Transcriptome analysis reveals that ClpXP
proteolysis controls key virulence properties of
Jessica K. Kajfasz,1Jacqueline Abranches1,2and Jose ´ A. Lemos1,2
Jose ´ A. Lemos
Received 21 June 2011
Revised27 July 2011
Accepted 30 July 2011
1Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14642, USA
2Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester,
NY 14642, USA
The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as
expression of virulence properties. However, with the exception of the Spx global regulator, the
molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans
are not well understood. Here, microarray analysis was used to provide novel insights into the
scope of ClpXP proteolysis in S. mutans. In a DclpP strain, 288 genes showed significant
changes in relative transcript amounts (P¡0.001, twofold cut-off) as compared with the parent.
Similarly, 242 genes were differentially expressed by a DclpX strain, 113 (47%) of which also
appeared in the DclpP microarrays. Several genes associated with cell growth were
downregulated in both mutants, consistent with the slow-growth phenotype of the Dclp strains.
Among the upregulated genes were those encoding enzymes required for the biosynthesis of
intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced
expression of glg and mle genes in DclpP and DclpX strains correlated with increased storage of
intracellular polysaccharide and enhanced malolactic fermentation activity, respectively.
Expression of several genes known or predicted to be involved in competence and mutacin
production was downregulated in the Dclp strains. Follow-up transformation efficiency and
deferred antagonism assays validated the microarray data by showing that competence and
mutacin production were dramatically impaired in the Dclp strains. Collectively, our results reveal
the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-
related traits that are influenced by ClpXP proteolysis.
Streptococcus mutans is a member of the oral microbiome
known for its close association with dental caries and,
occasionally, infective endocarditis. The niche in which S.
mutans thrives is the biofilm that forms on the enamel
surface of teeth (Loesche, 1986). The dental biofilm
environment is constantly and unpredictably changing
due to the eating habits of the human host, resulting in
large fluctuations in nutrient source and availability, pH,
and oxygen tension, among other stresses (Lemos & Burne,
2008). The remarkable ability of S. mutans to tolerate and
thrive during stressful conditions, particularly low pH, is
closely linked to its virulence in the oral cavity.
The Clp proteolytic complex is critical in maintaining
cellular homeostasis, particularly for organisms that must
continually endure environmental fluctuations (Frees et al.,
2004; Gottesman, 2003; Jenal & Hengge-Aronis, 2003;
Kajfasz et al., 2009). In S. mutans, Clp proteases are the
result of the association of the ClpP peptidase with one of
several Clp ATPases (ClpC, ClpE or ClpX), forming barrel-
shaped complexes that will target proteins for degradation
(Kajfasz et al., 2009; Lemos & Burne, 2002). Although S.
mutans also encodes ClpB and ClpL ATPases, these
proteins do not contain the recognition tripeptide that
permits interaction with ClpP, and are believed to function
mainly as molecular chaperones (Frees et al., 2004; Kajfasz
et al., 2009). When Clp ATPase subunits associate with
ClpP, the resulting protease performs an important protein
quality control role by targeting damaged or misfolded
proteins, threading them through its barrel for degradation
(Butler et al., 2006; Jenal & Hengge-Aronis, 2003). This
Abbreviations: CSP, competence-stimulating peptide; Erm, erythromycin;
IPS, intracellular polysaccharide; Kan, kanamycin; MLF, malolactic
fermentation; qRT-PCR, real-time quantitative reverse transcriptase
polymerase chain reaction.
The GEO Series accession number for the microarray data associated
with this study is GSE29871.
Three supplementary figures and two supplementary tables are available
with the online version of this paper.
Microbiology (2011), 157, 2880–2890
2880 052407G2011 SGM Printed in Great Britain
process is particularly important during stressful condi-
tions that increase the likelihood of misfolded or damaged
cellular proteins. Notably, Clp proteases also target
regulatory proteins, thereby keeping their numbers in
check, and providing a link between Clp proteolysis and
regulation of gene expression (Frees et al., 2004; Zuber,
Previously, we and others have demonstrated that ClpP
plays an important role in the expression of key virulence
attributes of S. mutans, including biofilm formation, cell
viability and acid tolerance (Chattoraj et al., 2010; Deng
et al., 2007; Kajfasz et al., 2009; Lemos & Burne, 2002; Zhang
et al., 2009). We also uncovered some surprising phenotypic
characteristics shared by strains bearing deletions in clpP or
clpX, including enhanced survival under short- and long-
term acidic conditions and increased sucrose-dependent
biofilm formation (Kajfasz et al., 2009). Additionally, we
showed that the Spx global regulator accumulates in S.
mutansstrainslacking functional ClpXP proteolysisandthat
inactivation of either one of the two Spx orthologues, SpxA
and SpxB, caused a reversion of many phenotypes observed
in S. mutans DclpX and DclpP strains (Kajfasz et al., 2009).
Thus, the underlying mechanisms by which ClpXP affects
virulence traits in S. mutans are intimately associated with
accumulation of the SpxA and SpxB proteins. However, not
all phenotypes associated with the clpP or clpX mutant
strains are expected to be linked to Spx accumulation, as
several distinct biological traits and regulatory circuits
controlled by Clp proteolysis in other bacterial species are
known to be Spx-independent (Frees et al., 2007).
Although ClpP proteolysis has been consistently implicated
in virulence (Frees et al., 2003; Gaillot et al., 2000; Ibrahim
et al., 2005), has shown promising results as a vaccine
candidate (Kwon et al., 2004) and is the target of two new
classes of antibiotics (Bo ¨ttcher & Sieber, 2008; Bro ¨tz-
Oesterhelt et al., 2005), a complete picture of the biological
role of Clp-dependent proteolysis in bacterial pathogenesis
has yet to emerge. As our previous study established a firm
connection between ClpXP proteolysis and cellular accu-
mulation of Spx proteins, our goals in this study were to
unveil the scope of the ClpXP regulon and to identify and
characterize virulence traits linked to functional ClpXP
proteolysis in both Spx-dependent and Spx-independent
manners. Microarray analysis provided insights into the
pleiotropic effects of deletions of clpP and clpX in S. mutans,
with expression of more than 10% of the genome altered in
each case. A follow-up physiological characterization of
selected virulence traits of S. mutans identified in the
microarrays validated the transcriptomic data and revealed
that ClpXP proteolysis is involved in intracellular polysac-
charide (IPS) production, malolactic fermentation (MLF),
competence development and bacteriocin production.
Bacterial strains and growth conditions. The strains used in this
study are listed in Table 1. S. mutans UA159 and its derivatives were
routinely grown in brain heart infusion (BHI) medium at 37 uC in a
5% CO2 atmosphere. When appropriate, kanamycin (Kan, 1 mg
ml21) or erythromycin (Erm, 10 mg ml21) was added to the growth
medium. For microarray analysis, S. mutans UA159 (wild-type) and
its Dclp derivatives were grown in BHI medium to mid-exponential
RNA extraction. RNA from S. mutans cells was isolated as described
previously (Abranches et al., 2006). Briefly, S. mutans cells grown to
the desired OD600were homogenized by repeated hot acid phenol/
chloroform extractions. The nucleic acid was precipitated with 1 vol.
cold 2-propanol and 0.1 vol. 3 M sodium acetate (pH 5) at 220 uC
overnight. RNA pellets were resuspended in nuclease-free H2O and
treated with DNase I (Ambion) at 37 uC for 30 min. The RNA was
repurified using an RNeasy mini-kit (Qiagen), including a second on-
column DNase treatment as recommended by the supplier. RNA
concentrations were determined in triplicate and samples were run on
an agarose gel to verify RNA integrity.
Microarray experiments. S. mutans UA159 version 1 microarray
slides were provided by the J. Craig Venter Institute Pathogen
Functional Genomics Resource Center (PFGRC; http://pfgrc.jcvi.org/
index.php/microarray). The microarray experiments and analysis
were as previously described (Abranches et al., 2006; Kajfasz et al.,
2010). Briefly, reference RNA was prepared from S. mutans UA159
cells that were grown in BHI medium to an OD600of 0.5, and used in
all hybridizations. cDNA samples generated from 2 mg RNA
originating from four independent cultures of each strain studied
were hybridized to the microarray slides, as was cDNA derived from
the reference culture. cDNA was coupled to Cy3-dUTP (test samples)
or Cy5-dUTP (reference samples; GE Healthcare). Mixtures of test
and reference cDNA were hybridized to the microarray slides for 16 h
at 42 uC in a MAUI (MicroArray User Interface) hybridization
chamber (BioMicro Systems). Hybridized slides were washed and
scanned using a GenePix scanner (Axon Instruments). Data were
analysed using the T4 software suite available at the PFGRC website.
Statistical analysis was carried out with BRB Array Tools (http://linus.
nci.nih.gov/BRB-ArrayTools.html) with a cut-off P-value of 0.001.
Additional details regarding array protocols are available at http://
pfgrc.jcvi.org/index.php/microarray/protocols.html. Microarray data
have been deposited in the NCBI Gene Expression Omnibus (GEO)
database (http://www.ncbi.nlm.nih.gov/geo) under GEO Series acces-
sion number GSE29871.
Real-time quantitative PCR. A subset of genes was selected to
validate the microarray data by real-time quantitative reverse
transcriptase PCR (qRT-PCR). Gene-specific primers (see Supple-
mentary Table S1, available with the online version of this paper)
were designed using Beacon Designer, version 2.0, software (Premier
Biosoft International). Reverse transcription and qRT-PCR were
carried out according to previously described protocols (Abranches
et al., 2006; Lemos et al., 2008). A Student’s t-test was performed to
verify significance of the PCR quantifications.
Construction of glg mutant strains. Standard DNA manipulation
techniques were used as previously described (Lemos & Burne, 2002;
Sambrook et al., 1989). The primers used to isolate the mutants are
listed in Supplementary Table S1. S. mutans strains lacking an
approximately 2.4 kb region of the genome encompassing 1160 bases
of the 39 end of glgB, all 1146 bases of glgC and 100 bases of the 59 end
of glgD were constructed using a PCR ligation mutagenesis approach
(Lau et al., 2002). Briefly, PCR fragments flanking the 59 end of glgB
and the 39 end of glgD were obtained and ligated to an erythromycin
resistance (Ermr) marker, and the ligation mix was used to transform
S. mutans UA159. Mutant strains were isolated on BHI plates
supplemented with Erm. Double DclpP (or DclpX) Dglg strains were
constructed by transformation of the single Dclp strains with the glg
Streptococcus ClpXP proteolysis
deletion PCR fragment obtained from the Dglg strain. The deletions
were confirmed as correct by PCR sequencing of the insertion site and
IPS determination. Accumulation of stored IPS was evaluated by
using a colorimetric assay that relies on the formation of an iodine–
polysaccharide complex (Busuioc et al., 2009). Briefly, bacteria were
streaked on Todd–Hewitt agar plates containing 2% glucose or 2%
sucrose and incubated for 48 h. The plates were then flooded with
5 ml iodine solution [0.2% (w/v) iodine in 2.0% (w/v) potassium
iodide solution]. IPS was detected by the formation of a brown
pigment, which was visible almost immediately upon adding the
Glycolytic pH minima. The ability of S. mutans strains to continue
to undergo glycolysis in an increasingly acidic environment was
evaluated by pH drop experiments (Belli & Marquis, 1991). Cultures
of S. mutans strains grown to exponential phase were harvested by
centrifugation, washed in ice-cold distilled water and resuspended in
10% culture volume of 50 mM KCl, 1 mM MgCl2salt solution. KOH
was used to titrate the suspensions to pH 7.2. Glycolysis was initiated
with the addition of 55.6 mM glucose, and the resulting fall in pH of
the suspension was monitored over a 30 min period. To study
differences in sugar storage, changes in pH were also monitored
immediately after cells were resuspended in salt solution, without
addition of glucose.
Long-term survival. The ability of the S. mutans Dglg strains to
survive a period of several days at low pH was assessed via long-term
survival assays, in which an overnight culture of cells was diluted 1:20
in tryptone-yeast extract (TY) medium containing excess glucose
(50 mM) (Kajfasz et al., 2009, 2010). The growth and pH of the
cultures were monitored at 37 uC and 5% CO2until stationary phase
was reached, at which point an aliquot was removed for serial dilution
and plating on BHI agar. Bacterial survival was assessed by plating the
cultures daily until growth was no longer detected.
Malolactic fermentation assay. The MLF assay was performed as
previously described (Martinez et al., 2010; Sheng & Marquis, 2007).
Briefly, overnight cultures were grown in BHI medium buffered to
pH 7 with 25 mM KPO4, then cells were harvested by centrifugation,
washed with 50 mM KCl, 1 mM MgCl2salt solution, and starved for
1 h at 37 uC in half the original culture volume of salt solution. After
the starvation period, cells were harvested by centrifugation and
resuspended in half the culture volume of potassium phosphate buffer
(20 mM, pH 7). To assay for MLF activity, the suspension was
adjusted to pH 4.0 with HCl to achieve the optimal pH for MLF
activity in S. mutans (Sheng & Marquis, 2007), and MLF was initiated
by the addition of L-malic acid to a final concentration of 50 mM.
Aliquots were removed immediately and 90 min following the
addition of L-malic acid, and assayed for the presence of L-malate
in the supernatant by using a
Biopharm). The values of L-malic acid metabolized were normalized
to cell dry weight of the samples.
L-malic acid detection kit (R-
Assay of genetic competence. Cultures were diluted 1:20 in
500 ml BHI medium containing 10% horse serum, and grown to an
OD600of0.15at37 uC ina5% CO2atmosphere,atwhich point200 ng
plasmid pMSP3535 (Bryan et al., 2000) was added. When desired, 5 ml
(at 1 mg ml–1) of synthetic competence-stimulating peptide (CSP) (Li
et al., 2001) was added to the cultures. The cultures were incubated
untilstationaryphase wasreached (#3 h forUA159,#4.5 hfor DclpX
and #5 h for DclpP). Transformants and total c.f.u. were enumerated
by plating appropriate dilutions on BHI agar plates with and without
Erm, respectively. The numbers of c.f.u. were counted after 72 h of
incubation, and transformation efficiency was expressed as the
percentage of transformants among the total viable cells.
Deferred antagonism assay. Bacteriocin production was measured
by assaying the ability of S. mutans to inhibit the growth of mutacin
IV- and mutacin V-sensitive species, Streptococcus gordonii and
Lactococcus lactis, respectively. Briefly, S. mutans cultures grown to an
OD600of 0.3 were spotted onto BHI agar and incubated for 24 h.
Following incubation, 500 ml of an overnight culture of S. gordonii or
L. lactis was added to 5 ml soft (0.75%) BHI agar, spread as an
overlay and incubated for another 24 h before zones of growth
inhibition around the S. mutans spots were measured.
Microarray analysis provides novel insights into
the scope of ClpXP proteolysis in S. mutans
To gain a better understanding of the phenotypes
previously observed in the DclpP and DclpX strains
(Kajfasz et al., 2009; Lemos & Burne, 2002), microarray
analysis was performed to compare the transcriptome of
mid-exponential-phase cultures of each mutant with that
of the parent strain. Prior to the microarray analysis, both
DclpP and DclpX were complemented by providing,
Table 1. Strains used in this study
Strain Relevant genotype Source or reference
JL30 (DclpP complement)
JL31 (DclpX complement)
JL26 (Dglg region)
JL27 (DclpP/Dglg region)
JL28 (DclpX/Dglg region)
L. lactis ATCC 11454
S. gordonii DL-1
clpP::NP (non-polar) Kan
JL1+pMSP3535 expressing clpP
JL2+pMSP3535 expressing clpX
glgB glgC glgD::Erm
Kajfasz et al. (2009)
Kajfasz et al. (2009)
J. K. Kajfasz, J. Abranches and J. A. Lemos
2882 Microbiology 157
respectively, the full-length clpP and clpX genes in trans
using the nisin-inducible plasmid pMSP3535 (Bryan et al.,
2000). Several phenotypes characteristic of the DclpP and
DclpX strains (Kajfasz et al., 2009), including slow growth
rates and aggregation in broth, were fully reversed by the
complementation (Supplementary Figs S1, S2 and S3). At
an assigned P-value of ¡0.001 and applying a twofold
change cut-off, there were 288 genes in the DclpP strain and
242 in DclpX that showed significant changes in relative
transcript amounts as compared with the parent strain.
The complete list of genes with altered expression in the
Dclp strains is provided in Supplementary Table S2. Of the
242 genes differentially expressed in DclpX, 113 (47%) also
appeared, following the same trend, in the DclpP micro-
arrays. The genes found to be differentially expressed in
only one of the two mutant strains are probably due to a
ClpP-independent function of ClpX, or to ClpP interac-
tions with another Clp ATPase partner (ClpC or ClpE). To
facilitate data interpretation, the genes that appeared on
these microarray analyses were grouped into functional
categories (Fig. 1). A subset of the differentially expressed
genes was selected and used for qRT-PCR analysis
(Supplementary Table S1) for validation of the microarray
data, and the results were consistent with the expression
trends observed in the microarrays.
Several genes encoding ribosomal proteins, translation
initiation factors and elongation factors were downregu-
lated in both mutants, consistent with the slow-growth
phenotype previously described for the two Dclp mutant
strains (Kajfasz et al., 2009). Interestingly, an overall
upregulation of genes involved in energy metabolism
(acoB, adh, mle, pckA, pfl, among others) and in sugar
uptake and metabolism (glg, gtfB, mal, msm and scr) was
observed in the mutants, suggesting an increased need for
ATP by these strains. Genes encoding putative peptidases
or endopeptidases (gcp, pepO, pepB, pepT) were found to
be upregulated in both DclpP and DclpX strains, a finding
that suggests an attempt by the cells to compensate for the
loss of ClpXP ‘housekeeping’ proteolysis.
Previous studies showed an increased capacity of the DclpP
and DclpX strains to form biofilms when grown with
sucrose (Deng et al., 2007; Kajfasz et al., 2009), suggesting
that glucan production is enhanced in these strains. Our
microarrays showed that expression of gtfB, encoding the
glucosyltransferase B enzyme responsible for establishing
the extracellular polysaccharide matrix along with gtfC and
gtfD (Ooshima et al., 2001), was upregulated more than
fourfold in DclpP, and more than sixfold in DclpX. Western
blot analysis using a polyclonal antibody against the S.
mutans GtfB protein (Wunder & Bowen, 2000) confirmed
that expression of GtfB was higher in DclpP and DclpX
(data not shown).
The DclpP and DclpX strains exhibit enhanced
accumulation of IPS
A common trend revealed by the microarray data was the
enhanced expression of the glg genes, coding for the
Fig. 1. Numbers of genes separated by functional categories that were differentially expressed by S. mutans DclpP (open bars)
and DclpX (solid bars) as compared with the parent strain UA159 with a twofold cut-off (P¡0.001).
Streptococcus ClpXP proteolysis
enzymes responsible for the production of glycogen-like
IPS. To investigate whether the DclpP and DclpX strains
have enhanced capacity to store IPS, we used an iodine-
staining colorimetric assay to visualize IPS accumulation.
Both DclpP and DclpX strains indeed showed enhanced
accumulation of IPS, as demonstrated by a darker brown
colour compared to the light brown colour observed in the
parent strain (Fig. 2). To investigate whether these
phenotypes in the Dclp strains would revert when the glg
operon was disrupted, we created double mutants by
inserting the glg mutation (constructed by deleting a 2.4 kb
region encoding all of glgC, as well as parts of glgB and
glgD) in the DclpP and DclpX strains. As expected, the Dglg,
DclpP/Dglg and DclpX/Dglg strains showed no apparent
incorporation of the iodine solution (Fig. 2). Similar results
were observed with plates containing 2% sucrose (data not
To further demonstrate that the clpP and clpX mutants
have enhanced IPS storage, we performed pH drop
experiments to evaluate the ability of these strains to
reduce the extracellular pH through glycolysis. When the
pH drop experiment was performed by the addition of
glucose to cells resuspended in salt solution, no differences
in kinetics and final pH were observed between the parent,
DclpP and DclpX strains (data not shown). To evaluate the
consumption of endogenous sugars, the pH drop experi-
ments were repeated without pH titration or the addition
of exogenous glucose, so that any drop in the extracellular
pH could be attributed to IPS utilization. In this case, both
the DclpP and the DclpX strains were able to reduce the pH
faster and to values considerably lower than those of the
parent strain (over 0.5 pH units lower) (Fig. 3). As
expected, the Dglg as well as the Dclp/Dglg double mutants
were unable to lower the pH without addition of
Increased production of IPS by the Dclp strains
does not appear to account for the enhanced
To test the hypothesis that enhanced IPS storage
contributes to the prolonged survival of S. mutans, we
repeated the long-term survival assay with the DclpP and
DclpX strains (Kajfasz et al., 2009), including the Dglg and
Dclp/Dglg strains that were unable to accumulate IPS.
When grown in TY medium containing 50 mM glucose at
37 uC in a 5% CO2 atmosphere, all strains attained a
similar final pH of approximately 4.2 within 18–24 h of
initiating the experiment. However, we did not see a
reversion of the enhanced-survival phenotype when a
deletion in the glg operon was added to the Dclp strains
(data not shown), suggesting that enhanced IPS storage
plays a minor role in the enhanced survival of the DclpP
and DclpX strains.
MLF activity is significantly enhanced in the
absence of ClpXP proteolysis
Our microarray analysis also revealed that the genes
(SMU.0138), were highly upregulated in both DclpP and
DclpX strains (.10-fold induction, Supplementary Table
S2). Recently, MLF has been identified as an important
buffering system in S. mutans and shown to protect the
cells against acid, oxidative and starvation stresses (Lemme
et al., 2010; Sheng & Marquis, 2007; Sheng et al., 2010).
The mle genes and enzyme activity are positively regulated
Fig. 2. Vizualization of IPS stores in S. mutans UA159 and its
derivatives. Agar plates inoculated with S. mutans UA159, DclpP,
DclpX, Dglg, DclpP/Dglg and DclpX/Dglg were flooded with iodine
solution. IPS was visualized by the formation of a brown pigment.
The image shown is a representative of three independent
Fig. 3. Glycolytic pH minima of S. mutans UA159 (black solid line),
DclpP (black dashed line), DclpX (black dotted line), Dglg (grey
solid line), DclpP/Dglg (grey dashed line) and DclpX/Dglg (grey
dotted line). pH drop experiments were performed without addition
of glucose to cells. The curves shown are representatives of at
least three independent experiments.
J. K. Kajfasz, J. Abranches and J. A. Lemos
2884 Microbiology 157
by addition of external L-malate and by low pH (Lemme
et al., 2010; Martinez et al., 2010; Sheng & Marquis, 2007).
In agreement with our microarray data, MLF activity was
minimal in the wild-type strain grown without addition of
L-malate at pH 7 but a .10-fold increase in activity was
seen in DclpP and DclpX strains grown under the same
conditions (mean MLF activity reported as mmol L-malate
decarboxylated during a 90 min period per mg cell dry
weight: S. mutans UA159, 1.196; DclpP, 12.64, P¡0.005;
DclpX, 13.53, P¡0.0005). When cultures were grown in the
presence of 25 mM L-malate, the DclpP and DclpX strains
also showed significantly enhanced ability to metabolize L-
malic acid at both neutral and acidic (pH 5.5) pH values
(data not shown).
ClpXP proteolysis regulates competence
development and bacteriocin production
The microarray data also revealed that transcription of a
number of genes known or predicted to be involved in
competence development and bacteriocin production were
downregulated in the Dclp strains (Table 2). Among the
known competence genes found to be downregulated were
the sigma factor comX, several genes of the late competence
comY operon and the newly described comR transcriptional
regulator (Mashburn-Warren et al., 2010). We assessed the
transformation efficiency of DclpP and DclpX with and
without the addition of exogenous CSP. In the absence of
CSP, we were unable to obtain a single transformant
colony of the DclpP and DclpX strains, whereas UA159 was
readily transformable (3.0561025% transformation effi-
ciency). Addition of exogenous CSP re-established com-
petence in DclpP and DclpX (1.261024and 261025%
transformation efficiencies, respectively), but dramatically
improved transformation efficiencies of the parent strain
(8.761023%). Fig. 4 shows a typical example of the
number of transformants obtained for each strain at the
same serial dilution.
Among the bacteriocin-related genes that appeared in the
microarray analysis were nlmA and nlmB, encoding the
non-lantibiotic mutacin IV (Qi et al., 2001), and cipB
(nlmC), encoding mutacin
Matsumoto-Nakano & Kuramitsu, 2006). We performed
a deferred antagonism assay, which revealed that the DclpP
and DclpX strains have lost the ability to antagonize growth
of S. gordonii or L. lactis by producing, respectively,
mutacin IV or mutacin V (Fig. 5).
V (Haleet al., 2005;
In the oral cavity, S. mutans must cope with large and
constant environmental fluctuations, including changes in
pH, oxygen tension, and nutrient source and availability
(Lemos & Burne, 2008). In such an environment,
cytoplasmic proteases that control the stability of regula-
tory proteins and prevent the accumulation of damaged
proteins are central to physiological homeostasis and
virulence. Clp-dependent proteolysis is of particular
relevance in Streptococcus, as this genus lacks other known
cytoplasmic proteases, such as Lon and ClpYQ (Kajfasz
et al., 2009). In fact, proteolysis mediated through ClpXP is
an important trait for stress tolerance, gene regulation and
virulence in several Gram-positive pathogens (Chastanet
et al., 2001; Chattoraj et al., 2010; Frees et al., 2003, 2004;
Gaillot et al., 2000; Ibrahim et al., 2005; Kwon et al., 2004).
Previously, we demonstrated that a number of virulence
properties of S. mutans, including stress tolerance, biofilm
formation and colonization in a rodent caries model, were
influenced by ClpXP proteolysis (Kajfasz et al., 2009).
Although we have shown that accumulation of the global
regulators SpxA and SpxB was associated with a number of
the phenotypes observed in the DclpP and DclpX strains
(Kajfasz et al., 2009), we reasoned that ClpXP would
probably control a number of additional physiological
properties of S. mutans in an Spx-independent manner.
Here, we used microarrays to better understand the global
effects of ClpXP proteolysis in S. mutans. The large number
of genes that were differentially expressed as compared
with the parent strain supports the hypothesis that ClpXP
is critical for S. mutans homeostasis, as the number of
differentially expressed genes represents over 12% of the S.
mutans genome (Ajdic ´ et al., 2002). The large number of
genes following the same trend in both mutants confirmed
the cooperative nature of ClpP and ClpX, and is in
agreement with the nearly identical phenotypes previously
observed for the DclpP and DclpX strains (Kajfasz et al.,
2009). Surprisingly, the genes found to be differentially
expressed in our DclpP arrays showed limited overlap with
a previous microarray analysis that used an S. mutans clpP
mutant generated using a markerless gene deletion system
(Chattoraj et al., 2010). Despite differences in growth
conditions, microarray platform and mutant construction,
the reasons for the small number of genes following the
same trend in the current and previous study are not
Some of the trends that were uncovered by the microarray
analysis support the relationship that we have previously
demonstrated between ClpXP proteolysis and the tran-
scriptional regulators SpxA and SpxB (Kajfasz et al., 2010),
which are targets of ClpXP proteolysis. For example, the
genes encoding Gor and TrxB, two enzymes involved in
oxidative stress responses regulated by Spx, were upregu-
lated in the Dclp strains. These same genes were down-
regulated in DspxA and DspxAB microarrays (Kajfasz et al.,
2010). Although this relationship between regulatory
mechanisms of ClpXP and Spx is clearly important, the
analysis described here also identified new genes and
pathways that are under ClpXP proteolytic control in an
Our microarray analysis identified a large number of
genes involved in sugar uptake and metabolism as being
upregulated in the Dclp strains. Among these were the
genes encoding enzymes responsible for the biosynthesis of
Streptococcus ClpXP proteolysis
IPS, a glycogen-like polymer that serves as a storage form
of carbohydrate (DiPersio et al., 1978). The synthesis of IPS
has been implicated in enhanced survival of S. mutans and
has been shown to contribute to caries formation (Gibbons
& Socransky, 1962; Spatafora et al., 1995; van Houte et al.,
1970). Previous work revealed that production of IPS via
glgA (encoding glycogen synthase) significantly extended
the survival of S. mutans during starvation, especially when
cells were grown in the presence of glucose (Busuioc et al.,
2009). The upregulation of several glg genes led us to
hypothesize that the enhanced long-term survival of DclpP
and DclpX (Kajfasz et al., 2009) could be associated with
increased IPS storage. Although we were able to dem-
onstrate that the DclpP and DclpX strains indeed accu-
mulate larger amounts of IPS when compared with the
parent strain, no phenotypic reversion was seen when the
Dclp/Dglg strains were subjected to long-term survival
experiments. These results do not rule out that increased
IPS stores play a role in the enhanced survival of DclpP and
DclpX but indicate that other factors may participate in the
enhanced survival of the Dclp strains. By comparing the
transcriptome of the Dclp strains with the previously
published Dspx strains (Kajfasz et al., 2010), we found no
indication that the upregulation of the glg genes in the
DclpP microarrays was linked to Spx regulation.
In a previous report, we showed that expression of mleS
and mleP (encoding the malolactic enzyme and malate
permease, respectively) was downregulated in the DspxA/
DspxB double mutant (Kajfasz et al., 2010). Here, we
showed that transcription of mleS and mleP was highly
upregulated (.10-fold) in both Dclp strains, suggesting
that the enhanced MLF activity of DclpP and DclpX may be
associated with the accumulation of Spx in the absence of
ClpXP proteolysis. Work is under way to specifically
address the possibility that the mle operon is under Spx
Although L-malic acid is not an energy source for S.
mutans, MLF is a major process for alkali generation and
for ATP synthesis by means of F-ATPase acting in the
synthase mode (Sheng & Marquis, 2007), a process that
confers protection against acid, oxidative and starvation
Table 2. Competence- and bacteriocin-related genes differentially expressed in the DclpP and DclpX strains
Expression relative to UA159
Locus tagGene name Definition
Rgg family, transcriptional regulator
Non-lantibiotic mutacin IV A
Non-lantibiotic mutacin IV B, GG-motif-containing peptide
Oligopeptide ABC transporter
ABC transporter, bacteriocin
ABC transporter, bacteriocin
Late competence gene
ABC transporter, bacteriocin
Putative bacteriocin, GG-motif-containing peptide
Putative immunity protein
Mutacin V, GG-motif-containing peptide
Late competence gene
Late competence gene
Late competence gene
Late competence gene
Sigma factor ComX
J. K. Kajfasz, J. Abranches and J. A. Lemos
2886 Microbiology 157
stresses (Lemme et al., 2010; Sheng & Marquis, 2007; Sheng
et al., 2010). Thus, it is possible that the enhanced acid
tolerance of the Dclp strains in vitro (Kajfasz et al., 2009)
may be linked to the increased MLF activity observed in
Another interesting finding was that a large number of genes
involved in competence development and bacteriocin
production were downregulated in the Dclp strains.
Coordinated competence development and production of
bacteriocin have been well documented, suggesting that S.
mutans utilizes competence-induced cell lysis to eliminate
competition and acquire DNA from neighbouring species
(Kreth et al., 2005, 2007; Steinmoen et al., 2003; van der
Ploeg, 2005). The repression of competence genes in the S.
mutans DclpP strain supports previous studies reporting
reduced genetic competence of an S. mutans clpP mutant
(Lemos & Burne, 2002). We assessed competence in DclpP
and DclpX and found that transformation efficiency of both
mutants was drastically reduced. Although only a few
competence- and bacteriocin-related genes were differenti-
ally expressed in our Dspx microarrays (Kajfasz et al., 2010),
studies with Bacillus subtilis and Streptoccocus pneumoniae
show a link between Spx regulation and competence
development. For example, the B. subtilis Spx negatively
regulates competence development by disrupting interac-
tions between the ComA~P response regulator and the C-
terminal domain of the RNA polymerase (Nakano et al.,
2003). Likewise, the S. pneumoniae SpxA1, a protein sharing
75% homology with the S. mutans SpxA, was shown to
regulate competence in S. pneumoniae by repressing
transcription of early competence genes (Turlan et al.,
2009). Thus, current evidence points towards a global model
in which Spx negatively regulates competence development
in Gram-positive bacteria, suggesting that the reduced
transformation efficiencies of the S. mutans DclpP and
DclpX strains may be linked to Spx accumulation.
Peptide bacteriocins are produced to inhibit the growth of
other microbes sharing the same ecological niche. The S.
mutans UA159 strain used in this study produces at least
two types of bacteriocins: mutacin IV, encoded by nlmA
and nlmB, and mutacin V, encoded by cipB (also known as
nlmC). Mutacin IV is active against members of the mitis
group of streptococci (Hale et al., 2005; Qi et al., 2001),
whereas mutacin V is active mostly against non-strep-
tococcal species (Hale et al., 2005). In addition to the genes
encoding mutacin IV and mutacin V, several other
bacteriocin-related genes, including immunity factors and
the locus encoding the ABC transporter required for
Fig. 4. Genetic competence of S. mutans UA159, DclpP and
DclpX. Plasmid (pMSP3535) was added to early exponential-
phase (OD600of 0.15) cells with or without the addition of CSP,
then plated at stationary phase. Images shown are representatives
of three independent experiments.
Fig. 5. Deferred antagonism assay. S. mutans
UA159 (wild-type, WT), DclpP and DclpX
were spotted onto agar plates and allowed to
grow for 24 h before an overlay with the test
strain was introduced. (a) Overlay with S.
gordonii DL-1 (mutacin IV sensitive). (b)
Overlay with L. lactis ATCC 11454 (mutacin
V sensitive). The images shown are represen-
tatives of three independent experiments.
Streptococcus ClpXP proteolysis
mutacin export, nlmET (Hale et al., 2005), were down-
regulated in both Dclp strains. Deferred antagonism assays
confirmed the microarray results and revealed that both
DclpP and DclpX were unable to inhibit growth of mutacin
IV- and mutacin V-sensitive strains. Despite the limited
overlap of genes found to be differentially expressed in our
DclpP microarrays and in a previous report (Chattoraj et al.,
2010), it is interesting to note that the data regarding
expression of bacteriocin-related genes and bacteriocin
production between both studies were in full agreement.
A general characteristic of secreted peptides in Gram-
positive bacteria is the presence of a highly conserved
double glycine (GG) motif preceding the site where specific
proteolytic cleavage of the signal peptide occurs during
peptide export (Ha ˚varstein et al., 1995). The S. mutans
UA159 genome encodes several putative peptides contain-
ing the conserved GG motif, including mutacin IV and V,
and CSP. Notably, several other putative GG-motif-
containing peptides were downregulated in the Dclp
strains, suggesting that ClpXP proteolysis may have a
broad role in biosynthesis or maturation of secreted
peptides. Collectively, these results identify ClpXP proteo-
lysis as a participant in the regulation of competence
development and bacteriocin production in S. mutans.
The coordinated activation of competence and bacteriocin
production has been linked to S. mutans virulence via
different mechanisms that are not mutually exclusive. For
example, in addition to killing bacterial competitors,
bacteriocin production has been linked to biofilm forma-
tion through extracellular DNA release and autolysis (Perry
et al., 2009a, b). Perry et al. (2009a) showed that
intracellular accumulation of unprocessed CipB (mutacin
V) mediates autolysis and proposed that CipB may be
involved in altruistic cell death of a subset of the
population (fratricide) during stressful conditions. It is
tempting to speculate that the unexpected enhanced
survival of DclpP and DclpX during acid stress (Kajfasz
et al., 2009) could, at least partially, be explained by a
reduction in normal levels of mutacin V-mediated
autolysis in these strains. In fact, we have previously
shown that the DclpP and DclpX strains undergo autolysis
at a slower rate than the parental strain (Kajfasz et al.,
2009). Our previous microarray data with S. mutans Dspx
strains did not offer additional insight regarding a direct
role of Spx with bacteriocin production. However, given
the relationship of Spx regulation with competence in
other organisms (Nakano et al., 2003; Turlan et al., 2009),
and the tight linkage between competence development
and bacteriocin production in S. mutans, we cannot rule
out that Spx may participate in the regulation of the genes
involved in bacteriocin production in a manner that has
yet to be determined.
In summary, the results presented here confirm the broad
scope of ClpXP regulation in S. mutans homeostasis and
identify virulence-related traits that are influenced by
ClpXP proteolysis, including IPS storage, MLF activity and
competence/bacteriocin production. Our data further
confirm the importance of Spx removal by Clp proteolysis
during unstressed conditions, as some of the identified
virulence traits affected by ClpXP are probably regulated in
an Spx-dependent manner. At the same time, it is clear that
ClpXP can also influence the expression of virulence traits,
such as IPS storage, in an Spx-independent manner.
Considering the high degree of conservation among Clp
proteins and given that ClpP-mediated proteolysis is an
established virulence factor for several Gram-positive
organisms (Frees et al., 2003; Gaillot et al., 2000; Ibrahim
et al., 2005; Kajfasz et al., 2009), our findings are likely to
have broad implications.
This study was supported by NIH-NIDCR award DE019783. J.K.K.
and J.A. were also supported by the NIDCR training in oral sciences
grant T32 DE007202.
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