Advances in Microbiology, 2012, 2, 208-215
http://dx.doi.org/10.4236/aim.2012.23025 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Biofilm Foptococcus mutans and
2,
icultural Science,
ai, Japan
2Laboratory ofnce, Tohoku University, Sendai, Japan
3Department of Oral Surgery, Health Sciences University of Hokkaido, Hokkaido, Japan
h; it is an infectious
sease of the oral cavity in which biofilms play a causative role. Control of biofilms has traditionally relied on
lt to remove biofilms by
sobrinus are important
causative a. They produce a homologous exocellular polysaccharide called glucan, which strongly ad-
amel surface. This is a review of oral microbial biofilm formation by S. mutans and other related bacte-
to can;
c
nd
cc
ica
of
for
t
i
S.
re highly associated with caries in hu
o f
ent
ur
at are toxic to most other bacter
s are exemplary and serve as a m
system for bacterial adhesion [3]. In this review, we
describe biofilm formation by S. mutans and related
bacteria.
1.1. Dental Plaque and Decalcification in Caries
Dental plaque is caused by a complex and dynamic proc-
esses that involves the progressive destruction of tooth
cteria. Acid forma-
mutans drives the
n the hydroxyapa-
by acid from the
ctors including the
concentration of the carbon source, the amount and ac-
e microflora, the flow rate of saliva,
he tooth surface. The major species
mutans and S. so-
eral mutans strep-
]. The mutans
l agents in dental
The etiology of caries disease is well established and
bacterial colonization appears to be an important step for
oral diseases, leading to biofilm formation [6-8]. Oral
biofilms mostly consist of multiple bacterial strains. It
has been recently shown that more than 700 bacterial
strains are present in dental plaque [9]. Specific patho-
gens, including Gram-negative bacteria, can cause seri-
ous clinical diseases. The microflora in healthy individu-
als is dominated by the presence of Gram-positive bacte-
*Corresponding author.
rmation by Stre
Related Bacteria
Junko Nishimura1, Tadao Saito1, Hiroshi Yoneyama2, Lan Lan Bai
Kazuhiko Okumura3, Emiko Isogai2*
1Laboratory of raduate School of AgrAnimal Products Chemistry, G
Tohoku University, Send
Animal Microbiology, Graduate School of Agricultural Scie
Received April 22, 2012; revised May 29, 2012; accepted June 8, 2012
ABSTRACT
Caries is a disease of human dentition characterized by the loss of mineralized surfaces of the toot
di
non-specific removal of plaque by mechanical means such as brushing, although it is difficu
this method. Caries is also a widespread infection in children. Streptococcus mutans and S.
gents of caries
heres to the en
ria.
Keywords: Caries; Streptococcus mutans; Strep
Glucosyltransferase
1. Introduction
Caries has been characterized as an ecological
in the mouth, involving infectious bacteria a
available sugar in drinks and foods. Streptoco
tans has been reported as the principal etiolog
of dental cavities and a normal inhabitant
plaque [1,2]. Oral microbial biofilms cause to
plex bacterial communities based on co-agglu
Specific important pathogens in the oral biofilm
mutans streptococci, particularly S. mutans and
nus, which a
coccus sobrinus; Biofilm; Exocellular Polysaccharide; Glu
ollision
readily
us mu-
l agent
dental
soluble polymers of glucose that aid in persist
zation of solid surfaces. These bacteria can s
enamel, dentine and cementum by ba
tion by cariogenic bacteria such as S.
dissolution of calcium and phosphate i
tite crystal structure. Decalcification
bacteria is dependent on a variety of fa
m com-tivity of the plaqu
and the nature of t
ination.
nclude
sobri-
mans. S.
orm in-
coloni-
vive at
ial spe-
odel
found in human dental plaque are S.
brinus, S. rattus, S. cricetus and sev
tococci are cariogenic in animals [4,5
streptococci are important etiologica
caries.
1.2. Role of Biofilms in Caries
mutans readily metabolizes dietary sucrose t
low pH values th
cies. Oral biofilm
C
opyright © 2012 SciRes. AiM

J. NISHIMURA ET AL. 209
ria, whereas patients with periodontitis have in
Gram-negative anaerobic rod-shaped bacteria [1
is communication between the multispecies in
and the fo
creased
. There
iofilms
tic and
or treat
flora is
bed, the
the de-
intake
within
. In ad-
cario-
acterial
e, non-
plaque
ces that
ergistic effect of oral bacteria in dental plaque t
root cause of dental caries.
(Figure 1). Cariogenic bacteria such a
and grow in the mouths of caries-ac
the low resting pH in the plaque env
vival in acidic environments depends
mutans and other acid-tolerant oral b
intracellular pH homeostasis using H
end product efflux (Figure 1). Caries
or below pH 4. Bowden and Hamilton
acid tolerance response: 1) increased
provides proton-efflux; 2) the shift t
comes optimum for glucose transport, t
way and proton impermeability; 3) de
the acid-sensitive, membrane-associ
enzyme IIs for the phosphotransfera
system is accompanied by an increas
tolerant, non-phosphotransferase transpo
creased specific activit
0]
b
rmation of biofilm can lead to antibio
nt
to
ate
s
nt
ith
b
c
ic
an
a
er an
ic
hich leads indirectly to the
ents
also
ot plaque, and it is actually t
ha
1.3. Metabolism by the Acidogenic Microflora
and Acid Tolerance
Lactic acid is generated in the presence of high sugar
concentrations, while low sugar concentrations lead to
the production of acetic acid, formic acid and ethanol
s S. mutans survive
tive patients due to
ironment [11]. Sur-
on the ability of S.
acteria to maintain
+/ATPase and acid
lesions can be near
[12] explained the
glycolytic activity
o a lower pH be-
he glycolytic path-
creased activity by
ated, sugar-specific
se sugar transport
e in the more acid
rt activity; 4) in-
y of the H+/ATPase responsible for
ower pH environments; 5)
ower values; and 6) a
tive metabolism as
.
lly, but not aerobi-
ng no amino acids
urce [13-15]. It is
suggested that this organism possesses glutamate dehy-
drogenase (GDH), glutamine synthetase (GS) and gluta-
mine oxoglutarate aminotransferase (glutamate synthase)
(GOGAT) to assimilate ammonia [15,16]. The gluta-
mate synthetic pathway that S. mu tans uses under an-
aerobic conditions is illustrated in Figure 2. This
oxygen resistance, resulting in failure to preve
oral diseases.
Normally, the composition of human oral micro
in a dynamic balance. When this balance is distur
normal oral flora becomes disordered, leading
velopment of dental plaque. High carbohydr
causes the accumulation of acidic metabolite
mature plaque, leading to a low pH environme
dition, the number of acid-tolerant bacteria w
genic potential will increase significantly or
virulence will be enhanced. In this circumstan
cariogenic plaque will change into cariogen
producing a large number of pathogenic subst
can cause the development of dental caries. In p
S. sanguinis and S. oralis are maintained und
logical balance. S. sanguinis decreases after m
logical imbalance, w
rticular, proton efflux from cells in l
eco-
ro-eco-
devel-
of acid
include
he syn-
t is the
increased capacity to maintain pH at l
shift to predominately homofermenta
indicated by increased lactate formation
1.4. Glutamate Biosynthesis by S. mutans
S. mutans can be cultured anaerobica
cally, on minimal medium containi
with ammonia as the sole nitrogen so
opment of dental caries. The causative ag
production are not limited to S. mutans, but
her bacteria in dental
Figure 1. Sugar metabolism and acid formation in cariogenic bacteria.

J. NISHIMURA ET AL. 210
Gluc os e
Phosphoenolpyr uv ate
Pyruvate Lactate
Acetyl- CoA
Oxaloacetate Citrate
Isocitrate
LDH
-Ketoglutarate Glu
Gl
t amate
utamine
PFL
PEPC
CS
ACN
ICDH GDH
GOGAT GS
Formate
NH
NH
3
3
mutans.
oxylase;
genase;
rate de-
gluta-
te ami-
trate in
onitase
der an-
ic con-
lutarate
activity
ogenic-
duricity
]. Aci-
number
at glu-
low pH
nt defi-
tamine
he par-
e trans-
lerance
1.6. Glucosyltransferase (GTF) a
Formation in S. mutans
S. mutans synthesizes EPS (glucans)
residues of sucrose by secreting g
(GTFs). It is well known that S. muta
GTFs (GTF-B, C, and D) (Table 1). G
mainly synthesize water-insoluble α
ter-soluble α-(1→6)-glucan. GTF-C
soluble and soluble glucan synthesis, w
by the genes gtfB, gtfC, and gtfD [21,
and GTF-S are consistent in these genes
between the genetics and the propertie
D has been shown by various resea
their reports, GTF-B, encoded by the
tans GS-5, was a strongly hydrophilic
1475 amino acids with a molecular
GTF-C was composed of 1375 am
(approx. 153 kDa) and was general
three small hydrophobic domains. Th
bp) was located immediately downs
gene. A third enzyme, GTF-D, was fo
lecular mass of 155 kDa and its kinetic
identical to the GTF-S enzyme. These
sucrase (GS), produced by Leucono
have high homology [26,27]. GTFs a
functional domains: an N-terminal glu
and a C-terminal glucan-binding do
N-terminal domain is noted as VR (v
the properties of the signal peptide a
according to changes in its compositi
The N-terminal domain catalyzes the tran
reaction using sucrose as a substrate,
molecular mass acceptors [29,30]. On
C-terminal domain of the GTFs binds
[29,31]. It seems that this domain pla
Figure 2. The glutamate synthesis pathway in
Abbreviations: PEPC, phoS. sphoenolpyruvate carb
ro
it
ara
α-ketoglutarate, a subs
ac
n
ob
tog
ir
id
19
ans has been demonstrated in a
suggested th
survival in
ta
lu
t
mat
to
1.5. Exocellular Polysaccharides (EPSs)
S. mutans is regarded as the main offending bacteria and
producer of exocellular polysaccharides (EPSs) called
glucans. EPSs are important in the first stage of caries de-
velopment because they promote co-aggregation and colo-
nization of cariogenic bacteria in biofilms.
Many Streptococcus strains can synthesize EPSs and
they are generally produced as adhesins. S. thermophilus
in the food manu-
ogurt starter.
mopolysaccharides
o whether they are
f sugar. S. mutans
produces glucan, a type of homopolysaccharide, while
esized by S. thermophilus
nd Glucan
from the glucosyl
lucosyltransferases
ns has at least three
TF-B and GTF-D
-(1→3)- and wa-
associates with in-
hich is controlled
22]. GTF-I, GTF-SI,
. The correlation
s of GTF-B, C, and
rchers [23-25]. In
gtfB gene on S. mu-
protein consisting of
mass of 166 kDa.
ino acid residues
ly hydrophilic with
e gtfC gene (4218
tream of the gtfB
und to have a mo-
parameters were
GTFs and glucan-
stoc mesenteroides,
nd GS have two
cansucrase domain
main (GBD). The
ariable region) as
nd core region vary
on and length [28].
sglycosylation
transferring to low
the other hand, the
to glucan polymers
ys a key role in the
glucan structure. In S. mutans and S. sobrinus, the
C-terminal domain has an acceptor site, called YG re-
peats, which binds both the 1,6-α-linked glucose residues
of dextran and the 1,3-α-linked glucose residues. It has
been also found YG repeats which were able to attach to
either substrates [32,33]. Shar et al. showed that GBD of
GTF I in S. downei was able to bind glucans with not
only alternating α-1,3 and α-1,6 links, but also mainly
α-1,3 or α-1,6 links [34] (Table 1 ). The detailed catalytic
PFL, pyruvate formate-lyase; LDH, lactate dehyd
CS, citrate synthase; ACN, aconitase; ICDH, isoc
hydrogenase; GDH, glutamate dehydrogenase; GS,
mine synthetase; GOGAT, glutamine oxoglut
notransferase (glutamate synthase).
organism can synthesize
the GDH reaction, using citrate synthase (CS),
(ACN) and isocitrate dehydrogenase (ICDH) u
aerobic conditions [15-17]. However, under aer
ditions, S. mutans is unable to synthesize α-ke
because PEPC, PFL and ACN enzymes lose the
in the presence of oxygen [18].
Two major S. mutans virulence factors are ac
ity (ability to produce acid via glycolysis) and aci
(ability to survive in a low pH environment) [
duricity in S. mut
of studies [2]. Recently, it has been
tamate metabolism is associated with
environments. Krastel et al. [20] reported a mu
cient in the glnQHMP operon encoding a g
transporter which survived at pH 3.5 better than
ent strain. These results suggest that the gluta
porter operon glnQHMP is involved in the acid-
response in S. mutans.
is the only “streptococcal” strain used
facturing process and is widely known as a y
Bacterial EPSs are divided into ho
and heteropolysaccharides according t
composed of one or several kinds o
heteropolysaccharides are synth
strains.

J. NISHIMURA ET AL. 211
Table 1. Composition of GTFs attern ns and bacteria.
PrProtein si
and glucan ps produced from S. mutad other relate
Strain otein ze
(
a.a.
)
Gene
R
atio of
g
lucan t
yp
e Re
f
erences
S. mutans GS-5 G1475 α-(1→3)
α-(1→ [23] TF B gtfB 87% 6) 13%
S. mutans GS-5 G1375 α-(1→
α-(1→6) 15% [24]
GS-5 GTF D 1430 α-(1→
α-(1→6) 70% [25]
L. mesenteriodes NRRL B-1299 1290 α-(1→3)
α-(1→6) [26]
NRRL B-1299 1508 α-(1→3) 5%
α-(1→ [27]
fe28 G1556 I α-(1→
α-(1→6) [34]
S. downei Mfe28 GTF S 1328 α-(1→
α-(1→6) 90% [34]
G1592 α-(1→[29,35]
nus B13N GTF-S1 gftU α-(1→
α-(1→3,6) [35,36]
1[35,37]
TF C gtfC 3) 85%
S. mutansgtfD 3) 30%
dsrA 15%
85%
L. mesenteriodes dsrB 6) 95%
S. downei MTF I gtf 3) 88%
12%
gftS 3) 10%
S. sobrinus 6715TF-I gtfIa 3)
S. sobri6)
S. sobrinus OMZ176 GTF-S2
S. sobrinus GTF-S
542 gftT
α-(1→3) 16%
α-(1→6) 73%
α-(1→3,6) 5%
[35,36] gftS
3
mechanisms and structures of GTFs and GS are
th
e g
ut
, G
tfI
).
fo
0.
an
dd
,6
it
o
ot
and α-1,6 linkages. On the other hand, GTF-S3 (155 kDa)
hydrolyzes sucrose and synthesizes water-soluble α-1,6
glucan (oligo-isomaltosaccharides). Hanada et al. [37]
showed that GTF-S2 has three repeated sequences of 51
to 52 amino acids, a partial repeat of 18 amino acids and
gtfS present in the region immediately upstream of the
gtfT gene. They identified the gtfU gene and GTF-S1
enzyme from the S. sobrinus strain B13N [36]. C-ter-
minal fragments of the GBD of S. sobrinus GTF-I were
tightly to a stretch
tion of individually
ompanied by the
nthesis was corre-
the time course of
sized glucans vary
re divided into four
ain glucosidic linkages:
α(1-3) glucosidic
utan); α(1-4) glucosidic bonds (reuteran); and
nds (alternan). Glu-
pes, the degree of
s, and their spatial
s very little knowl-
lucans to date.
ctures of EPS in
S. thermophilus
The sugar components of EPS synthesized from S. ther-
mophilus are mainly galactose, glucose, and rhamnose.
Furthermore, N-acetylgalactosamine, fucose, and ribose
are also components of EPS (Figure 3) [39-49]. Galac-
tose includes not only galactopyranose, but also galacto-
furanose. S. thermophilus synthesizes EPS via intracellu-
lar sugar nucleotide precursors (Figure 4). Metabolic
flux via sugar nucleotide precursors of EPS in S. the r-
not yet
at both
lucans,
ans for
TF-S1,
prepared and shown that GBD bound
of dextran chain through the combina
weak subsite/glucose interactions acc
entropy change. Recently, glucan sy
lated with its kinetic properties and
saccharide production [38].
The sizes and structures of synthe
according to the strain. Glucans a
types based on their different m
fully understood. Recently, it has been found
GTF-B and GTF-C are necessary to synthesiz
and it is important to activate GTF-B on S. m
formation of microcolonies [21].
1.7. Glucan from S. sobrinus
Four types of GTFs occur in S. sobrinus, GTF-I
GTF-S2, and GTF-S3, which are encoded by g
gtfT, and gtfS, respectively [35,36] (Table 1
level of homology is recognized between the
zymes and all members of GS proteins GH7
(175 kDa) synthesizes water-insoluble gluc
glucan). GTF-S1 (GTF-U) is activated by the a
water-soluble glucan (α-1,6 glucan with α-1,3
linkages) and GTF-S2 produces α-1,6 glucan w
glucan. These two enzymes can hydrolyze sucr
synthesize water-soluble glucan containing b
, gtfU,
A high α(1-6) glucosidic bonds (dextran);
bonds (m
ur en-
GTF-I
(α-1,3
ition of
branch
h α-1,3
se and
h α-1,3
both α(1-6) and α(1-3) glucosidic bo
cans vary due to branch linkage ty
branching, the length of branch chain
arrangement [22]. However, there i
edge about the detailed structure of g
1.8. Sugar Components and Stru

J. NISHIMURA ET AL. 212
→3)-- D -G alp-(1→4)-- D -G lcp-(1→4)-- D -Glcp-(1→6)- - D -G l cp-(1→
- D -G l cp-(1→4)
- D -G alf-(1→6)Strain: ST1 [39]
→3)-- D -G alp-(1→3)-- D -G alp-(1→3)-- L -Rhap-(1 →2) -- L -R hap-(1→2)-- D -G alp-(1
- D -G al f2Ac
0.4
-(1 →6)
→
Str ain: S3 [41]
→6)-- D -G al p-(1→6)-- D -G al p-(1→3)-- L -Rh ap-(1→4) -- D -G l cp-(1→6)-- D -G alf-(1→6) -
- L -Rha p-(1 →2)
-D -G lcp-(1→
Strain: EU20 [42]
→3)-- D -G alp-(1→4)-- D -G lcp-(1→
- D -G alp-(1→4)-- D -G lcp-(1→ 6)-- D -G lcp-(1→4)
Strai n: THS [40]
→3)--D-Galp-(1→3) --D-G lcp-(1 →3)--D-G al pNAc-(1→
-D-G alp-(1 →6)
→3)--D-Glcp-(1→3)--D-Glcp-(1→3)--D-G al f-(1 →
-D-Ga l p-(1→6)
Strain: Sfi39[46]; SY89, SY10 2[45 ]
→2)--D-G alp-(1 →3) --D-G a lp-(1 →3)--D-G alp-(1→3)--L-Rhap-(1→2) --L
-D-Galp-(1 →6)--D-Galp-(1→4)
-R ha p-(1→
Strain: MR-1C [47]L-F uc-(1 →3)
Str ain: OR901[ 48]; Rs , Sts [49]-D-Galp-(1 →6)--D-Galp-(1→
→2)--D-G alp-(1 →3) --D-G a lp-(1 →3)--D-G alp-(1→3)--L-Rhap-(1→2) --L-Rha p-(1→
4)
Strain: Sfi12 [46]
→2)--L-R ha f-(1→2)--D-Galp-(1→3)--D-Glcp-(1→3) --D-Ga l p-(1→3)- -L-R haf-(1→
-D-Galp-(1 →4)
Str ain: Sfi6[ 43]; Sfi20[4 4]; IMDO1,2, 3, NCF B859, 21[4 5]
Figure 3. Structures of exocellular polysaccharide produced from S. thermophilus.

J. NISHIMURA ET AL. 213
C
55
-P-P
C
55
-P
C
55
-P-P-Gal
C
55
-P-P-Gal-GalNAc
C
55
-P-P-Gal-GalNAc-Glc
C
55
-P-P-Gal-GalNAc-Glc
Gal
UDP
UDP-Gal
UDP-GalNAc
Pi
Elongated acceptor
(N+1 repeat units)
Acceptor
(N repeat units)
EpsE
EpsG
EpsF
EpsBCD+
other proteins
UDP UDP
UDP-Glc
UMP
UDP-Gal
EpsI
er ed by Stingele et al.
a
re
l enzymes
d th
protein for regulating gene
d, epsC and epsD encode prot
both strains. IS-elements and partial ORFs seem to pro-
vide genetic variability in S. thermophilus Sfi39.
2. Conclusion
Dental caries causes acid formation by cariogenic bacte-
ria such as S. mutans and results from the interaction of S.
mutans and other related bacteria by production of
biofilm on tooth surfaces. Exocellular glucosyltrans-
ferases (GTFs) produced by these bacteria play a key role
t least three GTFs,
whereas S. sobrinus has four. Branch linkage types, the
degree of branching, the length of branch chains vary
acc of GTFs and the strain. Engineer-
se would provide
eat oral diseases in
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Figure 4. The model of EPS biosynthesis on S. th
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