JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 1582–1588
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 5
Physiologic Effects of Forced Down-Regulation of dnaK and groEL
Expression in Streptococcus mutans?
Jose ´ A. Lemos, Yaima Luzardo, and Robert A. Burne*
Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610-0424
Received 25 October 2006/Accepted 5 December 2006
Strains of Streptococcus mutans lacking DnaK or GroEL appear not to be isolable. To better distinguish the
roles played by these chaperones/chaperonins in the physiology of S. mutans, we created a knockdown strategy
to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80% in strain SM13.
Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels
of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent
strain, had a strong tendency to aggregate in broth culture, and showed major changes in their proteomes.
Compared with the wild-type strain, SM12 and SM13 had impaired biofilm-forming capacities when grown in
the presence of glucose. The SM12 strain was impaired in its capacity to grow at 44°C or at pH 5.0 and was
more susceptible to H2O2, whereas SM13 behaved like the wild-type strain under these conditions. Phenotyp-
ical reversions were noted for both mutants when cells were grown in continuous culture at a low pH,
suggesting the occurrence of compensatory mutations. These results demonstrate that DnaK and GroEL
differentially affect the expression of key virulence traits, including biofilm formation and acid tolerance, and
support that these chaperones have evolved to accommodate unique roles in the context of this organism and
Streptococcus mutans, a bacterial pathogen associated with
human dental caries, thrives in multispecies biofilms on tooth
surfaces, where it is subjected to a continuous assault by host
defenses and to rapid and dramatic fluctuations in nutrient
availability, carbohydrate source, and pH. The organism has
evolved multiple physiologic and genetic adaptations to opti-
mize growth under these dynamic conditions, including the
capacity to scavenge and metabolize a wide variety of carbo-
hydrates and to adapt to a wide range of stresses, especially low
pH (15). The rapid and efficient responses to environmental
stimuli are considered to be critical to the persistence and
virulence of this organism, so efforts have been focused on
dissecting the molecular control of responses to low pHs, other
stresses, and nutrient flux. Through such studies, it has
emerged that the expression of the class I heat shock proteins
of S. mutans is highly responsive to intermittent and sustained
exposure to relevant environmental stresses (13, 18).
The ubiquitously distributed class I stress proteins, the
DnaK and GroEL molecular chaperones, are central to the
tolerance to environmental stresses and participate in a variety
of cellular processes including protein folding, protein trans-
location, and assembly and disassembly of protein complexes
(8, 11, 26). The GroEL and DnaK complexes, which include
GroES and DnaJ-GrpE, respectively, also regulate signal
transduction pathways by controlling the stability and activities
of transcriptional regulators and protein kinases (8, 11). In
many gram-positive bacteria, transcription of the groE (groES-
groEL) and dnaK (hrcA-grpE-dnaK-dnaJ) operons is negatively
controlled by HrcA, which binds to a highly conserved cis-
acting element (CIRCE) located in the regulatory regions of
these operons (28, 30). In some cases, the groE and dnaK
operons can also be under the negative control of CtsR, a
repressor that binds to a conserved direct-repeat sequence and
that was initially identified for its role in regulating clp gene
expression (5, 7).
Previously, we demonstrated that the transcription of the
dnaK operon in S. mutans is tightly controlled by HrcA (13,
18), which binds to two CIRCE elements located in the dnaK
promoter region. The expression of groE in S. mutans is under
the dual control of the HrcA and CtsR repressors, although the
repression by CtsR is not as strong as the repression by HrcA
(16). It was also demonstrated that the transcription of both
operons is rapidly induced by acid shock and other stresses and
that elevated levels of DnaK are maintained under acidic con-
ditions (13, 18). In Escherichia coli, GroEL is essential for
growth at all temperatures, while DnaK is essential only at
temperatures above 37°C and below 15°C (9, 10, 12). In the
gram-positive paradigm Bacillus subtilis, DnaK is essential only
at temperatures above 52°C (27). In S. mutans, attempts to
inactivate hrcA by inserting the strongly polar ?Km cassette
resulted in the isolation of only single-crossover insertions,
even at lower temperatures or in buffered medium, indicating
that the transcription of the downstream grpE-dnaK-dnaJ
genes, coding for the DnaK machinery, was essential for cell
viability. Similarly, strains lacking GroEL in S. mutans could
not be isolated.
To evaluate the role of HrcA as a repressor protein in
chaperone expression, an HrcA-deficient strain, SM11, was
constructed by allelic replacement of the 5? portion of the gene
with a polar kanamycin cassette (?Km) followed by the Strep-
tococcus salivarius urease promoter (PureI) (6, 18). The HrcA
mutant strain, which had constitutively elevated levels of
* Corresponding author. Mailing address: Department of Oral Bi-
ology, University of Florida College of Dentistry, P.O. Box 100424,
1600 SW Archer Road, Gainesville, FL 32610-0424. Phone: (352)
392-4370. Fax: (352) 392-7357. E-mail: firstname.lastname@example.org.
?Published ahead of print on 15 December 2006.
GroES-GroEL, presumably due to a loss of the HrcA repres-
sor, was more sensitive to acid killing and could not lower the
pH as effectively as the parent (18). The acid-sensitive pheno-
type was, at least in part, attributable to lower F-ATPase ac-
tivity (18). However, SM11 had only 50% of the DnaK protein
found in the parent strain, probably because transcription from
the S. salivarius PureI promoter was not as efficient as it was
from the cognate promoter. Thus, the behavior of SM11 sug-
gested that decreases in the levels of DnaK might be respon-
sible for decreased acid resistance, although this strain lacked
HrcA and overproduced GroES-GroEL. To distinguish the
roles played by molecular chaperones in the physiology of S.
mutans, we isolated strains that showed significant reductions
in the levels of DnaK or GroEL. The data presented here
demonstrate that the forced down-regulation of DnaK and
GroEL had substantially different effects on S. mutans and
confirmed that molecular chaperones play essential roles in
core physiologic responses and virulence-associated attributes.
MATERIALS AND METHODS
Bacterial strains and culture conditions. S. mutans UA159 and its derivatives
were routinely grown in brain heart infusion (BHI) broth in a 5% CO2aerobic
atmosphere at 37°C. When needed, kanamycin (1 mg ml?1) was added to the
medium. The ability to form stable biofilms was assessed by growing cells in wells
of polystyrene microtiter plates using biofilm medium (BM) (19). For acid
adaptation studies, cells were grown to an optical density at 600 nm (OD600) of
0.3 in BHI medium adjusted to pH 7.0 with KOH, harvested by centrifugation,
resuspended in fresh BHI medium that was adjusted to pH 5.0 with HCl, and
incubated for 2 h. Biofilms used for acid killing experiments and F-ATPase assays
were grown in BM for 48 h in wells of 24-well polystyrene tissue culture plates
(flat bottom). Cells were grown in continuous culture in a BioFloIII chemostat
(New Brunswick Scientific, Edison, NJ) in TY base medium (3% tryptone, 0.5%
yeast extract) containing 25 mM glucose, as previously described (13). The
dilution rate (D) of the culture was 0.3 h?1, corresponding to a generation time
of 2.3 h, and the pH of the vessel was maintained at either 7.0 or 5.0 by the
addition of 1 N KOH. The steady state was considered to be achieved when the
cultures were maintained under a particular growth condition for at least 10
DNA methods. Chromosomal DNA was prepared from S. mutans as previously
described (4). Restriction and DNA-modifying enzymes were obtained from Life
Technologies Inc. (Gaithersburg, MD) or New England Biolabs (Beverly, MA).
PCRs were carried out with 100 ng of S. mutans chromosomal DNA using Taq
DNA polymerase, and PCR products were purified by using the QIAquick kit
(QIAGEN). Plasmid DNA was introduced into E. coli by the calcium chloride
method (25). Mutants of S. mutans were generated by natural transformation
(23) with DNA from previously established strains or by using a PCR ligation
mutagenesis approach (14). Southern blotting was carried out under high-strin-
gency conditions as detailed elsewhere previously (25).
Construction of strains. To down-regulate dnaK expression, a 1.0-kbp frag-
ment containing the grpE-dnaK intergenic region and flanking portions of grpE
and dnaK were amplified and cloned into pGEM-5Zf(?) (Promega, Madison,
WI) to generate pJL71. In pJL71, a fragment containing 267 bp of the grpE-dnaK
intergenic region was replaced by an antibiotic cassette that contains the polar
?Km element (22) followed by the Streptococcus salivarius urease promoter
(PureI) (6). The resulting plasmid was then isolated and used to transform S.
mutans. By using this strategy, transcription of hrcA and grpE was still driven by
the cognate promoter (PhrcA), whereas dnaK and dnaJ were transcribed through
the weaker PureI promoter. To down-regulate groES-EL expression, a 40-bp
region containing the ?35 and ?10 sequences of the groE operon was replaced
by the ?Km-PureI cassette. Briefly, two 0.5-kb fragments flanking the ?35 and
?10 sequences of the groE promoter were amplified by PCR, ligated into the
?Km-PureI cassette, and used to transform S. mutans. Schematic diagrams
depicting the construction of strains SM12 and SM13 are shown in Fig. 1.
RNA methods. RNA was extracted by the hot acid-phenol method as described
elsewhere previously (1). Levels of dnaK, groEL, and clpP mRNAs were quan-
tified by real-time quantitative reverse transcriptase PCR (RT-PCR). Primer
design, RT-PCRs, real-time RT-PCR cycling conditions, data analysis, and qual-
ity control were performed as described elsewhere previously (1). A Student’s t
test was employed to analyze the significance of the real-time RT-PCR quanti-
Acid killing experiments. For acid killing of planktonic cells, strains were
grown in BHI medium to the desired growth phase, washed once with 0.1 M
glycine buffer (pH 7.0), and resuspended in one-half of the original volume of 0.1
M glycine buffer (pH 2.8) for up to 60 min. For acid killing of biofilm-grown cells,
cultures were grown in polystyrene plates in BM. Cells were incubated at 37°C in
FIG. 1. (A) Construction of dnaK (SM12) and groE (SM13) knockdown strains. To down-regulate dnaK and dnaJ, a fragment containing 267
bp of the grpE-dnaK intergenic region was replaced by a cassette containing the ?Km element and the PureI promoter. (B) To knock down groE
expression, a 40-bp region containing the native groE promoter was replaced by the ?Km-PureI cassette. For more details, see Materials and
VOL. 189, 2007 ROLE OF MOLECULAR CHAPERONES IN S. MUTANS 1583
a 5% CO2atmosphere for 48 h and then subjected to acid killing. Briefly,
planktonic cells were discarded, and the plates were blotted onto absorbent
paper. Biofilms were then incubated in 0.1 M glycine (pH 2.8) for up to 90 min.
For each time point, biofilms from two wells were independently resuspended in
the glycine solution by repeated pipetting, transferred into a 1.5-ml Eppendorf
tube, and dispersed by vortexing at high speed for 30 s. Dispersed cells were
serially diluted, plated in duplicates onto BHI plates, and incubated for 3 days
before colonies were counted. Cell viability at each time point was expressed as
the percentage of viable cells (CFU ml?1) at time zero.
ATPase assays. For F-ATPase assays, cells were permeabilized with toluene
and incubated with 5 mM ATP in ATPase buffer as previously described (2).
Samples were removed at various intervals and assayed for inorganic phosphate
release from ATP with the Fiske-Subbarow reagent (Sigma, St. Louis, MO).
ATPase activity was expressed as nmol of PO4min?1mg protein?1. The protein
concentration was determined using the BCA assay (Sigma) with bovine serum
albumin as the standard.
Protein electrophoresis and Western blotting. Whole-cell lysates for protein
analysis were obtained by homogenization in the presence of glass beads with a
Bead Beater (Biospec, Bartlesville, OK), as previously described (6). Protein
lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis, blotted onto Immobilon-P membranes (Millipore, Bedford, MA), and
subjected to Western blot analysis by using standard techniques. Membranes
were incubated with antibodies raised against purified, recombinant Streptococ-
cus pyogenes DnaK and GroEL proteins (17). Immune reactivity was detected by
incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin G fol-
lowed by detection with 4-chloro-1-naphthol. Two-dimensional (2D) gel electro-
phoresis was performed by Kendrick Labs, Inc. (Madison, WI), according to a
method described previously by O’Farrell (21). The protein concentration of
samples was determined using the bicinchoninic assay (Sigma).
RESULTS AND DISCUSSION
Forced down-regulation of groES-groEL and dnaKJ. Previ-
ous unsuccessful attempts to isolate strains completely lacking
GroEL or DnaK support that both proteins are essential for
the viability of S. mutans. To gain further insight into the roles
of DnaK and GroEL in the physiology of S. mutans, we created
a knockdown strategy to lower the levels of DnaK and GroEL,
resulting in strains SM12 and SM13, respectively (Fig. 1).
Western blotting with polyclonal antisera against purified, re-
combinant GroEL or DnaK from S. pyogenes was used to
confirm that the SM12 and SM13 strains had diminished levels
of DnaK and GroEL, respectively (Fig. 2). Densitometric anal-
ysis revealed that the levels of DnaK in SM12 were ?5% of
that found in S. mutans UA159 grown under the same condi-
tions, and GroEL levels in SM13 were about 20% of those
found in the wild-type strain. Interestingly, when we used the
same ?Km-PureI cassette in a previous study (18) to inactivate
the hrcA gene, the first gene in the dnaK operon, we observed
only a twofold reduction in DnaK levels. Thus, as we posited
previously (18), the grpE-dnaK intergenic region may play a
significant role in dnaK mRNA stability.
The strain producing low levels of DnaK, SM12, also showed
enhanced expression of GroEL (Fig. 2). In B. subtilis, the
GroES-GroEL complex has been shown to interact directly
with HrcA (20), increasing the binding affinity of the HrcA
repressor for the CIRCE element located in the dnaK and groE
promoter regions. In S. mutans, the groE operon is under the
control of HrcA, which binds to a CIRCE element in the
promoter region but also is subject to regulation by CtsR,
which binds a consensus heptad near the HrcA binding site
(16). It does not appear that the reason that GroEL levels are
increased in SM12 is due to effects on CtsR expression or
activity. In particular, we demonstrated previously that clpP is
under negative control by CtsR in S. mutans (16). Comparisons
of the levels of clpP mRNA by real-time PCR did not reveal
any differences between UA159 and SM12. Moreover, ctsR
levels were not altered in either SM12 or SM13 (data not
shown). In contrast, there is supporting evidence that S. mu-
tans DnaK has a significant role in modulating the activity of
HrcA. In particular, hrcA expression, which is autoregulated by
HrcA, was found to be increased approximately 150-fold in
SM12 but not in SM13 (data not shown). Western blot analysis
using a polyclonal antibody raised against S. mutans HrcA (18)
revealed higher levels of HrcA in the SM12 strain (data not
shown), suggesting that the DNA binding activity of HrcA may
be compromised, perhaps because DnaK is required for main-
taining HrcA in an active state.
To confirm that the differences in chaperone expression
observed at the protein level were due to changes in the tran-
scription of the genes, Real-time RT-PCR was used to quantify
groEL and dnaK mRNA in SM12 and SM13. Compared to the
parent stain, the levels of dnaK mRNA were diminished by
about 100-fold in SM12, and groEL mRNA was reduced about
eightfold in SM13. The results also indicated that groEL was
up-regulated in SM12, whereas dnaK expression levels were
not altered in SM13 (Fig. 3). Of note, real-time PCR quanti-
fication also indicated that dnaJ levels were reduced by approx-
imately 100-fold in SM12, consistent with our observation that
dnaJ is cotranscribed with dnaK. The mRNA measurements
are generally consistent with the protein measurements, al-
though differences in the magnitudes of the changes in protein
and mRNA in the mutants may reflect roles for the posttran-
scriptional control of the levels of DnaK and GroEL.
Characteristics of dnaK and groE knockdown strains. The
physiological characteristics of strains SM12 (dnaK knock-
down) and SM13 (groE knockdown) are shown in Table 1.
Both mutant strains grew slower than the wild type, formed
FIG. 2. Western blot analysis of DnaK and GroEL levels with poly-
clonal antibodies against S. pyogenes GroEL (1:500) (?-GroEL) and
DnaK (1:1,000) (?-DnaK). Total cell lysates (10 ?g per lane) were
obtained from mid-exponential-phase cultures grown in BHI medium.
FIG. 3. Real-time PCR quantification of dnaK and groEL mRNA.
Strains UA159 (wild type), SM12 (dnaK knockdown), and SM13 (groE
knockdown) were grown in BHI medium to the mid-exponential
phase. The data represent the means ? standard deviations from three
independent experiments. The asterisk indicates that the result was
statistically significant (P ? 0.01; Student’s t test).
1584LEMOS ET AL.J. BACTERIOL.
long chains, and had a strong tendency to aggregate in broth
culture. SM12 and SM13 had doubling times of 76 ? 11.5 min
and 90 ? 17.3 min, respectively, while the doubling time for
UA159 was 44 ? 3.6 min. Despite the slow-growth phenotype,
both SM12 and SM13 were able to reach the same final optical
density as the parent strain in approximately 22 and 28 h,
Biofilm formation assays were performed with BM supple-
mented with glucose at a final concentration of 20 mM. To
exclude the possibility that any observed differences were due
to variations in the growth capacities of the strains, cells were
incubated for 48 h, and the final OD600of the cultures in the
microtiter plates was assessed prior to the assay. The data
obtained indicated that although both mutants were capable of
reaching the same final OD600as the parent strain in BM, both
SM12 and SM13 strains had a reduced capacity to form bio-
films (Fig. 4).
2D gels revealed that the forced down-regulation of DnaK
and GroEL caused profound changes in the protein expression
patterns of S. mutans (Fig. 5). Compared to UA159, at least 9
proteins were up-regulated and 10 were down-regulated in the
dnaK knockdown strain (SM12). Consistent with the Western
blot results, DnaK was down-regulated and GroES and GroEL
were among the up-regulated proteins in SM12. The 2D pro-
tein pattern of the groEL knockdown strain (SM13) indicated
that at least 10 proteins were up-regulated and 12 proteins
were down-regulated in comparison to the parent strain. As
expected, the expression of GroES and GroEL was markedly
reduced in SM13, whereas DnaK levels were not affected. In S.
mutans, the inactivation of trigger factor (TF), a ribosome-
associated molecular chaperone, resulted in the up-regulation
of both GroES-GroEL and DnaK (29). The 2D protein profile
indicated that the expression of TF was not altered in the dnaK
and groEL knockdown strains of S. mutans. It was previously
demonstrated that TF has overlapping functions and cooper-
ates with the DnaK machinery (10). In E. coli, a deficiency of
both DnaK and TF results in a massive aggregation of cytosolic
proteins and growth in only a very narrow temperature range,
and these phenotypes were partially suppressed by the overex-
pression of GroES-GroEL (10).
Effects of DnaK and GroEL down-regulation on stress tol-
erance. The capacity of the mutant strains to grow on BHI agar
plates that had been adjusted to pH 5.0 with HCl was tested.
Both UA159 and SM13 could form normal-sized colonies on
acidified BHI after 48 h, whereas no growth of the DnaK
down-regulated strain was observed, even when the plates were
incubated for up to 7 days (Table 1). The capacity of the strains
to grow at temperatures above 37°C was tested. At 43°C,
growth rates of the parent strain were significantly reduced,
and extensive cell clumping was observed. SM13 showed very
poor growth yields at 43°C, whereas no growth was observed
when SM12 was incubated at this temperature (Table 1). Next,
we evaluated the capacity of the strains to form colonies in the
presence of H2O2. For that purpose, a uniform layer of cells
was spread onto BHI agar plates, and paper filter discs satu-
rated with 0.5% H2O2were placed onto the agar. The zone of
inhibition caused by H2O2diffusion was similar for the parent
and SM13 strains (approximately 1.22 ? 0.05 cm and 1.2 ?
0.08 cm, respectively), whereas SM12 was more sensitive, with
an inhibition halo of 1.52 ? 0.09 cm (Table 1). Collectively,
these results indicate a stress-sensitive phenotype of the dnaK
knockdown strain but reveal that the down-regulation of
GroEL has only a minor effect on the stress tolerance proper-
ties of S. mutans, although it should be taken into consider-
ation that there was a more dramatic reduction in the levels of
DnaK in SM12 than in the levels of GroEL in SM13.
To better assess the acid tolerance properties of SM12, acid
killing experiments were performed with biofilm-grown cells
that were exposed to pH 2.8. Unexpectedly, biofilm cultures of
SM12 were more resistant to acid killing (approximately 1 log)
than the parent strain (Fig. 6). Similar results were obtained
when mid-exponential- or stationary-phase planktonic cultures
were used (data not shown). At first, the impaired capacity to
grow at pH 5.0 and enhanced tolerance to acid killing of SM12
appeared to be contradictory. However, acid killing experi-
ments test the capacity of the strain to survive a lethal pH for
a limited period of time (up to 90 min) and then resume
growth when transferred to medium at a neutral pH. Because
the growth of SM12 was considerably slower than that of the
parent strain, one possible explanation for this unexpected
phenotype is that cells with a slower metabolism become less
susceptible to the damage caused by acidification. In fact, using
chemostat-grown cells, it was demonstrated that fast-growing
cells of E. coli were more sensitive to stresses than slow-grow-
TABLE 1. Characteristics of S. mutans dnaK knockdown (SM12) and groE knockdown (SM13) strainsa
(min) at 37°C
Growth characteristic(s) at 37°CGrowth at 43°C
H2O2inhibition zone (cm)
44 ? 3.6
76 ? 11.5
90 ? 17.3
Extensive clumping/long chains
Extensive clumping/long chains
1.22 ? 0.05
Sensitive (1.52 ? 0.09)
Like UA159 (1.12 ? 0.08)
aNG, no growth; ?, poor growth; ???, wild-type-like growth.
FIG. 4. Biofilm formation by S. mutans UA159 (wild type) and its
derivatives. Cultures were grown in a microtiter plate containing BM
supplemented with glucose at 37°C for 48 h. The graph shows the
averages and standard deviations for five independent experiments.
The asterisks indicate that the results were statistically significant in
comparison to UA159 (P ? 0.05; Student’s t test).
VOL. 189, 2007 ROLE OF MOLECULAR CHAPERONES IN S. MUTANS 1585
ing cells (3). To investigate this possibility, steady-state cultures
of the wild-type strain were grown in continuous culture under
two different dilution rates and subjected to acid killing. The
results obtained clearly indicated that cells grown at a lower D
of 0.1 h?1(generation time, 6.9 h) were more resistant to acid
killing than cells grown at a higher dilution rate (D of 0.3 and
a generation time of 2.3 h) (J. A. Lemos and R. A. Burne,
Effects of DnaK down-regulation on F-ATPase activity. In S.
mutans, the proton-translocating ATPase (F-ATPase) is con-
sidered to be the primary mechanism of proton extrusion to
maintain ?pH during growth in acidic conditions (24). Previ-
ously, a strain lacking the HrcA regulator (SM11), which dis-
played constitutively high levels of GroEL and lower levels of
DnaK, showed diminished F-ATPase activity when grown at
pH 5.0 in a continuous chemostat culture (18), although no
differences were noted at a neutral pH. It was speculated that
DnaK participates in the biogenesis of the F-ATPase complex
or in stabilizing this complex at a low pH. Although the dnaK
knockdown strain (SM12) isolated in this study also displayed
high levels of GroEL, the levels of DnaK were drastically
reduced in comparison to the SM11 strain. F-ATPase activity
of the wild-type and SM12 strains was assayed in permeabilized
cells from 48-h biofilms and in cells growing exponentially at
pH 7.0 or following acid adaptation at pH. 5.0. No differences
in the F-ATPase activities of the parent and SM12 strains were
observed when cells were grown in biofilms or in batch culture
at pH 7.0. In contrast, acid-adapted cells of SM12 showed
consistent reductions in F-ATPase activity compared to the
wild-type strain grown under the same conditions, although the
differences observed were not statistically significant (Fig. 7).
This finding coupled with our previous results with the SM11
strain (18) clearly support that the DnaK chaperone complex is
important for the biogenesis or stabilization of the F-ATPase
complex at a low pH, when the expression of the ATPase is
known to be elevated. The manifestation of the difference only
during growth at low pH values may be attributable to the
titration of DnaK to damaged proteins in acidic conditions
or perhaps because the enzyme requires DnaK, directly or
indirectly, for stability at a low pH. An alternative explana-
tion may be related to a role for DnaK in stabilizing a
transcriptional activator of the atp operon that is required
for efficient induction at a low pH. Real-time PCR did not
indicate differences in the mRNA levels of atpF (Fodomain,
b subunit) between the UA159 and SM12 strains of cells
growing exponentially in rich medium (data not shown), but
FIG. 5. 2D protein pattern of S. mutans UA159 (wild type), SM12 (dnaK knockdown), and SM13 (groE knockdown). Samples were grown in
BHI medium and harvested in the exponential growth phase (OD600? 0.5). Protein extracts (50 ?g per gel) were separated by isoelectric focusing
in the pI range of 4 to 8 in the first dimension and by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension.
The silver-stained proteins that exhibited more obvious differences in the SM12 and SM13 strains in comparison to UA159 are indicated. Proteins
with enhanced expression are indicated with double arrows, and proteins with reduced synthesis are indicated with black arrows. The filled triangle
indicates tropomyosin protein loaded as an internal control.
FIG. 6. Survival of S. mutans strains UA159 (wild type) (circles)
and SM12 (dnaK knockdown) (triangles) after acid challenge. Cells
from 48-h biofilms grown on the surface of polystyrene plates were
subjected to acid killing in 0.1 M glycine (pH 2.8). Cell viability at each
time point is expressed as the percentage of viable cells (CFU ml of
culture?1) at time zero. A Student’s t test indicated that the differences
observed at 60 min were statistically significant (P ? 0.001).
1586 LEMOS ET AL. J. BACTERIOL.
further studies will be needed to assess the way in which
DnaK impacts ATPase activity.
Instability of dnaK and groEL knockdown strains in culture.
The initial concern for growing SM12 and SM13 in the che-
mostat was that the strains would wash out because of their
slow-growth phenotype. After allowing cells to establish batch-
wise in the chemostat without pH control, both strains were
able to maintain constant and adequate cell densities at a
dilution rate of 0.3 h?1(generation time, 2.3 h) when the pH
of the vessel was kept at 7.0. After sampling steady-state cul-
tures growing at pH 7.0, cultures were acid shocked to pH 5.0
by titration with HCl over the course of about 1 min. Neither
SM12 nor SM13 was capable of tolerating such rapid acidifi-
cation of the vessel, and washout was evident shortly after the
acid challenge. To obtain steady-state cells of SM12 and SM13
growing at pH 5.0, the pH control of the vessel was adjusted to
5.0, and the culture was allowed to slowly lower the pH
through glycolysis. The wild-type strain was capable of lower-
ing the pH from 7.0 to 5.0 in a matter of hours, whereas SM12
and SM13 took as long as 48 h to drop the pH to 5.0.
Interestingly, when aliquots were collected from the chemo-
stat at pH 5.0 and plated onto BHI agar plates, both mutant
strains were able to form visible colonies after overnight incu-
bation, suggesting that the slow-growth phenotype was lost. A
closer examination of these cultures indicated that the strains
no longer aggregated in broth, although resistance to kanamy-
cin associated with the insertion of foreign DNA was retained.
The levels of DnaK and GroEL in cells obtained from the
chemostat kept at pH 7.0 or 5.0 were assessed by Western blot
analysis. SM12 cells grown at pH 5.0 demonstrated that the
reversion of the growth phenotypes was accompanied by the
restoration of DnaK levels (data not shown). In contrast, phe-
notypic reversion of SM13 was not associated with restored
levels of GroEL. PCR and sequence analysis of the mutated
regions did not reveal changes in the site where the foreign
DNA and promoter had inserted in either strain. The instabil-
ity of both mutants indicates that the suppression of the phe-
notype occurs rapidly when cells are cultivated under stressed
conditions and that reversion is associated with increases in the
levels of chaperones in the case of DnaK but that extragenic
suppression may be a factor for the reversion of both SM12
Concluding remarks. In this study, the down-regulation of
dnaK and groEL generated pleiotropic effects and confirmed
the essential nature of the two major molecular chaperones in
S. mutans physiology. Moreover, the results presented here
provide further evidence of molecular linkages between stress
responses and biofilm formation in S. mutans (15). Thus, it is
becoming clear that an appropriately regulated response by the
microorganism to the environmental stresses encountered dur-
ing the development and maturation of a biofilm have a pro-
found influence on the biofilm structure or whether biofilms
will form at all. Studies to dissect how chaperones affect the
expression or biogenesis of known virulence attributes involved
in biofilm formation are ongoing.
This study was supported by NIH-NIDCR award DE13239.
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