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Journal of Bacteriology, September 1999, p. 5389-5394, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Regulation of Cell Component Production by Growth
Rate in the Group B Streptococcus
Robin A.
Ross,1,*
Lawrence C.
Madoff,1,2 and
Lawrence C.
Paoletti1
Channing Laboratory1
and Division of Infectious Diseases,2
Department of Medicine, Brigham & Women's Hospital and Harvard Medical
School, Boston, Massachusetts 02115
Received 8 March 1999/Accepted 23 June 1999
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ABSTRACT |
Group B Streptococcus (GBS) is the leading cause of
bacterial sepsis and meningitis among neonates. While the capsular
polysaccharide (CPS) is an important virulence factor of GBS, other
cell surface components, such as C proteins, may also play a role in
GBS disease. CPS production by GBS type III strain M781 was greater
when cells were held at a fast (1.4-h mass-doubling time
[td]) than at a slow (11-h
td) rate of growth. To further investigate
growth rate regulation of CPS production and to investigate production
of other cell components, different serotypes and strains of GBS were
grown in continuous culture in a semidefined and a complex medium.
Samples were obtained after at least five generations at the selected
growth rate. Cells and cell-free supernatants were processed
immediately, and results from all assays were normalized for cell dry
weight. All serotypes (Ia, Ib, and III) and strains (one or two strains
per serotype) tested produced at least 3.6-fold more CPS at a
td of 1.4 h than at a
td of 11 h. Production of beta C protein
by GBS type Ia strain A909 and type Ib strain H36B was also shown to
increase at least 5.5-fold with increased growth rate (production at a
td of 1.4 h versus 11 h). The
production of alpha C protein by the same strains did not significantly
change with increased growth rate. The effect of growth rate on other cell components was also investigated. Production of group B antigen did not change with growth rate, while alkaline phosphatase decreased with increased growth rate. Both CAMP factor and beta-hemolysin production increased fourfold with increased growth rate. Growth rate
regulation is specific for select cell components in GBS, including
beta C protein, alkaline phosphatase, beta-hemolysin, and CPS production.
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INTRODUCTION |
Group B Streptococcus
(GBS), also known as Streptococcus agalactiae, is part of
the normal flora colonizing the respiratory, gastrointestinal, and
urogenital tracts of humans. It is also the leading cause of bacterial
sepsis and meningitis among neonates in the United States and is a
major cause of endocarditis and fever in parturient women (6,
29). Most GBS strains that cause human infection in the United
States are encapsulated by one of five antigenically distinct
polysaccharides (serotype Ia, Ib, II, III, or V) (10, 11, 34,
37). The capsular polysaccharide (CPS) of GBS is an important
virulence factor (1) and is the target of protective
antibodies. The CPS has antiphagocytic properties (4, 5,
18), and the degree of encapsulation correlates directly with the
virulence of the organism (28).
By definition, all strains of GBS produce Lancefield's group B
antigen, a cell wall-associated carbohydrate (23) that has been shown not to be a virulence factor of GBS (13, 19).
Other surface antigens produced by human isolates of GBS include the C
proteins alpha and beta and protein Rib (15, 22, 32, 38), which confer protective immunity and may have a role in GBS
pathogenesis (3, 15-17, 21, 32). The relative production
levels of CPS and surface proteins may affect their availability for
antibody binding (8). GBS also produces a number of enzymes
that may or may not be virulence factors, such as CAMP factor, a
phospholipase secreted by GBS that causes complete lysis of sensitized
sheep erythrocytes (SRBCs) (2). Partially purified CAMP
factor was found to be lethal for rabbits (30), and purified
CAMP factor, although not lethal in mice, did augment the virulence of
various GBS isolates (7). Production of beta-hemolysin by
GBS has been correlated with lung epithelial cell injury in vitro,
suggesting a possible pathogenic role of this enzyme in the invasive
step of early-onset GBS disease (24). The severe pneumonia
associated with early-onset disease could be caused in part by damage
of host cell membranes. In addition, increased hemolysin production was
associated with a 50% decrease in the lethal dose and an earlier time
of death in adult mice inoculated intranasally with GBS mutants producing a high level of beta-hemolysin compared with a nonhemolytic mutant (33).
Our laboratory has shown that production of CPS by GBS is regulated by
growth rate (27). GBS serotype III strain M781 grown in
continuous culture with modified chemically defined medium (MCDM)
produced sixfold more cell-associated CPS at mass-doubling times
(tds) of 0.8, 1.4, and 1.6 h than at
tds of 2.3 and 11 h. Cell growth rate was
determined to be the principal factor regulating CPS production, and
growth rate-dependent production of type III CPS occurred independently
of the growth-limiting nutrient. In this study we expand on these
initial findings to determine the extent of growth rate regulation of
(i) CPS of other GBS serotypes, (ii) different strains within selected
serotypes, and (iii) potential virulence (alpha and beta C proteins,
CAMP factor, and beta-hemolysin) and nonvirulence (group B antigen and
alkaline phosphatase) factors.
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MATERIALS AND METHODS |
Bacterial strains, strain maintenance, and culture medium.
S. agalactiae M781 (type III), A909 (type Ia), O90 (type
Ia), and H36B (type Ib) were isolated from patients with GBS disease (14, 15). Strain D66-7 (type III) was recently isolated from the vagina of a healthy woman and identified by established criteria (25); it has been passed three to five times in vitro. All
GBS strains were grown in MCDM under glucose limitation as described previously (27) except for the following changes: 5 g
of Casitone (Difco Laboratories, Detroit, Mich.) per liter was
substituted for vitamin assay Casamino Acids, tryptophan and glycine
were omitted, and thiamine was used at 1 mg/liter. GBS strains M781 and
H36B were also grown under unknown limitation in continuous culture
with Columbia broth (Difco Laboratories) supplemented with 8% glucose.
Continuous culture.
All experiments were conducted as
previously described, with a method of cultivating organisms in a
stable growth environment that allows us to study the effect of a
single environmental variable on the physiology and growth of the
organism. A growth vessel volume of 500 ml was maintained in a 1-liter
fermentor (Applikon, Foster City, Calif.). The sterile medium inflow
rate was adjusted to obtain the desired dilution rate (D)
and td. The tds used were 1.4 h (D = 0.49 h
1), 2.7 h
(D = 0.26 h1), 5.8 h (D = 0.12 h1), and 11 h (D = 0.06
h1). To ensure that a steady state had been reached,
samples were taken after at least five generations had elapsed from
initiation of fresh medium inflow.
Growth measurements.
Measurements were calculated as
previously described (27), except for modifications to cell
dry weight (CDW) determinations. After the final centrifugation, the
supernatant was decanted and pelleted cells were resuspended in the
residual liquid. The entire cell suspension was then transferred to a
preweighed 0.22-µm-pore-size polycarbonate filter. The tube was
rinsed four times (500 µl each) with water which was filtered through
a 0.22-µm-pore-size membrane and added to the same
0.22-µm-pore-size polycarbonate filter.
Determination of GBS cell wall components.
Cells removed
from the chemostat were washed, and cell walls were digested with
mutanolysin as described previously (27). Inhibition
enzyme-linked immunosorbent assay was used to quantify the amount of
each cell component as previously described (27), with the
following reagents: for cell-associated CPS types Ia, Ib, and III,
homologous serotype-specific rabbit antiserum (diluted 1:100,000)
(26, 35, 36), purified homologous CPS as the standard, and
homologous CPS coupled to poly-L-lysine as the coating antigen; for group B antigen, rabbit serum (diluted 1:100,000) raised
to group B antigen coupled to tetanus toxoid (19), purified group B antigen as the standard, and group B antigen coupled to human
serum albumin as the coating antigen; for alpha and beta C proteins,
mouse antiserum raised to alpha (diluted 1:1,000) and rabbit antiserum
raised to beta (diluted 1:50,000), and purified antigens as the
standards and coating antigens.
Determination of beta-hemolytic, alkaline phosphatase, and CAMP
factor activity.
Enzyme activities in GBS strain M781 cells
growing at tds of 1.4 and 11 h were
compared. We used two chemostats to simultaneously measure enzyme
activity at two different growth rates, one for each of the two
tds. In all enzyme activity assays, samples from the two chemostats were taken simultaneously.
For assessment of beta-hemolysin production, a 1-ml culture sample was
serially diluted in phosphate-buffered saline (PBS; 40 mM phosphate
[pH 7.0], 0.15 M NaCl) and plated on 5% sheep blood agar plates to
determine initial bacterial concentrations. In addition, a 120-ml
culture sample was taken, and the cells were sedimented by
centrifugation (20 min at 13,689 × g and 6°C). The pellet
was resuspended in residual liquid and transferred to a microcentrifuge
tube. The centrifuge bottle was washed with PBS, and the wash volume
was added to the microcentrifuge tube (approximately 1.5 ml). Cells
were sedimented in a microcentrifuge (4 min at maximum speed), washed
once in 1.0 ml of PBS, sedimented, and then resuspended in
filter-sterilized lysis buffer (60 mM phosphate [pH 7.0], 0.15 M
NaCl, 146 mg of MgCl2 [20 mM Mg2+], 1%
soluble starch [Difco Laboratories], 1% T-500 dextran [Sigma Company, St. Louis, Mo.] 3% Tween 20, 1% glucose). Acid-washed glass
beads (0.5 ml; Sigma) were added to microcentrifuge tubes, which were
then shaken with a dental amalgamator (Foremost Dental Manufacturing,
Englewood, N.J.) (3 min at high speed) (39). Samples were
incubated for 30 min at 37°C to achieve maximum bacterial lysis. Cell
debris was sedimented in a microcentrifuge (4 min at maximum speed).
The pellet was resuspended in PBS to a final volume of 1.0 ml and
processed as described above to determine bacterial concentration. The
lysate was used to prepare two sets of diluted supernatants (serial
twofold dilutions in PBS). Defibrinated SRBCs were prepared in a
96-well microtiter plate. Two hundred microliters of 1% SRBC was
sedimented by centrifugation (10 min at 2,800 rpm [model TJ-6
centrifuge; Beckman Instruments, Inc., Palo Alto, Calif.]), and the
supernatant was decanted. One set of serially diluted bacterial
extracts (150 µl/dilution) was mixed with SRBCs until the pellets
were resuspended. The mixture was transferred to another 96-well
microtiter plate (incubation plate). The other set of serially diluted
bacterial extracts was transferred (150 µl/well) to the incubation
plate. Controls, both positive (0.1% sodium dodecyl sulfate in PBS)
and negative (PBS alone), were prepared in duplicate with and without
blood, transferred to the incubation plate (150 µl/well), and
incubated at 37°C for 1 h before debris was sedimented (10 min
at 2,800 rpm [TJ-6 centrifuge]) and 100 µl of the supernatant was
transferred to a fresh microtiter plate. The absorbance of each sample
was determined at 540 nm. A hemolytic titer was defined as the
reciprocal of the dilution of sample producing 50% hemoglobin release.
The absorbance value for 50% hemoglobin release was calculated for
each assay as the average A540 of the positive
control (100% hemoglobin release) minus the average
A540 of the negative control (background
absorbance) divided by 2. Sample absorbance readings were adjusted for
background with the absorbances from the matched serially diluted
samples without SRBCs. Efficiency of bacterial lysis was measured by
comparison of the bacterial concentration before and after lysis.
For alkaline phosphatase and CAMP factor activity, culture samples were
normalized for CDW by dilution with Tris buffer (0.01
M Tris HCl, 0.01 M MgCl
2, 0.15 M NaCl [pH 7.4]). One milliliter
of each
normalized sample was centrifuged (10 min at 2,800 rpm
[TJ-6
centrifuge]). Pelleted cells were analyzed for alkaline
phosphatase
activity, and the supernatant was used in CAMP factor
analysis. Enzyme
assays were performed as described
below.
Alkaline phosphatase activity was measured with a modification of a
previously described method (
31). Cells were resuspended
in
1 ml of 0.85% NaCl, and 0.5 ml of this suspension was added
to 0.5 ml
of a fresh substrate solution and mixed. A negative
control was
prepared by the addition of 0.5 ml of 0.85% NaCl to
0.5 ml of a fresh
substrate solution that consisted of
p-nitrophenyl
phosphate
(1 mg/ml) in 0.04 M glycine (pH 10.5). A standard curve
was generated
with commercial alkaline phosphatase solution (50
U/ml; Sigma) in 0.04 M glycine (pH 10.5). All samples, including
the standard, were
incubated at 37°C for 15 min without shaking.
Cells were removed by
centrifugation, and the supernatants were
filtered through a
0.22-µm-pore-size membrane. Test samples and
the negative control
were transferred to the microtiter plate
(200 µl/well), and the
absorbance at 405 nm was measured. Alkaline
phosphatase activity is
reported as units of enzyme activity per
milligram of CDW and is the
mean of four
samples.
The amount of CAMP factor produced by GBS grown in continuous culture
at different growth rates was determined by a modification
of a
published method (
9). SRBCs (2 × 10
4) were
washed three times and resuspended in 100 µl of PBS. A
sphingomyelinase solution was prepared by soaking one Beta Lysin
disk
(Remel, Lenexa, Kans.) in 0.5 ml of Tris buffer. The SRBC
suspension
(20 µl) was added to 4 ml of Tris buffer and 40 µl
of the
sphingomyelinase solution, mixed, and incubated 5 min at
30°C.
Supernates from CDW-normalized samples were filtered through
a
0.22-µm-pore-size membrane, and 50 µl was transferred to microtiter
plate wells containing 50 µl of sensitized SRBCs, mixed, and
incubated
30 min at 30°C. For the negative control, 50 µl of Tris
buffer
was used. Absorbance at 490 nm was measured immediately after
3 s of high-speed shaking. The absorbance values reported are
the
mean of 12 replicates. The titer was defined as the reciprocal
of the
dilution resulting in the
A490 value at the
midpoint of
the exponential portion of the
A490
curve (0.25 for both
curves).
Statistics.
Instat version 2.0 software (Graphpad Software,
Inc., San Diego, Calif.) was used to analyze data to determine
significance (P < 0.05).
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RESULTS |
Biomass production.
The mean CDW values (± standard errors of
the means [SEM]) determined for each serotype and strain grown in
MCDM with glucose limitation are presented in Table
1. The mean CDW values for GBS grown in
Columbia broth supplemented with 8% glucose at
tds of 11 h and 1.4 h were 1.69 ± 0.17 and 2.34 ± 0.11 mg/ml, respectively, for strain M781 and
2.16 ± 0.01 and 2.37 ± 0.01 mg/ml, respectively, for strain
H36B.
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TABLE 1.
Biomass produced by different GBS serotypes and strains
during continuous culture growth in MCDM and glucose limitation
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Production of CPS.
All five GBS strains grown in MCDM with
glucose limitation produced significantly more CPS (P < 0.01, unpaired t test) at the fast
td of 1.4 h than at the slower
td of 11 h (Table
2). The increase in net CPS production
ranged from 3.6-fold (strain D66-7) to 14.9-fold (strain A909).
Columbia broth supplemented with 8% glucose (growth limitation
unknown) produced a similar pattern of increase in specific CPS
production with a td of 1.4 h: strain M781
(serotype III) produced 7.5-fold more CPS and strain H36B (serotype Ib)
4.4-fold more than at the slower growth rate (data not shown).
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TABLE 2.
Production of specific CPS by different GBS serotypes and
strains grown in continuous culture using MCDM and glucose limitation
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For GBS type Ia strain A909 (Fig.
1A) and
type Ib strain H36B (Fig.
2A) examined
over a range of increasing growth rates,
CPS production increased with
increasing growth rate. Both strains
exhibited a significant linear
trend in the change in the amount
of CPS produced with increasing
td (
P < 0.01). In addition to
both strains producing significantly more CPS at a
td of 1.4 h
than 11 h, strain H36B
produced significantly more CPS at a
td of
1.4 h than 5.8 h (
P < 0.01, Tukey-Kramer
multiple comparison
test) that was not exhibited by strain A909.
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FIG. 1.
Effect of growth rate on production of specific CPS (A)
and beta (B) and alpha (C) C proteins by GBS type Ia strain A909. Cells
were grown in continuous culture in MCDM with glucose limitation. Shown
in the bar graph are means of three samples, with error bars
representing SEM. CPS and beta C protein produced at a
td of 1.4 h compared with 11 h,
P < 0.05; all other combinations were not significant
at P > 0.05.
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FIG. 2.
Effect of growth rate on production of specific CPS (A)
and beta (B) and alpha (C) C proteins by GBS type Ib strain H36B. Cells
were grown in continuous culture in MCDM with glucose limitation. Shown
in the bar graph are means of three samples, with error bars
representing SEM. CPS produced at a td of
1.4 h compared with production at 5.8 or 11 h, P < 0.01; beta C protein produced at a td of
1.4 h compared with production at 5.8 or 11 h, P < 0.05; all other combinations were not significant at
P > 0.05.
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Production of alpha and beta C proteins.
The production of
alpha and beta C proteins with changing growth rate was investigated in
strains A909 and H36B. We found that 5.5-fold more beta C protein was
produced by strain A909 cells (Fig. 1B) at the fast
td of 1.4 h than at the slower
td of 11 h (P < 0.05,
Tukey-Kramer multiple comparison test). Similarly, strain H36B (Fig.
2B) produced 7.9-fold more beta C protein at the fast than at the
slower growth rate (P < 0.05, Tukey-Kramer multiple
comparison test). Both strains exhibited a significant linear trend in
the change in the amount of CPS produced with increasing
td (P < 0.01).
In contrast to the production of beta C protein, production of alpha C
protein by strains A909 and H36B did not change significantly
(
P > 0.05, Tukey-Kramer multiple comparison test)
between any
of the growth rates tested (
tds of
1.4, 2.7, 5.8, and 11 h) (Fig.
1C and
2C). A Western blot of these
samples confirmed the relative
levels of alpha C protein detected by
enzyme-linked immunosorbent
assay (data not
shown).
Group B antigen production.
Growth rate did not alter the
production of group B antigen in type III strain M781 (Table
3). The mean ± SEM values of
specific group B antigen produced at tds of 11 and 1.4 h were 26.7 ± 3.5 and 30.9 ± 4.9 µg of
antigen/mg of CDW, respectively (P = 0.52, unpaired
t test, n = 10 and 12, respectively).
Beta-hemolysin.
A faster growth rate had a positive effect on
beta-hemolysin production by GBS strain M781. Cells grown at a
td of 1.4 h produced a significant 4.2-fold
greater amount of beta hemolysin activity (titer of 50 ± 7 [mean ± SEM]) than cells grown at a td
of 11 h (12 ± 6) (P < 0.05, Mann-Whitney
test) (Table 3).
CAMP factor.
Strain M781 produced 4.1-fold more CAMP factor
when grown at a td of 1.4 h than when grown
at a td of 11 h (Fig.
3 and Table 3), a result confirmed by a
cohemolysis assay in which the zones of cohemolysis on a blood agar
plate for the samples from cells grown at tds of
1.4 h and 11 h were 3 to 3.5 mm and 1 to 1.5 mm, respectively. However, there was no difference in CAMP factor activity
at different growth rates as determined by the slope of the linear
portion of their titration curves (0.31 absorbance units/reciprocal
dilution of sample) (Fig. 3).
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FIG. 3.
Lysis of SRBCs by CAMP factor from GBS type III strain
M781 grown in continuous culture. Amounts and activities of CAMP factor
produced at growth rates of 1.4 h (open squares) and 11 h
(closed diamonds) were compared.
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Alkaline phosphatase.
The amount of alkaline phosphatase
produced by strain M781 increased with decreasing growth rate (Table
3). There was an approximately 1.1-fold increase in the amount of
enzyme produced by cells grown at a td of
11 h compared with those grown at a td of
1.4 h. The mean ± SEM values of alkaline phosphatase at tds of 1.4 and 11 h were 1.34 ± 0.03 and 1.48 ± 0.03 U/mg of CDW (P = 0.01, unpaired
t test), respectively. This change, though statistically
significant, is unlikely to be biologically significant.
| |
DISCUSSION |
In our initial investigation of the regulation of CPS production,
we concluded that growth rate regulates CPS production in GBS type III
strain M781 (27). We have continued these studies to
determine the scope and specificity of growth rate regulation of CPS
production and the production of other GBS cellular factors, both
virulent and nonvirulent. By using continuous culture methods to study
the physiology of GBS, we have been able to separate and define
parameters that are interdependent during batch culture growth, such as
nutrient concentration and growth rate. Continuous culture permits
cells to grow indefinitely in an unchanging environment, resulting in a
metabolically homogeneous population of cells. The range of growth
rates studied (td from 1.4 to 11 h) cannot be directly correlated with in vivo growth rates for GBS because these
values are not known, but they can be indirectly correlated with the
range of growth rates for bacteria growing in the oral cavity
(20).
Regulation of CPS production by growth rate was common to all serotypes
and strains of GBS tested and was not dependent on the growth medium
used. Various serotypes and strains of GBS, in addition to type III
strain M781, were tested with a chemostat. For all serotypes and
strains tested (type III strains M781 and D66-7, type Ia strains A909
and O90, and type Ib strain H36B), there was a significant increase in
CPS production when cells were grown at a td of
1.4 rather than 11 h (Table 2). Even a recent vaginal isolate
(strain D66-7) produced more specific CPS at a
td of 1.4 h, an indication that an increase
in CPS production is not restricted to laboratory strains that have
been passed a number of times on laboratory medium. It is interesting
that a GBS strain believed to be a constitutive high producer of CPS, strain O90, produced significantly more CPS at a
td of 1.4 h than at a
td of 11 h. The results from continuous
culture studies using supplemented Columbia broth indicated that
increased production of specific CPS with increased growth rate is not
dependent on a component of MCDM.
Production of other cell surface antigens varied, and no pattern
related to their properties as virulence factors was noted (Table 3).
Production of group B antigen, not considered a virulence factor
(13, 19), did not significantly change with growth rate as
was the case with alkaline phosphatase. Although the change in
production was statistically significant, a 1.1-fold change for this
enzyme most likely is not biologically significant. For both strains
H36B and A909, beta C protein production followed a pattern similar to
that for CPS production in the same samples (Fig. 1A, 1B, 2A, and 2B).
However, this was not the case for production of alpha C protein by the
same strains (Fig. 1C and 2C). These results indicate that the
mechanisms of regulation of alpha and beta C protein production are
different. Alpha and beta C proteins have been shown by others to have
unrelated amino acid sequences and to be produced independent of each
other (12). From these results, we conclude that beta C
protein production is regulated by growth rate, while production of
alpha C protein, alkaline phosphatase, and group B antigen is not.
Furthermore, growth rate regulation of production of GBS cell surface
antigens is specific, since not all surface antigens were produced
optimally at a fast td nor were they all
virulence factors.
Another interesting finding was the growth rate regulation of CAMP
factor. Cells grown at a td of 1.4 h
produced more CAMP factor than those grown at a
td of 11 h (Fig. 3), but the activity of
the enzyme produced at each td did not vary.
Beta-hemolysin activity also increased significantly with increased
growth rate. These results suggest a different type of growth rate
regulation for the production of these components than for the
production of CPS, CAMP factor, beta-hemolysin, and beta C protein.
In summary, growth rate regulation of CPS by GBS is not unique to
strain M781 serotype III but is exhibited by other clinically important
serotypes as well. This type of regulation affects production of some
(beta C protein, CAMP factor, and beta hemolysin) but not all (alkaline
phosphatase, group B antigen, and alpha C protein) GBS components,
demonstrating that GBS can specifically modulate cellular components in
response to growth rate. Future studies will determine the molecular
mechanisms by which growth rate regulates GBS antigen production.
| |
ACKNOWLEDGMENTS |
We thank Claudia Gravekamp for her contributions to the work
involving alpha and beta C proteins and Ken Johnson for his
technological expertise.
This work was supported by NIH-NIAID grant AI-25152.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Channing
Laboratory, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-0726. Fax: (617) 731-1541. E-mail: rross{at}channing.harvard.edu.
| |
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Journal of Bacteriology, September 1999, p. 5389-5394, Vol. 181, No. 17
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
