JOURNAL OF BACTERIOLOGY, May 2009, p. 2973–2984
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 9
The LiaFSR System Regulates the Cell Envelope Stress Response in
Prashanth Suntharalingam, M. D. Senadheera, Richard W. Mair,
Ce ´line M. Le ´vesque, and Dennis G. Cvitkovitch*
Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada
Received 4 November 2008/Accepted 13 February 2009
Maintaining cell envelope integrity is critical for bacterial survival, including bacteria living in a complex
and dynamic environment such as the human oral cavity. Streptococcus mutans, a major etiological agent of
dental caries, uses two-component signal transduction systems (TCSTSs) to monitor and respond to various
environmental stimuli. Previous studies have shown that the LiaSR TCSTS in S. mutans regulates virulence
traits such as acid tolerance and biofilm formation. Although not examined in streptococci, homologs of LiaSR
are widely disseminated in Firmicutes and function as part of the cell envelope stress response network. We
describe here liaSR and its upstream liaF gene in the cell envelope stress tolerance of S. mutans strain UA159.
Transcriptional analysis established liaSR as part of the pentacistronic liaFSR-ppiB-pnpB operon. A survey of
cell envelope antimicrobials revealed that mutants deficient in one or all of the liaFSR genes were susceptible
to Lipid II cycle interfering antibiotics and to chemicals that perturbed the cell membrane integrity. These
compounds induced liaR transcription in a concentration-dependent manner. Notably, under bacitracin stress
conditions, the LiaFSR signaling system was shown to induce transcription of several genes involved in
membrane protein synthesis, peptidoglycan biosynthesis, envelope chaperone/proteases, and transcriptional
regulators. In the absence of an inducer such as bacitracin, LiaF repressed LiaR-regulated expression, whereas
supplementing cultures with bacitracin resulted in derepression of liaSR. While LiaF appears to be an integral
component of the LiaSR signaling cascade, taken collectively, we report a novel role for LiaFSR in sensing cell
envelope stress and preserving envelope integrity in S. mutans.
In any microorganism, the first and major cellular structure
to be impacted by threats from the environment is the cell
envelope. This structure is vital for survival since it protects the
cell from the environment, counteracts the inner high turgor
pressure, acts as a permeability barrier, and provides a sensory
platform that transmits information from the cell’s surround-
ings ultimately to its genome (15). Hence, maintaining cell
envelope integrity in the face of environmental insults by re-
sponding to cell envelope stress is critical for bacterial survival.
Two-component signal transduction systems (TCSTSs) are
among the primary sensory-regulatory mechanisms that medi-
ate bacterial adaptation processes (e.g., countering envelope
stress) in response to environmental perturbations (5, 21).
These systems modulate the expression of genes, encoding
products crucial to cell survival, via a cytoplasmic response
regulator (RR), upon receipt of an external stimulus detected
by a membrane-bound histidine kinase (HK) sensor (21). Sig-
nal transduction is mediated through a phosphorelay cascade
from an autophosphorylated His residue located in the acti-
vated HK sensor to a conserved Asp residue in the cognate
RR, altering the RR’s affinity to bind to promoter regions of
target genes and regulating their expression (21).
A prototypical gram-positive TCSTS that orchestrates cell
envelope stress response is the Bacillus subtilis LiaRS system
(24). This system is transcriptionally activated by exposure to
alkaline shock, organic solvents, detergents, secretion stress,
and notably lipid II cycle inhibitors such as the antibiotics
vancomycin and bacitracin, the bacteriocin nisin, and cationic
antimicrobial peptides (39, 51); hence, its cogname, lipid II-
interacting antibiotics LiaRS. Lipid II contains the complete
peptidoglycan (PG) subunit linked to the membrane-embed-
ded lipid carrier C55-isoprenyl phosphate (36, 60). The mole-
cule “flips” between the cytoplasmic and extracellular faces of
the cell membrane in a dynamic process (referred to as the
lipid II cycle) essential for translocating PG precursors for cell
wall biosynthesis (36, 60). The Lipid II cycle is considered the
rate-limiting step of PG polymer biosynthesis and, conse-
quently, the subject of intense scrutiny in the development of
novel inhibitors that target or exploit this process (9).
B. subtilis LiaRS is widely disseminated in Firmicutes (low
G?C gram-positive) bacteria, and homologs have been char-
acterized in Lactococcus lactis and Staphylococcus aureus as
part of the complex regulatory network that counteracts cell
envelope stress (24, 29, 37). However, the nature of the enve-
lope stress signal and the regulon genes controlled by this
system diverges based on the organism. While liaRS homologs
in both L. lactis (cesSR) and S. aureus (vraSR) are transcrip-
tionally induced by lipid II cycle inhibitors, the latter S. aureus
system is unique in responding to a wider array of cell envelope
antibiotics including teicoplanin, ?-lactams and D-cycloserine
(29, 37, 70). Moreover, in B. subtilis, LiaR regulates the tran-
scription of its own liaIHGFSR operon and another operon
encoding a second TCSTS (24). In contrast, recent transcrip-
* Corresponding author. Mailing address: Dental Research Insti-
tute, Faculty of Dentistry, University of Toronto, 124 Edward St.,
Toronto, Ontario M5G 1G6, Canada. Phone: (416) 979-4917, x4592.
Fax: (416) 979-4936. E-mail: email@example.com.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 27 February 2009.
tome profiling of S. aureus and L. lactis exposed to lipid II cycle
inhibitors identified 46 VraSR-dependent and 23 CesSR-de-
pendent genes (29, 37), many of which are presumably in-
volved in cell envelope biogenesis or stress-related functions.
The physiological role (especially the envelope stress response
function) of B. subtilis LiaRS homologs in streptococci is, how-
ever, poorly understood.
Streptococcus mutans is considered to be one of the major
pathogens associated with human dental caries. Life in the oral
cavity is typically characterized by fluctuating environmental or
physiochemical factors that include changes in the availability
of nutrients, pH, oxygen, the presence of bacteriocins, and
antimicrobial compounds; all of which strongly influence the
survival of S. mutans within the plaque ecosystem. Hence,
among 13 TCSTSs identified in the S. mutans UA159 genome,
four (ComDE, CiaRH, VicRK, and LiaSR) have to some ex-
tent been characterized and shown to play a prominent role in
regulating environmental stress tolerance and other diverse
phenotypes conducive to persistence (3, 4, 7, 32, 33). The
present study describes the S. mutans cell envelope stress re-
sponse via LiaSR TCSTS, a system previously shown to be
involved in tolerating acidic pH and biofilm formation (32).
This TCSTS was originally referred to as HK11/RR11 by Li
et al. (32) and was recently renamed LiaSR by Chong et al.
(12), owing to its close homology to the B. subtilis LiaRS
TCSTS (24). A recent transcriptome comparison by Perry et al.
(49) between a liaR mutant and its UA159 progenitor strain
identified 174 LiaR-dependent genes in S. mutans biofilm ver-
sus planktonic growth, including many genes with functions in
protein translation, energy metabolism, transport, and stress
tolerance. These authors also reported several LiaR-depen-
dent gene products involved in cell envelope functions and
cells derived from liaR-deficient biofilms revealed a distinctive
round morphology, in contrast to the rod-shaped cells typical
of the wild-type UA159 (49), thus implying a role for LiaR in
the cell envelope and/or cell shape of S. mutans.
Herein, we define the transcriptional organization of the
LiaSR TCSTS in S. mutans strain UA159 as part of a penta-
cistronic liaFSR-ppiB-pnpB operon. We show that liaSR and
the 5? proximally encoded liaF assist in the tolerance of S.
mutans to a variety of environmental threats, including stres-
sors that specifically target the cell envelope. Under noninduc-
ing conditions, liaF was shown to have a negative role on liaRS
transcription, whereas expression of liaR was induced by in-
hibitors that compromised cell membrane integrity or hindered
lipid II-mediated cell wall biosynthesis. Moreover, the liaFSR
system was shown to upregulate gene products involved in cell
wall PG matrix biosynthesis and membrane protein biogenesis,
thus expanding our understanding of how S. mutans can re-
spond to cell envelop stress and elucidating a novel role for the
LiaFSR system in responding to cell envelope stress in S.
MATERIALS AND METHODS
S. mutans strains and growth conditions. The S. mutans strains used in the
present study are listed in Table 1. S. mutans UA159 and its mutant derivates
were routinely grown in solid/liquid Todd-Hewitt-yeast extract (THYE) medium
and incubated as standing cultures at 37°C in air with 5% CO2. When required
erythromycin (10 ?g/ml) was supplemented in the medium for the selection and
growth of mutant strains. Insertional deletion mutants of the liaFSR genes in S.
mutans UA159 background were constructed by PCR ligation mutagenesis and
allelic replacement as previously described (30). Primers used to construct and
confirm the gene deletions are listed in Table S1 in the supplemental material.
DNA sequencing was also performed to further confirm correct in-frame inser-
tion of the antibiotic cassette into the target gene. Quantitative real-time reverse
transcription-PCR (qRT-PCR) experiments showed no lia-specific gene expres-
sion in these mutants. Relative expression analysis of mutant and wild-type
cDNAs using primers specific for downstream genes ruled out negative polar
effects during mutagenesis of the lia genes (data not shown). Growth kinetic
analysis were performed using a microbiology workstation (Bioscreen C Lab-
systems, Helsinki, Finland) equipped with BioLink software to monitor the
turbidity (i.e., the optical density at 600 nm [OD600]) readings translated to
growth curves (26). The following stressors affecting growth rates were tested as
previously described (54): acid (pH 5.5), 5% (vol/vol) ethanol, 0.4 M NaCl, and
0.006% (vol/vol) H2O2.
Cell envelope antimicrobial susceptibility assays. The MIC and minimum
bactericidal concentration (MBC) of cell envelope inhibitors were examined in S.
mutans parent UA159 and mutant strains. Cell envelope inhibitors tested in-
cluded the antibiotics vancomycin, D-cycloserine, bacitracin, and ?-lactam anti-
biotics (i.e., penicillin G and oxacillin); the bacteriocin nisin; and cell membrane-
interfering compounds chlorhexidine and sodium dodecyl sulfate (SDS) (all
obtained from Sigma-Aldrich Canada, Ltd., Oakville, Ontario, Canada). The
methodology was based on McBain et al. (40), with the following modifications:
(i) 100 ?l of mid-log phase bacterial cells adjusted to an OD600of ?0.01 (?105
CFU/ml) was added to a 96-well microtiter plate containing THYE medium
supplemented with twofold serial dilutions of cell envelope inhibitors; (ii) bac-
terial growth after 48 h was spectrophotometrically measured by using an ELISA
microtiter plate reader (model 3550; Bio-Rad Laboratories, Richmond, CA) at
an absorbance of 490 nm (OD490) and; (iii) relative cell density percentages were
calculated by using the following equation: (OD490of culture in the presence of
each concentration of antibiotics)/(OD490of culture in the absence of antibiot-
ics) ? 100. The MIC was determined as the lowest product concentration needed
to ensure that culture did not grow to over 10% of the relative cell density. MBC
testing was carried out using MIC microtiter plates. Briefly, aliquots of 20 ?l
were taken from each well (including the MIC endpoint) and spot plated onto
THYE agar and incubated for 48 h. MBCs were determined as the lowest
concentration of biocide at which ?5 CFU were observed after 48 h of incuba-
tion at 37°C and 5% CO2. Two independent MIC or MBC determinations were
carried out in duplicate for each trial. Susceptible strains were defined as strains
with at least twofold lower MIC or MBC levels than the wild-type UA159
Cell preparation for gene expression analysis. To study gene expression of S.
mutans cells treated with cell envelope inhibitors, total RNA was isolated from
cultures grown in THYE medium supplemented with various envelope antimi-
crobials. Briefly, overnight cultures were diluted 1:20, grown to mid-log phase
(OD600? 0.4), and treated with or without different concentrations of antimi-
crobials for 10, 30, or 60 min. RNA was extracted from treated or untreated cells
and converted to cDNA as previously described by Senadheera et al. (54). To
determine gene expression levels at different growth stages, total RNA was
harvested from S. mutans cultures grown in THYE to early-log (OD600? 0.15),
mid-log (OD600? 0.4), and stationary (OD600? 1.0) phases.
qRT-PCR analysis was performed by using the Mx3000P QPCR system (Strat-
agene, La Jolla, CA) and Quantitect SYBR-Green PCR kit (Qiagen, Missis-
sauga, Ontario, Canada) as previously described (54). qRT-PCR primers specific
for target genes are listed in Table S2 in the supplemental material. A relative
expression ratio (i.e., the fold induction) was derived from the primer efficiency
(Eff) and cycle threshold (CT) values of the target gene in the experimental
condition (CT-experimental) versus control samples (CT-control) according to
TABLE 1. S. mutans strains used in this study
Source or reference
UA159Wild-type strain; Erms
J. Ferretti, University
UA159 ?liaF::erm; Ermr
UA159 ?liaS::erm; Ermr
UA159 ?liaR::erm; Ermr
UA159 ?liaSR::erm; Ermr
UA159 ?liaFSR::erm; Ermr
aErms, erythromycin sensitivity.
2974SUNTHARALINGAM ET AL.J. BACTERIOL.
FIG. 1. Organization and properties of liaFSR-ppiB-pnpB locus genes. (a) Genetic map of S. mutans liaFSR-ppiB-pnpB operon and homologs
in representative streptococci and characterized Firmicutes bacteria. Arrows labeled “P” represent identified or putative promoter regions (“*”
indicates LiaR-like autoregulated promoters), while circles labeled “T” represent putative transcriptional terminators or a weak terminator
(dashed circle) (24, 37, 39, 70). Homologs and orthologs to the liaFSR-ppiB-pnpB genes are shown as black, dark gray, light gray, hatched black
lines, and spotted locus boxes, respectively. Genes flanking the loci of interest are represented as white boxes. For clarity the loci boxes are not
drawn to scale. Abbreviations of bacterial species: Smu, S. mutans strain UA159; Spy, S. pyogenes strain M1 GAS; Spn, S. pneumoniae strain
TIGR4; Lla, L. lactis subsp. cremoris strain MG1363; Sau, S. aureus subsp. aureus strain Mu50; and Bsu, B. subtilis subsp. subtilis strain 168. The
gene names in the presented bacterial species are according to GenBank entries of the published genomes. (b) Conserved domains and (putative)
functions of liaFSR-ppiB-pnpB operon genes.
VOL. 191, 2009S. MUTANS LiaFSR CELL ENVELOPE STRESS RESPONSE SYSTEM 2975
the 2???CTmethod (35). The fold expression change was calculated according to
the method of Pfaffl et al. (50) using the following formula: fold change ?
[Efftarget gene (CT-control ? CT-experimental)]/[Eff16S rRNA (CT-control ? CT-experimental)].
Expression results were normalized relative to the S. mutans internal standard
16S rRNA gene whose expression was invariant under the experimental assay
conditions tested. Relative gene expression was calculated in triplicate from at
least three independent experiments (n ? 3). Statistical analysis was performed
by using Student t test. A gene was considered significantly altered in expression
when P ? 0.05 and expression was up- or down-regulated ?2.0-fold.
Reverse transcription-PCR. To detect polycistronic transcripts, total RNA was
isolated from mid-log-phase S. mutans UA159 cultures, DNase treated, and
converted to cDNA (54). PCR was performed with 700 ng of cDNAs as described
by the manufacturer (MBI Fermentas, Canada). Primers for the amplification of
cDNAs are listed in Table S3 in the supplemental material. Controls without RT
enzyme were included in all experiments. The same reverse transcription-PCR
primers were used for colony PCR as a positive control to directly amplify
chromosomal DNA from single colonies of S. mutans UA159 as described by
Poyart et al. (52). Reverse transcription and colony PCR products were resolved
on a 1.2% agarose gel.
5?-RACE PCR analysis. To detect the S. mutans liaF transcriptional start site
(TSS), 5?-RACE (rapid amplification of cDNA ends) PCR was performed es-
sentially as described by Sambrook and Russell (53) using two primer pairs
(Table S3 in the supplemental material and see Fig. 3a). Total RNA (10 ?g) was
harvested from mid-log-phase (OD600? 0.4) S. mutans UA159 cultures, treated
with RQ1 DNase I (Promega, Madison, WI), and then used to synthesize cDNA
using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and the
RACE outer primer as recommended by the supplier. The double-stranded
cDNA-RNA complex was treated with RNase H and RNase T1(Roche Molec-
ular Biochemicals, Indianapolis, IN), purified, and eluted by using a Qiagen PCR
purification kit to a final volume of 30 ?l. cDNA (12.5 ?l) was used for poly(dG)
and poly(dT) 3?-end tailing with terminal deoxyribonucleotidyltransferase, as
recommended by the manufacturer (Amersham Pharmacia Biotech). Tailed
DNA (1 ?l) was used for PCR amplification with poly(dC) or poly(dA) tail
primer combined with the RACE PCR inner primer to generate a PCR product.
This PCR amplicon was purified and sent for nucleotide sequence analysis
(AGTC Corp., Toronto, Ontario, Canada).
RESULTS AND DISCUSSION
liaFSR forms part of a pentacistronic operon in S. mutans.
The S. mutans SMu485 (liaF)-SMu486 (liaS)-SMu487 (liaR)-
SMu488-SMu489 locus spans 4,282 bp in size (position 454530
to 458812 bp; National Center for Biotechnology Information
[NCBI]). LiaFSR orientation, conserved topological organiza-
tion, domains, and putative functions are shown in Fig. 1.
Although not explored in the present study, the two remaining
genes downstream of liaR (i.e., SMu488 and SMu489), have
been renamed ppiB and pnpB, respectively, reflecting their
presumed enzymatic peptidyl-prolyl cis/trans isomerase and
polynucleotide phosphorylase properties.
To determine whether the liaFSR-ppiB-pnpB genes consti-
tute a pentacistronic operon, we used reverse transcriptase
PCR and further analyzed the nucleotide sequence to identify
potential transcriptional terminators. Sequence analysis sug-
gested a distinct operon encoding a eukaryotic-type serine/
threonine protein kinase (PknB), its phosphatase counterpart
(PppL), and a transcriptional terminator located ?318 bp 5? of
liaF (22). Reverse transcriptase PCR analysis utilizing primers
specific for the pknB and liaF coding regions indicated that liaF
was not transcriptionally linked with the upstream pppL-pknB
operon (data not shown). Eleven base pairs downstream of the
pnpB open reading frame (ORF), a 59-bp inverted repeat
(?G ? ?29.4 kcal/mol) was followed closely by a stretch of 12
A-T-rich nucleotides, indicating good potential for this area to
form a rho-independent transcriptional terminator (14) and
suggesting that liaFSR-ppiB-pnpB is a transcriptionally discrete
locus. Reverse transcription-PCR was again utilized to confirm
the presence of a five-gene operon. Analysis of the lia tran-
scripts using this method indicated that a pentacistronic tran-
script encompassing the liaFSR-ppiB-pnpB genes was gener-
ated (Fig. 2). Moreover, Northern blot experiments using liaF
or liaS gene specific probes further established the presence of
an ?4.3-kb pentacistronic mRNA (data not shown).
To precisely map the promoter region upstream of liaF, the
TSS was determined by using 5? RACE PCR. The ?1 TSS was
identified 95 bp downstream of the NCBI annotated ATG
translational start site (Fig. 3a). No RACE PCR product im-
plicating additional TSSs upstream of the currently identified
?1 start point was obtained. A B. subtilis ?A-type putative ?10
promoter box (TAcAAT) was located 7 bp upstream from the
?1 TSS, and a weakly conserved ?35 promoter motif
(gTGAaA) separated by 17 bp was identified (Fig. 3a). Since
our results were inconsistent with the NCBI annotated liaF
translational start site, we determined the optimal ATG start
codon of liaF ORF to be located 24 bp 3? from the TSS
(position 454653 bp) with a putative ribosomal binding site
(RBS) motif 5 bp 5? from the initiation codon (Fig. 3a). The
identified TSS reduced the predicted size of the liaF ORF by
123 bp. Moreover, the new liaF start codon aligns with the
annotated start codons of some its closest homologs/ortho-
logues in Streptococcus species and B. subtilis liaF.
Recently, Martínez et al. (37) identified a L. lactis CesR
binding motif and showed that streptococcal, lactococcal, and
staphylococcal LiaR/CesR/VraR proteins phylogenetically
cluster together. The CesR motif appears to be conserved in
the genomes of S. aureus and S. pneumoniae and is notably
present upstream of the liaSR homologs in these species (37).
A CesR-like binding motif was also detected upstream of the S.
mutans liaF and other LiaR-regulated genes, although the
pseudo-CesR box is located at positions ?82 to ?62 compared
to ?46/?72 of L. lactis CesR-dependent genes (Fig. 3b) (37).
FIG. 2. Reverse transcriptase and colony PCR products to detect
transcripts from the liaF-pnpB operon. Lanes 1, 4, 7, 10, and 13 rep-
resent colony PCR products of UA159 wild type (positive controls
[“?”]). Lanes 2, 5, 8, 11, and 14 represent reverse transcriptase PCR
products using no reverse transcription (negative control [“–”]) sam-
ples of UA159 cDNA. Lanes 3, 6, 9, 12, and 15 represent reverse
transcriptase PCR products using UA159 cDNA. Regions of the liaF-
pnpB operon detected by the reverse transcriptase PCR primers are
2976 SUNTHARALINGAM ET AL.J. BACTERIOL.
The existence of this potential cis element suggests positive
autoregulation, as with other LiaSR homologs (24, 37, 70).
Growth kinetics under stress. To examine the importance of
the lia gene products in S. mutans’ stress tolerance, we con-
structed isogenic in-frame deletions of the liaF, liaS, and liaR
genes. The mutants were designated SMULiaF, SMULiaS,
and SMULiaR, respectively (Table 1), and used for growth
kinetic measurements to expand upon previous studies that
focused exclusively on liaSR (7, 32, 65). While overnight cul-
tures of UA159, SMULiaS, and SMULiaR strains grew as
uniformly buoyant and turbid solutions, SMULiaF cells con-
sistently aggregated and settled at the bottom of the test tube
(data not shown). Although the liaS and liaR mutants dis-
played similar growth rates relative to wild type in THYE at
pH 7.5, the growth rate and yield of SMULiaF was markedly
impaired in this medium, as well as under a number of other
FIG. 3. TSS and sequence analysis of liaF gene. (a) DNA sequence surrounding the NCBI predicted LiaF translational start codon (highlighted
in black). The identified TSS is marked (?1 and underlined) and the putative ?10 and ?35 promoter motifs are boxed. Predicted RBS and
translational initiation codon are in bold and highlighted in gray. It should be noted that there is no typical RBS motif located upstream of the
NCBI annotated translational start codon. Lowercase nucleotides are mismatches to established RBS and B. subtilis ?A-type promoter boxes
consensus sequences (11, 42). 5? RACE-PCR primers used for mapping the TSS are represented by arrows. (b) Identification of the L. lactis
CesR-like IR binding motifs (boxed) (37) in the putative promoter regions of S. mutans strain UA159 liaF, spxA, SMu1727, and SMu751 genes
together with liaF homologs in L. lactis subsp. cremoris strain MG1363 (llmg1650), S. pyogenes strain MGAS315 (spyM3_1368), and S. pneumoniae
strain TIGR4 (sp0385). The position of CesR-like binding motif relative to (putative) promoter regions and the nucleotide similarity to CesR
consensus are shown. Conserved residues are in boldface; mismatches to consensus are in lowercase. Also shown is the putative ?10 promoter
(double underlined) or ?35 promoter motifs (single underlined). H ? A?T?C; D ? A?T?G. Bioinformatic sequence analysis was carried out
using the MacVector 7.2 software (Oxford Molecular).*, Although no CesR-like binding motif was detected immediately preceding SMu753 gene,
a CesR sequence was detected upstream of the (presumably cotranscribed) SMu751 gene.
VOL. 191, 2009S. MUTANS LiaFSR CELL ENVELOPE STRESS RESPONSE SYSTEM2977
stressors, including acidity at pH 5.5, 5% ethanol and 0.4 M
sodium chloride (Fig. 4a, growth yield data not shown). Inter-
estingly, under H2O2-induced oxidative stress, the growth of all
lia mutants was enhanced relative to the wild-type UA159
strain, as judged by the decreased lag time (Fig. 4b).
Since SMULiaR did not share the same level of sensitivity to
acid, ethanol, and high osmolarity stressors as that seen with
SMULiaS, the possibility of in vivo cross talk between LiaS and
other noncognate RR(s) or between LiaR and other phospho-
donors is likely (63). Li et al. (32) and Chong et al. (12) have
previously suggested the possibility of cross talk between the
LiaS sensor with one or more RRs by comparing phenotypic
variations (e.g., acidic pH resistance and bacteriocin mutacin
IV production) between the liaS and liaR mutants.
liaFSR mutants are sensitive to cell wall lipid II inhibitors
and cell membrane-disrupting agents. S. mutans LiaSR ho-
mologs in L. lactis (CesSR), S. aureus (VraSR), and B. subtilis
(LiaRS), respond to stress elicited by cell envelope interfering
antibiotics, primarily lipid II-interacting inhibitors such as van-
comycin and bacitracin (18, 37, 39). To determine whether the
S. mutans LiaSR system is part of the cell envelope stress
response, the tolerance of wild-type UA159 and liaFSR mu-
tants to cell envelope biosynthesis inhibitors was assessed by a
microtiter plate-based MIC and MBC assays. Table 2 displays
antibiotic susceptibility profiles of UA159 parent and liaF, liaS,
and liaR mutants tested with a panel of eight cell envelope
inhibitors, each active at different steps in PG biosynthesis.
Similar to antibiotic resistance studies of L. lactis cesR and S.
aureus vraSR mutants (18, 29, 37), S. mutans liaFSR-deficient
mutants were approximately two- to eightfold more sensitive
than the parent strain UA159 to vancomycin and bacitracin,
antibiotics that interfere with Lipid II recycling. These mutants
also had at least a two- to fourfold reduction in MICs and
MBCs relative to UA159 when exposed to compounds that
compromise the integrity of the cell membrane (i.e., the pore-
forming chlorhexidine and the detergent SDS). On the other
hand, the lia mutants were not sensitive to nisin, a lantibiotic
that both disrupts bacterial membranes through pore forma-
FIG. 4. Growth rates and lag times of S. mutans strains exposed to stress conditions. (a) Growth rates of S. mutans strains exposed to specific
stressors are presented as doubling times (min). Stressors: THYE medium supplemented with neutral (pH 7.5), acid (pH 5.5), 5% (vol/vol) ethanol
(EtOH), and 0.4 M NaCl. (b) Lag times for S. mutans strains to reach an OD600of ?0.1 when exposed to 0.006% (vol/vol) hydrogen peroxide
(H2O2)-mediated oxidative stress. The results were obtained from three independent experiments conducted with replicates of five for each strain.
Significant differences in growth rates or lag times compared to UA159 parent are indicated as calculated by using the Student t test (***, P ?
0.001;**, P ? 0.01;*, P ? 0.05). Error bars represent the standard errors.
TABLE 2. Susceptibility profiles of S. mutans strains exposed to cell envelope-interfering antimicrobialsa
Lipid II inhibitor Non-lipid II inhibitorCell membrane-disrupting agents
VANBACNIS DCSPENG OXA % SDS
MICMBC MIC MBC MIC MBCMICMBC MICMBC MIC MBCMIC MBCMIC MBC
aVAN, vancomycin; DCS, D-cycloserine; BAC, bacitracin; NIS, nisin; CHX, chlorhexidine; PENG, penicillin G; OXA, oxacillin. *, Reductions (2-fold ?*? or ?4-fold
?**?) in MIC/MBC compared to UA159 parent are indicated. All concentrations are in micrograms per milliliter.
2978 SUNTHARALINGAM ET AL.J. BACTERIOL.
tion and blocks the lipid II moiety of PG biosynthesis (67). It
has been shown that exposure of an L. lactis cesR mutant to
nisin resulted in a twofold decrease in the 50% lethal dose
relative to its parent strain (37). In L. lactis, the organism that
synthesizes nisin, the CesSR system may well be part of the
self-protection resistance mechanism to this lantibiotic.
MIC/MBC assays conducted with non-lipid II interacting
?-lactams (penicillin G and oxacillin) that prevent the later
transpeptidation step of PG biosynthesis, as well as
cycloserine, which competitively inhibits the completion of the
PG subunit pentapeptide side chain, revealed no effect on the
sensitivities of lia mutants relative to parent (Table 2) (10).
Unlike S. mutans LiaSR, resistance to these antibiotics in S.
aureus partly involves the homologous VraSR system (8, 18,
29). Indeed, oxacillin showed drastic (at most 64-fold) reduc-
tion in the MICs of a vraSR mutant compared to the parent
methicillin-resistant S. aureus strain (8).
Similar to results from growth kinetics under environmen-
tal stress, the antibiotic-mediated stress response of isogenic
liaF and liaR mutants exhibited enhanced sensitivity to van-
comycin, bacitracin, and chlorhexidine that was noticeably
different from that of the liaS HK mutant. One explanation
for the greater liaR deficiency relative to the liaS mutant
strain may be that the LiaR signaling cascade is activated
independently of phosphorylation by its cognate LiaS sensor
(64). In fact, the LiaR homolog in S. aureus (VraR) is
capable of undergoing in vitro phosphorylation by acetyl
phosphate, although the rate is much slower than direct
phosphorylation by it cognate sensor VraS (6). Another
route of LiaR phosphorylation could result from cross talk
with other sensor HK(s) such as CiaH or VicK, both of
which are implicated in cell envelope stress tolerance (7).
Biswas et al. (7) recently demonstrated tolerance of S. mu-
tans liaS mutants to vancomycin, bacitracin, and other ?-lac-
tam cell envelope antibiotics. Although our findings pertaining
to bacitracin and ?-lactam susceptibility of the liaS mutant
agreed with those of Biswas et al. (7), lia mutants exposed to
vancomycin in our study displayed at least a twofold decrease
in MBC sensitivity relative to the wild type. This inconsistency
may be due to the disk diffusion assay with measurement of
zones of inhibition used by the former group (7) compared to
our microdilution method to establish MIC/MBC and the
growth stage of tested strains. In addition, it should be noted
that disparities between disk diffusion methodology and the
determination of MIC have been reported in the clinical liter-
ature for several antibiotics (28, 41).
LiaR responds to lipid II-interfering and cell membrane-
perturbing antimicrobials. Since liaFSR mutant strains exhib-
ited increased susceptibility to lipid II-interfering antibiotics,
the role of these inhibitors in inducing transcription of the lia
operon was investigated by qRT-PCR. Accordingly, liaR ex-
pression was examined using mid-log-phase UA159 cultures
treated with or without inhibitory and subinhibitory concen-
trations of several cell envelope antimicrobials for 10 min. Of
the antimicrobial agents tested, vancomycin, bacitracin, nisin,
and chlorhexidine acted as liaR inducers in a concentration-
dependent manner (Fig. 5). All of these compounds interfere
with the lipid II-cycle except chlorhexidine (a cell membrane
pore-forming antiseptic) whose molecular effect on the lipid II
cycle is currently unknown. While the expression levels of liaR
remained elevated at high concentrations of vancomycin and
nisin, its transcription was decreased down to slightly induced
levels (?1.7-fold) or repressed levels (?0.5-fold) under the
highest concentrations of bacitracin and chlorhexidine (Fig. 5).
Since exposure of UA159 cells to the latter compounds present
at 10? the MIC, revealed only 2 and 12% decreases in cell
counts, respectively (data not shown), it is likely that the re-
duction in liaR transcripts was due to an active mechanism
rather than biocide-mediated cell death.
Exposure to subinhibitory and inhibitory concentrations of
bacitracin for up to 60 min suggested that peak induction of
liaR expression occurred between 10 and 30 min after bacitra-
cin treatment (Fig. 5). This result was consistent with the
temporal response of liaSR homologs in L. lactis and S. aureus
exposed to related lipid II cycle inhibitors (29, 37). Other cell
envelope antimicrobials, such as the ?-lactams (penicillin G
and oxacillin) or D-cycloserine, were unable to significantly
stimulate liaR expression in the concentration ranges of 0.5?
to 10? the MIC, respectively (data not shown). Similar lipid II
inhibitor concentration-dependent induction profiles were il-
lustrated in the homologous B. subtilis LiaRS and L. lactis
CesSR systems (37, 39). Relative to other inducers, bacitracin
was utilized in further qRT-PCR experiments due to its high
induction potential at subinhibitory concentrations. In addi-
tion, UA159 cells treated with different concentrations of bac-
itracin displayed nearly equivalent expression levels for the
liaF and liaS genes as liaR, supporting coinduction and cotrans-
cription of all lia genes (data not shown).
liaFSR transcription is growth phase-dependent. In B. sub-
tilis, the LiaR-dependent liaIHGFSR promoter (PliaI) was
shown to be induced at the onset of the stationary phase in the
absence of an external stress stimulus and appeared to be
tightly controlled by at least five regulators involved in the
transition into stationary phase (25), a growth state typified by
a nutrient-depleted and hostile environment (43). To investi-
gate whether S. mutans LiaSR is also induced by the transition
into stationary-phase liaF, liaS, and liaR expression levels at
the mid-log and stationary growth phases were compared to
that of early-log-growth-state cells. Notably, lia gene expres-
sion levels were repressed at least 10-fold in stationary phase
(liaF ? 0.1 ? 0.02, liaS ? 0.1 ? 0.03, and liaR ? 0.1 ? 0.02)
and 2-fold in mid-log phase (liaF ? 0.5 ? 0.02, liaS ? 0.5 ?
0.01, and liaR ? 0.4 ? 0.001) relative to early-log growth state
(mean fold increase ? the standard error; n ? 3). While these
data suggested growth phase-dependent regulation of the lia
genes, they also emphasized the involvement of LiaSR in early-
log-phase growth marked by high growth rates, as well as
increased cell division, cell separation, and PG biosynthesis.
LiaFSR positively regulates cell envelope biogenesis, chap-
erone/proteases, and transcription factors. B. subtilis LiaRS
TCSTS is genetically and functionally linked to the upstream
liaF gene product to constitute a three-component signaling
system (24). We envisioned a similar role with S. mutans Lia-
FSR, since this gene cluster is conserved, coexpressed, and
transcriptionally inducible by envelope-specific antimicrobials,
resembling the B. subtilis LiaFRS model. To elucidate the
underlying regulon components involved in S. mutans LiaFSR-
mediated cell envelope stress tolerance, cDNAs derived from
UA159 and SMULiaFSR cells were used to study the expres-
sion levels of several genes encoding the following products:
VOL. 191, 2009S. MUTANS LiaFSR CELL ENVELOPE STRESS RESPONSE SYSTEM2979
membrane-targeted or secreted proteins; PG biosynthesis, re-
modeling, and modification enzymes; cell envelope chaperone/
proteases; and transcriptional attenuators. The functions of
these selected genes and their fold expression in UA159 and
SMULiaFSR cells are listed in Table 3. The target genes were
based either on previously identified LiaR-regulated genes
(49) or on homologs and/or orthologs to S. aureus VraR or L.
lactis CesR-upregulated genes stimulated by cell envelope in-
hibitors (29, 37). Moreover, putative L. lactis CesR-like bind-
ing motifs were detected 5? proximal to most of the predicted
TSSs of these genes (Fig. 3b and data not shown for other
genes), except for htrA and ftsH, where the promoter boxes or
pseudo-CesR binding sequence were difficult to define.
Treatment of UA159 cultures with 0.5? the MIC bacitracin
concentration increased all target gene expression by 2- to
10-fold relative to control cultures, whereas expression re-
mained uninduced in the liaFSR-deficient mutants (Table 3).
The result above confirmed LiaFSR-dependent upregulation
of the tested genes, suggesting a multifaceted response by this
system to bacitracin inhibition of PG synthesis. The induction
of murB, dagK, and SMu707c genes can be interpreted as an
attempt by S. mutans cells to boost the rate of PG synthesis and
murein remodeling to restore stress-induced damaged or miss-
ing cell wall material. In S. aureus, Utaida et al. (59) reported
that bacitracin-challenged cells induced pbpB, sgtB, murA, and
bacA gene expression to increase the rate of PG synthesis. Our
observed induction of rgpG may also suggest increased levels of
cell wall polysaccharide modification and resistance to bacitra-
cin. Tsuda et al. (57) had previously shown that the presence of
rhamnose-glucose polysaccharide in the cell wall confers resis-
tance to bacitracin by S. mutans cells, although the precise
mechanism is unknown.
An elevated expression level of the envelope chaperone/
proteases ftsH and htrA implies that damaged, misfolded, or
aggregated proteins accumulate in the cell envelope, triggering
the activation of genes encoding these compensatory proteins.
In fact, several genes belonging to the cell envelope protein
biogenesis/repair, chaperonin, or protease functional catego-
ries (e.g., htrA, ftsH, prsA, mrsA1, and hslO) were noted to be
upregulated in S. aureus, S. pneumoniae, and L. lactis exposed
to cell-wall-active antibiotics (19, 37, 47, 59). Pechous et al.
(47) have proposed that, in S. aureus, the inhibition of PG
synthesis by cell wall antibiotics interferes with the incorpora-
tion of integral proteins covalently attached to PG, which are
linked to PG at the lipid II stage (48). These authors specu-
lated that the resulting accumulation of unincorporated cell
wall proteins in the cell membrane and the subsequent disrup-
tion of normal envelope functions and protein translocation
may lead to the activation of repair, chaperonin, and protease
genes (47). A similar process could be operating in S. mutans
cells exposed to bacitracin stress with the induction of htrA and
Likewise, the upregulation of trigger factor (ropA), ftsY, and
SMu1727 (oxaA-like) genes involved in the biogenesis, trans-
FIG. 5. Fold expression of liaR gene expression in S. mutans UA159 wild-type strain exposed to cell envelope biosynthesis inhibitors.
Concentration-dependent induction of liaR expression exposed to cell envelope inhibitors for 10, 30, and 60 min. The inhibitor and concentrations
utilized (in ? the MIC) are shown on the x axis. VAN, vancomycin; BAC, bacitracin; NIS, nisin; and CHX, chlorhexidine. The results were from
at least three independent experiments, and error bars represent the standard errors.
2980SUNTHARALINGAM ET AL. J. BACTERIOL.
location, and correct insertion of integral membrane or se-
creted proteins into the cell membrane may be viewed as an
attempt by S. mutans cells to maintain the integrity of mem-
brane-associated or secreted polypeptides. This could be ac-
complished by replacing damaged or denatured peptides
and/or increasing the synthesis and deployment of envelope
stress-related repair enzymes, chaperones, and proteases. In S.
mutans, both FtsY and trigger factor have been demonstrated
to play important roles in stress response such as acid and high
salt-osmolarity tolerance (20, 66). Martínez et al. (37) also
established a link between envelope integrity and the L. lactis
CesR-mediated induction of membrane protein biogenesis or
protein secretion genes (e.g., ppiB and oxaA2) in their study of
lactococcal cells exposed to a lipid II inhibitor.
Lastly, the transcriptional modulators SMu753 and SpxA
(SMu2084c) are highly induced by the LiaFSR system, and
homologs/orthologs of these genes play a role in envelope
stress tolerance in other bacteria (Table 3). SMu753 encodes a
TABLE 3. qrt-PCR expression ratios and function of selected envelope-related genes in S. mutans strains
Mean qRT-PCR fold
expression ? SEa
Synthesis of membrane/
Trigger factor (ropA) 1.7 ? 0.10.8 ? 0.01Ribosome-associated peptidyl-prolyl cis/trans
isomerase (PPIase) foldase with chaperone
and acid stress resistance functions
Membrane-associated receptor component of
the signal recognition particle system
responsible for targeting nascent peptides to
the cell membrane
OxaA-like precursor protein required for the
insertion of integral membrane proteins into
ftsY 2.1 ? 0.2 1.0 ? 0.1 20, 44
SMu1727 3.2 ? 0.40.7 ? 0.1pFam02096
Cell wall PG biosynthesis,
murB2.4 ? 0.20.8 ? 0.03 Essential UDP-N-acetylenolpyruvoylglucosamine
reductase enzyme involved in the second step
of the “sugar building block” necessary for
Diacylglycerol kinase catalyzes the formation of
phophatidic acid by phosphorylating
diacylglycerol; also involved in stress
resistance and presumed to have C55-isoprenyl
kinase activity involved in PG synthesis
Endolysin containing a ?-1,4-N-
acetylmuramidase domain, possibly involved
in diverse cell wall functions, including PG
restructuring and turnover, cell separation,
Involved in the first step of S. mutans cell wall
rhamnose-glucose polysaccharide synthesis by
to a lipid carrier
dagK (dgk) 3.3 ? 0.30.9 ? 0.1
SMu707c 2.3 ? 0.2 1.3 ? 0.1 62
rgpG 2.0 ? 0.1 0.9 ? 0.0358, 68
Cell envelope chaperone/
ftsH 2.5 ? 0.040.9 ? 0.1Universally conserved membrane bound
metalloprotease and chaperone; in E. coli,
FtsH maintains inner membrane stability by
processing/degrading specific membrane
proteins and transcription factors
Highly conserved cell wall-associated serine
protease and chaperone; S. mutans HtrA is
involved in stress resistance, maturation of
extracellular and surface attached proteins,
biofilm formation, and genetic competence
htrA (degP) 2.9 ? 0.6 0.8 ? 0.003 2, 16, 27
Transcriptional regulatorsSMu753 10.6 ? 1.4 0.5 ? 0.1 Hypothetical membrane protein that contains
E. coli phage shock protein C (PspC) domain;
E. coli PspC is a transcriptional regulator via
protein-protein interactions with other phage
Conserved regulator in gram-positive bacteria
that interacts with RNA polymerase
haloenzyme; in B. subtilis, Spx is a regulator
of the disulfide stress response to alleviate
damage caused by thiol oxidation
spxA (SMu2084c) 6.8 ? 0.90.7 ? 0.1 45
aqRT-PCR expression ratios of selected genes in mid-log-phase S. mutans UA159 parent and SMULiaFSR strains, treated for 10 min with 0.5? the MIC of
bacitracin versus untreated control cells. Results were obtained from three independent experiments.
VOL. 191, 2009S. MUTANS LiaFSR CELL ENVELOPE STRESS RESPONSE SYSTEM 2981
membrane protein with a conserved phage shock protein C
(PspC) domain, a major component of the E. coli PspABC
DEF envelope stress response system (13). The Psp system is
induced in response to ethanol, heat, osmotic shock, and bac-
teriophage infection, with PspC playing a role in modulating
expression of its own phage shock operon and other stress
resistance genes (1, 13). Whether the SMu753 gene product
has a similar function in S. mutans remains to be determined.
The transcriptional factor SpxA is orthologous to the L. lactis
SpxB and B. subtilis Spx regulators, both of which are inducible
by cell-wall-active inhibitors and involved in envelope stress
tolerance (17, 61). Although the S. mutans spxA gene is clearly
LiaFSR dependent, its role in the resistance to envelope stress
warrants further investigation.
LiaF is integral to and represses LiaSR-mediated signal
transduction. The B. subtilis transmembrane protein LiaF, part
of the LiaFRS three-component signaling system, acts as a
potent inhibitor of LiaR-dependent gene expression in the
absence of inducing conditions (24). To determine whether the
S. mutans LiaF played a similar role on LiaSR signal transduc-
tion, we studied the role of each lia component in regulating
LiaSR-dependent spxA and SMu1727 transcription under bac-
itracin-induced and control conditions. While the absence of
either liaR or liaS resulted in a 2- to 10-fold decrease in spxA
and SMu1727 expression under inducing conditions, deficiency
in liaF drastically increased their transcription with or without
an inducer (Fig. 6). Specifically in strain SMULiaF, spxA and
SMu1727 expression was upregulated 2.5- and 5.5-fold, respec-
tively, under inducing conditions, as well as 8- and 16-fold
under noninducing conditions (Fig. 6). These results suggest a
functional role for LiaF in repressing LiaSR-dependent induc-
tion of spxA and SMu1727 in the absence of an inducer. Since
a lower inhibitory effect is apparent under inducing conditions,
it is likely that the presence of bacitracin alleviated LiaF re-
pression of spxA and SMu1727 by enhancing the transcription
Under uninduced conditions, the lack of liaF caused 13- and
15-fold increases in liaR and liaS expression relative to wild-
type levels, respectively (data not shown). Since antibiotic re-
sistance cassettes (e.g., erythromycin resistance [Ermr]) in-
serted into the chromosome of insertion-deletion mutant
strains can exhibit downstream polar effects, we measured
the downstream gene expression in strains SMULiaS and
SMULiaR versus the wild type. In SMULiaR, ppiB transcrip-
tion was increased by nearly 3.4-fold, whereas strain SMULiaS
showed approximate 3.7- and 3.6-fold increases in liaR and
ppiB expression compared to UA159 parent (data not shown).
Notably this ?4-fold increase in expression of downstream
genes (due to insertion of the Ermrcassette) is still drastically
lower than the 13- to 15-fold increase of liaS and liaR expres-
sion in the liaF mutant, suggesting a high likelihood for liaF-
mediated repression of liaSR. Although the above results may
not directly demonstrate LiaF’s negative effect on liaSR tran-
scription, more studies are warranted to examine its precise
interaction with LiaS and/or LiaR components to confer re-
pression of liaSR transcription. Interestingly, both an overex-
pression of liaSR (as possibly witnessed in a liaF-deletion
strain) and diminished liaR expression (as observed in a liaR
mutant) exhibited growth defects in the presence of a number
of environmental stressors, including envelope-specific antimi-
crobials. Hence, we speculate that S. mutans liaFSR transcrip-
tion is tightly regulated and expresses its locus and regulon
genes at the optimal time and transcript level to secure enve-
Conclusion. Sensing and responding to physical and chem-
ical threats that may compromise the functional integrity of the
bacterial cell envelope are vital to the survival of a bacterium.
The present study describes a role for the LiaFSR system in
responding to cell envelope stress in S. mutans. Although this
system is involved in tolerance to a variety of stressors includ-
ing pH, NaCl, SDS, H2O2, etc., the LiaFSR appears to be a cell
envelope damage-sensing system rather than a direct stress-
sensing mechanism. In the former case, it is possible that these
environmental threats could have deleterious effects on enve-
FIG. 6. Fold expression of spxA and SMu1727 genes in S. mutans lia mutant strains normalized to wild-type UA159 response under 0.5? the
MIC bacitracin inducing (a) and noninducing (b) control conditions. In panels a and b, the fold expression ratios of spxA and SMu1727 genes in
UA159 are set at 1.0 under both inducing and control conditions. The results were obtained from three independent experiments, and error bars
represent standard error.
2982 SUNTHARALINGAM ET AL.J. BACTERIOL.
lope macromolecular structures (55), compromising envelope
integrity and activating the LiaFSR system, as well as regulon
genes involved in combating stress-related damage. While Lia-
FSR in S. mutans responded to a variety of cell envelope active
antimicrobials (e.g., bacitracin, vancomycin, nisin, and chlo-
rhexidine), we also showed its regulatory role in inducing genes
encoding PG synthesis and remodeling enzymes in addition to
membrane protein synthesis and envelope chaperone/pro-
teases, a finding consistent with a pathway that responds to PG
or envelope protein damage. Moreover, our study has high-
lighted similarities (e.g., lipid II inhibitors as inducers) and
major differences (e.g., growth phase regulation, mutant phe-
notypes displayed, and regulon genes) between the S. mutans
LiaFSR and its analogous system in B. subtilis. Overall, this
variance in the S. mutans LiaFSR attributes may reflect its
adaptation to its particular niche in the oral cavity, compared
to the soil-inhabiting B. subtilis. The results from the present
study enhance our understanding of how S. mutans can sense
and respond to environmental threats that cause cell envelope
stress. Such progress in elucidating the components underlying
mechanism can eventually lead to the design and synthesis of
drug targets to efficiently control bacterial pathogenesis.
This study was supported by NIH grant 5R01DE013230-08 and
CIHR grant MT-15431 to D.G.C. D.G.C. is a recipient of a Canada
Research Chair position. P.S. is a CIHR Strategic Training Fellow
supported by training grant STP-53877 and a Harron Scholar.
We thank Elena Voronejskaia for technical assistance with the
Northern blot analysis.
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