JOURNAL OF BACTERIOLOGY, Apr. 2009, p. 2060–2068
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 7
Role of Clp Proteins in Expression of Virulence Properties of
Jessica K. Kajfasz,1Alaina R. Martinez,1Isamar Rivera-Ramos,1,2Jacqueline Abranches,1,2
Hyun Koo,1,3Robert G. Quivey, Jr.,1,2and Jose ´ A. Lemos1,2*
Center for Oral Biology,1Department of Microbiology and Immunology,2and Eastman Department of Dentistry,3University of
Rochester Medical Center, Rochester, New York 14642
Received 12 November 2008/Accepted 16 January 2009
Mutational analysis revealed that members of the Clp system, specifically the ClpL chaperone and the
ClpXP proteolytic complex, modulate the expression of important virulence attributes of Streptococcus mutans.
Compared to its parent, the ?clpL strain displayed an enhanced capacity to form biofilms in the presence of
sucrose, had reduced viability, and was more sensitive to acid killing. The ?clpP and ?clpX strains displayed
several phenotypes in common: slow growth, tendency to aggregate in culture, reduced autolysis, and reduced
ability to grow under stress, including acidic pH. Unexpectedly, the ?clpP and ?clpX mutants were more
resistant to acid killing and demonstrated enhanced viability in long-term survival assays. Biofilm formation
by the ?clpP and ?clpX strains was impaired when grown in glucose but enhanced in sucrose. In an animal
study, the average number of S. mutans colonies recovered from the teeth of rats infected with the ?clpP or
?clpX strain was slightly lower than that of the parent strain. In Bacillus subtilis, the accumulation of the Spx
global regulator, a substrate of ClpXP, has accounted for the ?clpXP phenotypes. Searching the S. mutans
genome, we identified two putative spx genes, designated spxA and spxB. The inactivation of either of these genes
bypassed phenotypes of the clpP and clpX mutants. Western blotting demonstrated that Spx accumulates in the
?clpP and ?clpX strains. Our results reveal that the proteolysis of ClpL and ClpXP plays a role in the
expression of key virulence traits of S. mutans and indicates that the underlying mechanisms by which ClpXP
affect virulence traits are associated with the accumulation of two Spx orthologues.
Streptococcus mutans, a common inhabitant of dental bio-
films, is considered a major etiologic agent of human dental
caries. The abilities of this organism to form biofilms on the
tooth surface, metabolize a wide range of carbohydrates, and
tolerate rapid and frequent environmental fluctuations are key
virulence attributes of this bacterium (3, 29). In addition to
dental caries, S. mutans is often a cause of subacute bacterial
endocarditis, a life-threatening inflammation of heart valves.
Bacteria in dental plaque are subjected to a wide range of
stresses. The intermittent ingestion of food by the host results
in dramatic variations in nutrient availability and pH. In addi-
tion, bacteria also have to cope with significant fluctuations in
oxygen tension and osmolarity. One of the consequences of
exposure to environmental stresses is the accumulation of ab-
normal proteins due to increased errors in transcription and
translation (9, 22, 24). The capacity to maintain protein ho-
meostasis by stabilizing proteins that perform essential func-
tions and by refolding or degrading misfolded or aberrant
proteins is central for the viability and growth of bacterial
pathogens in the host (9, 17, 22, 24).
In the gram-negative bacterial paradigm Escherichia coli,
there are four classes of energy-dependent cytoplasmic pro-
teases: Lon, FtsH, ClpAP/XP, and ClpYQ (also known as
HslUV) (22). In Lon and FtsH, a single protein encodes both
the ATPase and proteolytic sites, whereas the ATPase and
proteolytic domains are encoded by different proteins in the
ClpAP/XP and ClpYQ classes. While the metalloprotease
FtsH is ubiquitous in eubacteria, low-GC gram-positive bacte-
ria (GPB) lack the Lon protease (9, 17). In addition, the
available genome sequences of streptococci indicate that they
also lack ClpYQ that is found in the genome of some GPB,
including the pathogenic Staphylococcus aureus (9, 18). There-
fore, it has been proposed that ClpP complexes are more
central for stress tolerance and global regulation in strepto-
cocci than in other bacterial groups (17). In the ClpP proteo-
lytic system, ClpP must associate with a Clp ATPase partner
that possesses nucleotide binding domains characteristic of the
AAA? superfamily of ATPases to form a functional complex
(9, 17). In addition to forming protease complexes, Clp
ATPases can also perform essential housekeeping functions,
including protein reactivation activities typical of molecular
In addition to cellular protein quality control, bacterial pro-
teolysis also plays an important role by controlling the stability
of regulatory proteins (17, 22, 24). Many bacterial stress re-
sponses are triggered by inhibition of the degradation of stress-
induced transcriptional regulators or sigma factors or by the
degradation of repressors of stress gene expression (17, 24).
Among the regulators targeted by Clp proteolysis is Spx, sup-
pressor of clpP and clpX, a global transcriptional regulator of
oxidative stress commonly found in low-GC GPB (42). Studies
with Bacillus subtilis revealed that Spx is a substrate of Clp
proteolysis (35) and that the accumulation of Spx was respon-
* Corresponding author. Mailing address: Center for Oral Biology,
Box 611, 601 Elmwood Ave., University of Rochester Medical Center,
Rochester, NY 14642. Phone: (585) 275-1850. Fax: (585) 276-0190.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 30 January 2009.
sible for the pleiotropic phenotypes associated with clpP or
clpX mutations (34). Similar observations were made for Lac-
tococcus lactis, for which the inactivation of an Spx homologue
alleviated the effects conferred by the clpP and clpX mutations
The genome of S. mutans UA159 encodes orthologs of five
Clp ATPases (ClpB, ClpC, ClpE, ClpL, and ClpX), and a
single ClpP peptidase (2, 30). Among the five Clp ATPases,
ClpC, ClpE, and ClpX have the recognition tripeptide that
allows the proteins to interact with ClpP, while ClpB and ClpL
are expected to function as protein chaperones independent of
ClpP (17). Previously, we showed that a strain lacking ClpP
exhibited impaired growth under stress conditions, formed
long chains, had a strong tendency to aggregate in culture, had
reduced genetic transformation efficiencies, and showed major
defects in its capacity to form biofilms in medium containing
glucose as the primary carbohydrate source (30). More re-
cently, it was demonstrated that clpP mutants exhibited in-
creased sensitivity to several environmental stress challenges
and that ClpP is critical in the organism’s adaptive response to
oral care products such as sodium fluoride, hydrogen peroxide,
and chlorhexidine (4, 13).
Despite the progress that has been made in demonstrating
the relevance of Clp proteolysis in S. mutans, the molecular
mechanisms underlying these phenotypes remain mostly unde-
termined. To better define the role of the Clp system in S.
mutans, clpB, clpC, clpE, clpL, clpP, and clpX mutants were
constructed by allelic replacement. Phenotypic characteriza-
tion of the mutants has shown that the ?clpL, ?clpP, and ?clpX
strains presented the most striking phenotypes compared to
those of the parent strain. In addition, we have shown that
many phenotypes typical of clpP or clpX mutations were, in
great part, associated with the accumulation of two putative
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains used in this
study are listed in Table 1. S. mutans UA159 and its derivatives were grown in
brain heart infusion (BHI) medium at 37°C (except for growth under heat stress
at 42°C) in a 5% CO2atmosphere. When appropriate, kanamycin (1 mg ml?1),
spectinomycin (1.5 mg ml?1), or erythromycin (10 ?g ml?1) was added to the
medium. The BHI medium was adjusted to pH 5.5 with HCl where indicated.
Growth curves were determined by measuring the change in the optical density
at 600 nm (OD600) in BHI broth. To evaluate the capacity of the mutant strains
to grow under different stress conditions, the strains were grown overnight in
BHI broth and diluted 20-fold in fresh BHI broth buffered to pH 7 and incubated
at 37°C (control) or 42°C (heat stress) or in BHI broth buffered to pH 5.5 (acid
Construction of mutant strains. Standard DNA manipulation techniques were
used as previously described (30, 38). The primers used to isolate the mutants are
listed in Table 2. S. mutans strains lacking the various clp genes (clpB, clpC, clpE,
clpP, and clpX) or putative spx genes (smu1142c or smu2084c) were constructed
using a PCR ligation mutagenesis approach (27). Briefly, PCR fragments flank-
ing each target gene were obtained and ligated to a nonpolar kanamycin (Kmr),
erythromycin (Emr), or spectinomycin (Spr) resistance marker, and the ligation
mix was used to transform S. mutans UA159. A strain lacking clpL was made by
the insertion of an Emrmarker in a naturally occurring BglII restriction site
located 324 bp from the start codon. Mutant strains were isolated on BHI plates
supplemented with the appropriate antibiotic(s). Double and triple mutants were
also constructed by further transformations using chromosomal DNA isolated
from single mutants. The deletions were confirmed as correct by PCR sequenc-
ing of the insertion site and flanking sequences.
Long-term survival. The ability of the S. mutans strains to survive a period of
several days was assessed via a long-term survival assay, in which an overnight
culture of cells was diluted 1:20 in tryptone-yeast extract (TY) medium contain-
ing excess glucose (50 mM). The growth of the cultures was monitored until
stationary phase was reached, at which point an aliquot was removed for serial
dilution and plating on BHI agar. The cultures were incubated in TY medium
containing 50 mM glucose at 37°C and 5% CO2for several days, with serial
dilutions of the cultures plated daily until growth was no longer detected. The
plates were incubated for 48 h before colonies were counted.
Acid-killing experiments. For acid killing, strains were grown in BHI medium
to mid-logarithmic growth phase (OD600? 0.5), washed once with 0.1 M glycine
buffer (pH 7), and resuspended in 1/5 of the growth volume in 0.1 M glycine
buffer (pH 2.85) for up to 90 min. Every 30 min, aliquots were serially diluted,
plated on BHI plates and incubated for 48 h before colonies were counted.
Autolysis assay. An autolysis assay was performed as described previously (1).
Briefly, S. mutans strains were grown in BHI medium to late exponential phase
(OD600? 0.7), harvested, and washed twice with phosphate-buffered saline. The
cells were then resuspended to an OD550of 0.9 in 20 mM potassium phosphate
buffer (pH 6.5), containing 1 M KCl, 1 mM CaCl2, 1 mM MgCl2, and 0.4%
sodium azide. Autolysis was measured at 30-min intervals at 44°C using a Bio-
screen C growth monitor (Oy Growth Curves AB Ltd., Helsinki, Finland) by
measuring the OD540. Sterile mineral oil was added over the cell suspension to
create an anaerobic environment, and the plate was shaken for 15 s prior to each
Biofilm assay. The ability of S. mutans strains to form biofilms was assessed by
growing cells in wells of polystyrene microtiter plates using a semidefined biofilm
medium (BM) (33). The wells of the plates were first coated for 30 min with 100
?l of sterile, clarified, pooled human saliva. Strains grown in BHI medium to an
OD600of approximately 0.5 were diluted 1:100 in BM containing either 1%
glucose or 1% sucrose and added to the wells of the microtiter plate. The plates
were incubated at 37°C in a 5% CO2atmosphere for 24 h. After incubation, the
plates were washed twice with water to remove planktonic and loosely bound
bacteria, and adherent cells were stained with 0.1% crystal violet for 15 min. The
bound dye was eluted with 33% acetic acid solution, and biofilm formation was
then quantified by measuring the optical density of the solution at 575 nm.
Western blot analysis. To detect Spx levels in the clpP and clpX mutants,
whole-cell protein lysates were prepared by the homogenization of cells in the
presence of glass beads with a Bead Beater (Biospec, Bartlesville, OK). The protein
concentration of the samples was determined using the bicinchoninic acid assay.
The protein lysates were separated by 12% sodium dodecyl sulfate-polyacrylam-
ide gel electrophoresis, blotted onto Immobilon-FL membranes (Millipore, Bed-
ford, MA), and subjected to Western blot analysis by using standard techniques.
The membranes were incubated with antibody raised against Bacillus subtilis Spx
(a generous gift from Peter Zuber, Oregon Health & Science University, Bea-
verton, OR). Immune reactivity was visualized by the incubation of the blot with
ECL detection reagents (GE Healthcare, Buckinghamshire, United Kingdom)
and immediate exposure to BioMax MS film (Kodak, Rochester, NY).
TABLE 1. S. mutans strains used in this study
JL15 (?clpP ?spxA)
JL16 (?clpP ?spxB)
JL17 (?clpX ?spxA)
JL18 (?clpX ?spxB)
JL20 (?clpP ?spxA
JL19 (?clpX ?spxA
clpP::nonpolar Km, spxA::Sp
clpP::nonpolar Km, spxB::Em
clpX::nonpolar Km, spxA::Sp
clpX::nonpolar Km, spxB::Em
clpP::nonpolar Km, spxA::Sp,
clpX::nonpolar Km, spxA::Sp,
aStr, streptomycin resistance.
VOL. 191, 2009S. MUTANS Clp PROTEINS2061
Animal studies. To test the infectivity of the clpP and clpX mutants in vivo, we
conducted a study to evaluate the ability of the strains to colonize the teeth of the
animals using a rodent model of dental caries (7). Briefly, specific-pathogen-free
Sprague-Dawley rats were infected by means of a cotton swab with the ?clpP and
?clpX strains and UA159StR, a spontaneous streptomycin-resistant strain (12).
At weaning, pups aged 21 days were randomly placed into three groups of eight
and infected for two consecutive days with actively growing ?clpP, ?clpX, or
UA159StRstrains. The rats were fed a highly cariogenic diet (Diet-2000 con-
taining 56% sucrose) and 5% sucrose water (wt/vol) ad libitum. On experimental
days 4 and 10, the animals were screened for the successful infection of each
strain by oral swabbing and plating on mitis-salivarius agar containing the ap-
propriate antibiotic. The experiment proceeded for 16 days, at the end of which
the animals were euthanized by CO2asphyxiation and the lower jaws removed
for microbiological assessment (25). The number of mutans streptococci recov-
ered from the animals was expressed as CFU ml?1of jaw sonicate. The data were
subjected to analysis of variance for significance and Grubbs’ test to detect
outliers. This study was reviewed and approved by the University of Rochester
Committee on Animal Resources.
Growth characteristics of clp mutants. To assess the func-
tion of S. mutans Clp proteins in homeostasis and the expres-
sion of virulence properties, deletions of clpB, clpC, clpE, clpL,
clpP, and clpX were created by replacing the entire corre-
sponding gene with an antibiotic resistance cassette, or, in the
case of the clpL mutant, with an Emrresistance marker at the
clpL 5? end (Table 1). Because there is precedent in the liter-
ature that ClpC and ClpE may have overlapping functions and
one could compensate for the loss of the other (17), a ?clpC
?clpE double mutant was generated. Similarly, ClpB and ClpL,
which lack the ClpP recognition tripeptide, appear to have
similar function in S. aureus (17). Thus, a ?clpB ?clpL double
mutant was also obtained.
Striking phenotypes were immediately noticed in the clpP
and clpX mutant strains; both of these strains formed unusually
long chains and had a strong tendency to clump in broth (data
not shown), characteristics that were previously observed in
the clpP insertional mutant (30). Standard growth curves in
BHI medium (37°C, 5% CO2atmosphere) revealed a slow-
growth phenotype for the ?clpP and ?clpX strains (Fig. 1).
Moreover, the ?clpP and ?clpX strains failed to reach the same
final growth yield as the other strains. The ?clpL mutant also
TABLE 2. Primers used for gene inactivation
aRestriction sites used to facilitate ligation are highlighted in bold.
FIG. 1. Growth curves of S. mutans UA159 (f), ?clpB (Œ), ?clpC
(?), ?clpE (?), ?clpL (F), ?clpP (?), and ?clpX (‚) strains in BHI
medium at 37°C. The curves shown are the averages with standard
deviations of the results from three independent experiments.
2062KAJFASZ ET AL. J. BACTERIOL.
consistently demonstrated a longer lag phase than the parent
but was able to reach a similar final yield as the parent before
entering stationary phase (Fig. 1). The growth curves of the
?clpCE strain indicated that the double mutant did not differ
from its ?clpC and ?clpE counterparts, i.e., the ?clpCE strain
could grow as well as its parental strain (data not shown). The
slow-growth phenotype of the ?clpL strain was retained in the
?clpBL double mutant strain (data not shown).
We also tested the capacity of each clp deletion mutant to
grow at 42°C or in medium acidified to pH 5.5. Under both
conditions, the growth of the ?clpP and ?clpX strains was
slower and the final growth yield was considerably lower com-
pared to that of the wild-type strain (data not shown). More-
over, the growth of the ?clpP and ?clpX strains under aerobic
conditions (using a rotary shaker, 150 rpm) was severely im-
paired (data not shown). Compared to that of the parent
strain, there were no obvious differences in the growth of the
?clpB, ?clpC, ?clpE, and ?clpL strains under the stress con-
Clp mutations affect long-term survival. In a long-term sur-
vival assay, the viability of the ?clpL strain was significantly
lower (an approximately 3-log reduction after 48 h) than that
observed for UA159, whereas no significant differences were
observed for the ?clpB, ?clpC, and ?clpE strains. Surprisingly,
there was an approximate 2-log enhancement in survival for
both the ?clpP and ?clpX strains (Fig. 2). Additionally, the
?clpP and ?clpX strains survived until day 4 of the experiment,
while no other strain had detectable colonies after day 3. All
strains tested were capable of lowering the pH of the culture to
?4.2 after 24 h of incubation, with the exception of the ?clpP
(24-h pH, ?4.4) and ?clpX (24-h pH, ?4.3) strains. After 48 h,
there was no substantial difference in the average pH of the
?clpX strain versus that of the parent UA159 strain, but the
?clpP strain maintained a slightly higher pH (?0.2 pH unit)
throughout the duration of the experiment. Because the ?clpP
and ?clpX strains were unable to lower the pH as rapidly and
to the same values of the other strains, it was possible that the
enhanced survival of these mutants was linked to differences in
the growth yield and final pH. To test this hypothesis, we
repeated the experiment, but this time, upon entering station-
ary phase, we used 1 N HCl to artificially lower the pH of the ?clpP
and ?clpX cultures to the same pH value attained by the
parent. The culture pH remained stable for the duration of the
experiment for all strains tested. The results obtained were
nearly identical to those observed when the final culture pH
was not adjusted (data not shown), strongly suggesting that the
enhanced survival of the ?clpP and ?clpX mutants was not due
to differences in the pH.
The clpL, clpP, and clpX mutations affect acid challenge
survival. The differences noted in the ?clpL, ?clpX, and ?clpP
strains in the long-term survival assays led us to further char-
acterize the acid tolerance of these strains by performing acid-
killing experiments. Consistent with the long-term survival re-
sults that showed that the viability of the clpL mutant was
significantly reduced, the ?clpL strain was more sensitive to
acid challenge compared to the parent strain (Fig. 3). Also in
agreement with the long-term survival data, we observed an
enhanced ability of the ?clpP and ?clpX strains to survive acid
challenge compared to that of UA159. At the final time point,
the mutant strains displayed an approximate 2-log (?clpX) to
4-log (?clpP) enhancement of survival compared to that of
UA159. Strikingly, the ?clpP mutant suffered very little cell
death over the course of the experiment.
Mutations in clpP and clpX reduce S. mutans autolysis. The
results of the long-term and acid-survival assays led us to ques-
tion if the enhanced survival of strains bearing mutations in
clpP and clpX could be due to reductions in autolytic rates.
This hypothesis was confirmed as the ?clpP and ?clpX strains
underwent autolysis at a slower rate than the parental strain
Clp proteins have a large role in biofilm formation. The
capacity of the clp deletion strains of S. mutans to form biofilms
in the presence of glucose or sucrose was evaluated. When
provided with glucose, the ?clpP and ?clpX strains showed
significantly less biofilm mass than the parent strain, whereas
no significant differences were observed in the ?clpB, ?clpC,
?clpE, and ?clpL strains (Fig. 5). In sucrose, biofilm formation
by the ?clpL, ?clpP, and ?clpX mutants was significantly en-
hanced, and a small reduction in biomass was observed in the
FIG. 2. Long-term survival of S. mutans UA159 (f), ?clpB (Œ),
?clpC (?), ?clpE (?), ?clpL (F), ?clpP (?), and ?clpX (‚) strains in
TY medium supplemented with 50 mM glucose at 37°C. Aliquots of
culture were first plated on BHI agar when stationary growth phase
was reached (day 0) and again each day until no surviving colonies
were detected. The results presented are averages and standard devi-
ations of results from three independent experiments.
FIG. 3. Acid killing of S. mutans UA159 (f), ?clpL (F), ?clpP (?),
and ?clpX (ƒ) strains. Aliquots were plated on BHI agar immediately
upon suspension in 0.1 M glycine (pH 2.85) and after 30, 60, and 90
min of incubation. The curves shown are the averages with standard
deviations of the results from three independent experiments.
VOL. 191, 2009S. MUTANS Clp PROTEINS 2063
?clpE strain (Fig. 5). To exclude the possibility that the differ-
ences observed were due to small variations in the growth
capacity of the strains, the biofilm data were normalized by the
The double inactivation of clpCE or clpBL did not alter
phenotypes associated with the single mutants. As mentioned
above, studies conducted in other organisms have suggested
redundant roles between the ClpC/ClpE and ClpB/ClpL
ATPases (17). Biofilm and long-term survival assays with the
?clpCE and ?clpBL mutants indicated that the double mutant
strains did not differ phenotypically from the single mutant
counterparts (data not shown). Although we cannot exclude
that there may be functional redundancies among the chaper-
ones, our experimental conditions failed to identify phenotypes
that were caused or enhanced by the clpCE or clpBL double
The inactivation of clpP or clpX decreases the infectivity of S.
mutans. The characterization of the clp mutants revealed that
the inactivation of either clpP or clpX resulted in increased
resistance to acid killing and the enhanced capacity to form
biofilms in sucrose. Considering that the ability to form bio-
films in the presence of sucrose and the capacity to survive
under low-pH conditions are critical for S. mutans to colonize
the oral cavity and cause disease, one might reason that mu-
tations in clpP or clpX render a strain more virulent. To test
this possibility, we evaluated the capacity of the ?clpP and
?clpX strains to colonize the teeth of pathogen-free rats. How-
ever, the average recovered CFU from recipient animals in-
fected with the wild-type strain was slightly higher than the
average recovered CFU from the clpP and clpX mutants. Al-
though both mutants showed the same trend, the differences
observed were not statistically significant (P ? 0.05 by analysis
of variance) (Fig. 6).
The inactivation of putative spx genes alleviates the ?clpP
and ?clpX phenotypes. Searching the S. mutans UA159 ge-
nome (2), we identified two proteins (Smu1142c and
Smu2084c, herein designated SpxA and SpxB, respectively)
that shared significant homology with B. subtilis Spx (80 and
73% similarities, respectively) and contain the CxxC redox
disulfide motif (see the supplemental material). To gain insight
into the role of these proteins in S. mutans and to assess
whether any of the phenotypes associated with the ?clpP and
?clpX mutations were Spx-dependent, both genes were deleted
and a series of double mutants (the ?clpP ?spxA, ?clpP ?spxB,
?clpX ?spxA, and ?clpX ?spxB strains) were constructed by
transforming the ?clpP and ?clpX single mutants with chro-
mosomal DNA isolated from the ?spxA and ?spxB mutant
strains. The inactivation of either spxA or spxB in the ?clpP or
?clpX strains reversed the tendency of these mutants to aggre-
gate in broth (Fig. 7). The inactivation of the spxA gene in the
?clpP or ?clpX strains completely restored the growth defect
that was initially observed in these strains, whereas the inacti-
vation of spxB partially restored the growth defect of the ?clpP
strain but not of the ?clpX strain (Fig. 7). Also, the inactivation
of spxA or spxB altered phenotypes seen in the ?clpP and
?clpX mutants during long-term survival (Fig. 8) and acid-
killing assays (Fig. 9). More specifically, the ?clpP ?spxA strain
survived similarly to the ?clpP strain until day 3 of the exper-
iment. However, different than the ?clpP single mutant but
similar to the parent strain, the ?clpP ?spxA strain had no
survivors on day 4 (Fig. 8). Interestingly, the inactivation of
FIG. 4. Autolysis of S. mutans UA159 (f), ?clpP (?), and ?clpX
(ƒ) strains. A typical result representative of three independent ex-
periments is shown.
FIG. 5. Biofilm formation by S. mutans UA159 (wild type) and its
derivatives. Cultures were grown in a microtiter plate containing BM
supplemented with 1% glucose or 1% sucrose at 37°C for 24 h. Biofilm
formation was normalized by total growth to exclude apparent differ-
ences due to the growth abilities of each strain. Absorbance at 575 nm
of crystal violet is shown as averages with standard deviations of the
results from three independent experiments. ?, P ? 0.05; ??, P ? 0.005
FIG. 6. Colonization of S. mutans UA159, ?clpP, and ?clpX strains
on the teeth of rats after 16 days. The symbols shown represent the
recovered bacterial colonies from each individual rat, while the line
represents the mean recovery per bacterial strain. Grubbs’s test did not
indicate the presence of outliers among each strain.
2064KAJFASZ ET AL. J. BACTERIOL.
spxB in the ?clpP strain not only reversed the original pheno-
type of ?clpP, but the viability of the ?clpP ?spxB double
mutant was significantly lower than that observed for the wild-
type strain under the same conditions (Fig. 8). In the ?clpX
mutant, the inactivation of either spxA, spxB, or both elimi-
nated the increased viability of the ?clpX mutant, and the
viabilities of the ?clpX ?spxA and ?clpX ?spxB strains were
reduced by 1 day compared to that of its parent strain (Fig. 8).
In the acid-killing experiments, the deletion of spxA or spxB in
the ?clpP or ?clpX strain reversed the stress-resistance phe-
notype observed in the ?clpP and ?clpX single mutants (Fig.
9). In addition to the ?clp ?spx double mutants, triple mutant
strains (?clpP ?spxAB and ?clpX ?spxAB) were created. While
there were no difficulties in isolating a ?clpX ?spxAB strain,
the ?clpP ?spxAB mutant was recovered only when plates were
incubated in an anaerobic atmosphere, suggesting that the
strain is unable to cope with oxidative stress. Thus, because the
?clpP ?spxAB triple mutant was able to grow only in an an-
aerobic environment, this strain was not included in any fur-
ther experiments. The characterization of the ?clpX ?spxAB
triple mutant indicated that the strain displayed a behavior
similar to the ?clpX ?spx double mutant strains during long-
term survival and acid-killing assays (Fig. 8 and 9). Collectively,
these data indicate that the introduction of a mutation in spx,
either on spxA or spxB, resulted in the complete or partial
reversal of the ?clpP or ?clpX phenotypes.
Spx accumulates in the ?clpP and ?clpX strains. As the Spx
protein is highly conserved in low-GC GPB, we used antibodies
raised against B. subtilis Spx to assess Spx levels in the ?clpP
and ?clpX strains. As expected, Spx clearly accumulated in
stationary-phase lysates of the ?clpP and ?clpX mutants even
though a band was not detected in the parent strain (Fig. 10).
The inactivation of spxA or spxB resulted in the disappearance
of the Spx protein, suggesting that the protein band detected in
the ?clpP and ?clpX extracts was a result of the accumulation
of both SpxA and SpxB.
Previous reports have demonstrated that ClpP participates
in key physiologic processes of S. mutans, including stress tol-
erance and biofilm formation (4, 13, 30). Here, we expanded
our analysis by systematically deleting each clp gene (clpB,
clpC, clpE, clpL, clpP, and clpX) identified in the S. mutans
genome. Our results revealed that members of the Clp system,
specifically ClpL, ClpP, and ClpX, modulate the expression of
important virulence attributes of S. mutans.
Although it lacks the recognition tripeptide that permits
interaction with ClpP, the ?clpL strain showed several impor-
tant phenotypes. The clpL mutant displayed an enhanced ca-
pacity to form biofilms in sucrose, had reduced viability in a
long-term survival assay, and was more sensitive to acid killing
than the parent strain. In a previous report, the S. mutans ClpL
protein was induced nearly fourfold in continuous cultures
grown at pH 5 compared to that of cells grown at pH 7 (32). A
role for ClpL in the acid tolerance of lactic acid bacteria is
further illustrated by studies with Lactobacillus species. For
Lactobacillus reuteri, clpL was upregulated during acid shock,
FIG. 7. The inactivation of spxA or spxB alleviates the slow-growth and aggregation phenotypes of the ?clpP and ?clpX strains.
VOL. 191, 2009S. MUTANS Clp PROTEINS2065
and a clpL mutant was significantly more sensitive to low pH
than the parental strain (40). Similarly, a proteomic approach
identified ClpL as upregulated during the acid adaptation of
Lactobacillus bulgaricus (14).
The ?clpP and ?clpX strains displayed several phenotypes in
common, including those that support previous findings with a
clpP insertional mutant such as a strong tendency to clump in
broth, slow growth rates, poor growth yields, and impaired
growth under low-pH or elevated-temperature conditions (30).
In another study, an S. mutans clpP knockout mutant was also
more sensitive to stress challenges, including growth at low pH
or in the presence of compounds commonly used in products
for oral care (13). With the exception of an S. aureus clpX
mutant that showed improved heat tolerance (16), the inacti-
vation of clpP or clpX of low-GC GPB has resulted in stress-
sensitive phenotypes (15, 21, 37). Therefore, it was surprising
that the S. mutans clpP and clpX mutants showed enhanced cell
viability at a nonlethal pH and increased capacity to survive
lethal acidification. There are two, not mutually exclusive, pos-
sible explanations for this result. First, the ?clpP and ?clpX
mutants are intrinsically more resistant to acid killing due to
downstream effects in gene regulation caused by the loss of
proteolytic control of regulatory proteins targeted by ClpXP,
such as the Spx regulator (see below for more detail). Another
possibility is that strains with slower metabolisms, such as the
?clpP and ?clpX strains, become less susceptible to damages
caused by acidification. This appears to be the case for a DnaK
knockdown strain of S. mutans that was completely unable to
grow at pH 5 but more resistant to acid killing (31). In support
of this hypothesis, it has been demonstrated that fast-growing
E. coli cells were more sensitive to stress than slow-growing
cells (5). Another result that may tie in to either of these
possibilities is the reduced autolysis seen for the ?clpP and
?clpX mutants that indicates that ClpXP proteolysis may reg-
ulate the stability or activity of autolytic enzymes in the cell.
In a previous report, we observed that a clpP insertional
mutant showed major defects in its capacity to form biofilms in
medium containing glucose (30). However, Deng and col-
leagues demonstrated increased biofilm formation of an S.
mutans clpP mutant grown in sucrose (13). Here, we confirm
the findings above, as biofilm formation by the ?clpP strain was
impaired in the presence of glucose but enhanced when su-
crose was used as the sole carbohydrate source. The ?clpX
strain also showed a reduced capacity to form biofilms in glu-
FIG. 8. Long-term survival of S. mutans strains bearing mutations
in both the clp and spx genes in TY medium supplemented with 50 mM
glucose at 37°C. Aliquots of culture were first plated on BHI agar when
stationary growth phase was reached (day 0) and again each day until
no surviving colonies were detected. The results presented are a rep-
resentative of three independent experiments.
FIG. 9. Acid killing of S. mutans strains bearing mutations in both
the clp and spx genes. Aliquots were plated on BHI agar immediately
upon suspension in 0.1 M glycine (pH 2.85) and after 30, 60, and 90
min of incubation. The curves shown are the averages with standard
deviations of the results from three independent experiments.
FIG. 10. Western blot analysis of Spx levels in S. mutans UA159
and its derivatives. The total cell protein (25 ?g per lane) of the
stationary-phase lysates was separated by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis and subjected to Western blotting using
an anti-B. subtilis Spx antibody diluted 1:250.
2066KAJFASZ ET AL. J. BACTERIOL.
cose and an enhanced capacity in sucrose, suggesting that
ClpXP proteolysis regulates biofilm formation in S. mutans. In
S. mutans, there are two distinct mechanisms to colonize tooth
surfaces, sucrose-independent and sucrose-dependent mecha-
nisms (3). Sucrose-independent mechanisms are considered to
play an important role in the initial interactions between the
bacteria and components of the enamel pellicle (28). The su-
crose-dependent pathway involves the production of extracel-
lular polymers of glucose, glucans, formed through the action
of three glucosyltransferase enzymes and glucan binding pro-
teins (3). Notably, the production of glucans via glucosyltrans-
ferase enzymes is directly associated with the development of
dental caries in rats (41). The increased capacity of the ?clpL,
?clpP, and ?clpX strains to form biofilms when grown in su-
crose suggests that glucan production is enhanced in these
strains. Of note, microarray analysis indicated that the expres-
sion of gtfB, responsible for establishing the extracellular poly-
saccharide matrix along with gtfC, was upregulated more than
fourfold in the ?clpP and ?clpX strains (J. K. Kajfasz and J. A.
Lemos, unpublished data).
A correlation between Clp proteolysis and bacterial patho-
genesis has been previously proposed (9, 17). A number of
studies have demonstrated that ClpP proteolysis is required for
virulence and the progression of disease by bacterial patho-
gens, including gram-positive organisms (16, 20, 23, 26). Im-
munization with ClpP was shown to elicit an immune response
that protected mice against pneumococcal infections (10, 26).
The relevance of ClpP complexes in pathogenesis has been
further illustrated by the discovery of new drugs that target
ClpP of GPB. In one study, acyldepsipeptides were shown to
bind specifically to ClpP, leading to uncontrolled proteolysis by
ClpP that resulted in self-digestion of the bacteria (8). More
recently, cell-permeable trans-?-lactones were shown to spe-
cifically bind to ClpP, causing the complete inhibition of the
peptidase activity of ClpP in S. aureus (6). The enhanced ca-
pacity to form biofilms in sucrose and increased resistance to
acid killing of the S. mutans ?clpP or ?clpX strains suggested
that the inactivation of clpP or clpX could potentially increase
the virulence of this dental pathogen. The finding of a hyper-
virulent ?clpP strain was unforeseen and could make targeting
ClpP to control diseases potentially unsafe. To test whether the
inactivation of clpP or clpX increased the infectivity of S. mu-
tans, a study was conducted to evaluate the ability of the mu-
tants to implant on the teeth of rats. The results obtained from
this study did not indicate that the inactivation of clpP (or
clpX) enhanced the infectivity of S. mutans. On the contrary, a
small reduction in the levels of infection was observed for both
One of the key roles of bacterial proteolysis is to control the
stability of regulatory proteins involved in a variety of cellular
processes, including stress responses, sporulation, cell division,
and transition between growth phases (17, 24). In B. subtilis,
the inactivation of the transcriptional regulator Spx, a known
target of ClpXP proteolysis, suppressed phenotypes associated
with clpP or clpX mutations, including poor growth, genetic
competence, and sporulation frequency (34). Similar findings
have been made in L. lactis and S. aureus (19, 36), suggesting
that the linkage of Spx accumulation with ClpXP phenotypes
may be conserved between low-GC GPB. Here, we showed
that the mutation of either of two spx orthologs, named spxA
and spxB, was responsible, at least in part, for the attenuation
of phenotypes associated with the loss of ClpXP proteolysis in
S. mutans. Both SpxA and SpxB contain the characteristic
CxxC redox disulfide motif and share significant homology to
B. subtilis Spx (80% and 73%, respectively). Thus, the under-
lying mechanisms by which ClpXP affect virulence traits in S.
mutans appear to be, in great part, associated with the accu-
mulation of two Spx orthologues. While the high levels of
homology and the comigration of SpxA and SpxB coupled with
the very low concentrations of Spx in the cell lysates made it
difficult to demonstrate that both Spx proteins are subject to
ClpXP degradation, our physiology experiments demonstrated
clearly that both SpxA and SpxB contribute to the phenotypes
characteristic of the ?clpP and ?clpX strains.
Despite the strong association of Spx accumulation with
phenotypes resulting from clpP and clpX mutations, the effects
of global regulation by Spx in pathogenic bacteria have been
largely overlooked. In L. monocytogenes, an spx homologue
was found upregulated during the intracellular growth of the
bacteria, suggesting that Spx might play a role in intracellular
invasion, survival, or both (11). More recently, Spx was shown
to affect growth, general stress tolerance, and biofilm forma-
tion in S. aureus (36). Notably, unsuccessful attempts to isolate
a clpP spx double mutant in S. aureus were reported, suggesting
that the strain was not viable (36). The finding of the dual Spx
regulators is particularly novel and may have broader implica-
tions since two or more copies of putative spx genes are also
found in the genome of related streptococci, enterococci, and
lactococci. Studies to understand how the Clp and Spx regu-
lons interact and exert an effect on phenotypes associated with
virulence are ongoing.
We thank Kathleen Scott-Anne for assistance with rat experiments,
and Peter Zuber for kindly providing the anti-Spx antibody.
This study was partially supported by the NIDCR training program
in oral science grant T32DE007202 and by grant DE017425 to R.G.Q.
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