glycine)
was
73%, which suggested that the glycine
concentration tested
was too high. VC(OD) was 10%, which suggested
that the optical
densities used in the trials were too low. VC(CF) was
14%, which
led to trials at higher values. VC (pH) was 3%, which
prompted
a decrease in the pH of the EPB.
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TABLE 1.
Effects of glycine concentration, cell density when
glycine is added, concentration factor of cells, pH, and
MgCl2 concentration in EPB on the electrotransformation
efficiency of S. salivarius ATCC 25975 with pNZ123a
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|
Modifications of variables that were suggested by the results of the
experiment described above were tested in a second experiment
(Table
2). In this case, there was little
difference between
(3.0 × 10
4 Tf/µg)
and
(2.9 × 10
4 Tf/µg) which
suggested that the factors were within optimal levels.
VC(0) was
33%, while VC(glycine) was 70%. The change in the sign
of the VC
value from the previous experiment suggested that the
overlapping
values were optimal; therefore, 10% glycine was used.
VC(OD) was
reduced to 9%, suggesting that the optical densities
were within an
acceptable range. Therefore, in subsequent experiments,
glycine was
added to cultures when the OD
660 of the culture was
0.5. VC(pH) was
44%, which led to a further decrease in the EPB
pH.
Finally, a VC(MgCl
2) of
15.5% led to omission of
magnesium
from the EPB.
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TABLE 2.
Effects of glycine concentration, cell density when
glycine is added, concentration factor of cells, and MgCl2
concentration in EPB on the transformation efficiency of S. salivarius ATCC 25975 with pNZ123a
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The results of the final multifactorial experiment, in which four
factors were tested, are shown in Table
3.
was 6.7 ×
10
4 Tf/µg and
was 5.5 × 10
4 Tf/µg, which suggested that optimal conditions were
again found
within the test parameters. Interactions between certain
factors
were indicated by elevated VC values; e.g., VC(
, kV) was
93%,
and VC(pH, hours in glycine) was
94%. Linear experiments
were
designed to optimize each pair of variables. Treatment for 1 h
in the presence of 10% glycine and subsequent washing and
resuspension
of cells in pH 4.5 buffer resulted in the greatest number
of transformants
(data not shown). The results of experiments in which
the relationship
between the resistance setting and the voltage applied
was examined
were more difficult to interpret, as shown in Fig.
1. When the
resistance was 400
, there
was a clear peak in transformability
at 1.25 kV, and the efficiency
decreased at higher or lower voltages.
The greatest number of
transformants was obtained at 1.45 kV and
200
; however, the number
of transformants obtained decreased
sharply as the voltage was
increased or decreased. The next highest
number of transformants was
obtained at a voltage of 1.60 kV,
with high numbers on either side.
Thus, additional experiments
were performed with a resistance of 200
and a voltage of 1.60
kV. The viability of
S. salivarius
in response to electroporation
was 50% when the starting concentration
was 10
10 cells/ml, measured as described above.
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TABLE 3.
Effects of electroporation parameters, length of
incubation in the presence of 10% glycine, and pH of EPB on the
transformation efficiency of S. salivarius
ATCC 25975a
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FIG. 1.
Relationship between the number of transformants
obtained per milligram of pNZ123 DNA and the voltage of the
electroporator. Electroporation was performed with 200 ng of added DNA
at the voltages indicated and at levels of resistance of 100 ( ),
200 ( ), and 400 ( ). Tf, transformants.
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|
The relationship between the amount of plasmid DNA present in the
electroporation cuvette and the number of transformants
obtained is
shown in Fig.
2. The highest number of
transformants
was obtained with 1 µg of pNZ123.
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FIG. 2.
Relationship between the amount of pNZ123 DNA in the
electroporation mixture and the number of transformants obtained.
Electroporation was performed as described in Materials and Methods.
Tf, transformants.
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|
The optimal conditions for preparation of cells and electroporation
were as follows. Cultures used to prepare competent cells
were
initiated by adding 1% overnight precultures. Glycine was
then added
to a final concentration of 10% when the culture OD
660 was
0.5. The cells were incubated for 1 h at 37°C and collected
by
centrifugation. They were washed twice in ice-cold EPB (5 mM
potassium
phosphate [pH 4.5], 0.4 M sorbitol, 10% glycerol), concentrated
50-fold, frozen in an ethanol-dry ice bath, and then stored at
80°C
until they were used. Cells were thawed on ice, combined
with 1 µg of
plasmid, and transferred to a prechilled 1-mm electroporation
cuvette,
and electroporation was performed with the standard settings
(1.60 kV
and 200
). After the electric pulse, the cells were
diluted in 1 ml
of ice-cold growth medium containing 0.4 M sorbitol
and incubated for
3 h at 37°C. Aliquots were spread onto agar
plates, and
transformants were counted after 48 to 72 h of incubation.
Using
this procedure, we reproducibly obtained approximately 10
5
S. salivarius ATCC 25975 transformants/µg of pNZ123 DNA.
After
close to 2 years of storage at
80°C,
S. salivarius
cells retained
their electroporation competence, and their
transformation efficiency
was not significantly different from the
transformation efficiency
of freshly prepared cells (Table
4). We also tested the procedure
with
other strains of
S. salivarius and found that all of the
strains could be transformed, but the transformation efficiencies
were
lower with strain ATCC 13419 and fresh isolate 30.1 and greater
with
strain ATCC 7073 than with
S. salivarius ATCC 25975 (Table
4). The transformation efficiencies could be improved by optimizing
the
electroporation conditions for individual strains.
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TABLE 4.
Efficiency of transformation of pNZ123 into
electroporation-competent S. salivarius ATCC 25975 and
other strainsa
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Plasmid stability and double transformation.
The stabilities
of plasmid markers in transformants were measured as described above.
After growth in the absence of selective pressure, pDL278 and pNZ123
were maintained in all S. salivarius transformants tested
after 20, 40, and 60 generations.
It was possible to introduce both pDL278 and pNZ123, which produced a
double transformant of
S. salivarius (Fig.
3). The efficiency
of pNZ123 introduction
was the same whether the cells contained
pDL278 or not. However, the
efficiency was reduced drastically
if electroporation was conducted
with both plasmids at the same
time or if pDL278 was introduced into
cells harboring pNZ123 (1
to 2 transformants/µg of DNA). Whether
antibiotic selection was
simultaneous or sequential made no difference
to transformation
efficiency.
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FIG. 3.
(A) Southern blot analysis of plasmid preparations from
spectinomycin-resistant transformants probed with plasmid pDL278. Lane
1, pDL278: lane 2, S. salivarius ATCC 25975; lane 3, L. lactis LM 0230; lane 4, P. acidilactici
SMQ-250; lane 5, S. sanguis ATCC 10556; lane 6, S. sobrinus 6715; lane 7, S. vestibularis ATCC 49124; lane
8, S. thermophilus ATCC 19258; lane 9, S. thermophilus DT1 (now known as S. thermophilus
SMQ-310); lane 10, S. salivarius ATCC 25975 Spr
Cmr. (B) Southern blot analysis of plasmid preparations
from chloramphenicol-resistant transformants probed with plasmid
pNZ123. Lane 1, pNZ123; lane 2, S. salivarius ATCC 25975;
lane 3, L. lactis LM 0230; lane 4, S. vestibularis ATCC 49124; lane 5, S. mutans XS123; lane
6, S. thermophilus DT1 (now known as S. thermophilus SMQ-310); lane 7, S. salivarius ATCC 25975 Spr Cmr.
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Transformation of other bacteria.
The technique described
above was applied to eight other bacterial species with pNZ123 and
pDL278. The results of these experiments are shown in Table
5. Each strain tested was transformed
with at least one of the plasmids. The presence of specific plasmids in
the various transformants was confirmed by Southern hybridization in
which pNZ123 or pDL278 was the probe, as described above (Fig. 3). The
sizes of the recovered plasmids corresponded to the original sizes of
the transforming plasmids, indicating that the plasmids had not
undergone any overt size modification following transformation into and
replication in the new host.
Although
S. mutans XS123 was not transformed with pDL278 by
electroporation techniques used for other strains of
S. mutans (
1), we were able to transform this strain with
the technique
described in this paper. A technique for introducing DNA
into
S. sobrinus via mobilization has been described
(
1a) because
electroporation of this bacterium is very
difficult (
10). The
transformation efficiency which we
measured was very low; however,
the method described in this paper can
serve as a starting point
to improve efficiency. This technique has
also been used to introduce
DNA into
Lactobacillus species
when all other avenues had been
exhausted (
9a), further
supporting the general utility of the
technique.
In this study we developed a new method for preparing
electroporation-competent cells and found that it may be applied to
a
variety of gram-positive bacteria. The technique was optimized
for
S. salivarius ATCC 25975, which had never been transformed
previously despite numerous attempts with a wide variety of
protocols.
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ACKNOWLEDGMENTS |
Medical Research Council of Canada grants MT6979 and MT11276
supported this study, and M.F. is a scholar of the Fonds de la Recherche en Santé du Québec.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Groupe de
Recherche en Écologie Buccale, Faculté de Médecine
Dentaire, Université Laval, Québec, Canada G1K 7P4. Phone:
(418) 656-2131, ext. 5502. Fax: (418) 656-2861. E-mail:
Michel.Frenette{at}greb.ulaval.ca.
Present address: Lilly Research Laboratories, Eli Lilly and
Company, Indianapolis, IN 46285.
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Applied and Environmental Microbiology, September 1999, p. 3800-3804, Vol. 65, No. 9
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Abstract
Introduction
