The LiaFSR system regulates the cell envelope stress response in Streptococcus mutans.
ABSTRACT 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.
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ABSTRACT: Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/ homeostasis and affects stress responses and biofilm formation by S. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically as compared to the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. RealTime-PCR and Western blot analysis revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by S. mutans. TEM analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant, TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in S. mutans.Microbiology 01/2013; · 3.06 Impact Factor
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ABSTRACT: Spx of Bacillus subtilis is a redox-sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram-positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol-specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease-resistant products were used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD-expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT-qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.MicrobiologyOpen. 07/2013;
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ABSTRACT: The genetic bases for antibiotic tolerance are obscure. Daptomycin (DAP) is a lipopeptide antibiotic with bactericidal activity against enterococci. Using time-kill assays, we provide evidence for the first time that a deletion of isoleucine in position 177 of LiaF, a member of the three-component regulatory system LiaFSR involved in the cell-envelope response to antimicrobials, is directly responsible for a DAP-tolerant phenotype and is likely to negatively affect response to DAP therapy.Antimicrobial Agents and Chemotherapy 03/2013; · 4.57 Impact Factor
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
UA159 Wild-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 SYSTEM2975
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
2976SUNTHARALINGAM 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 SYSTEM 2977