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Applied and Environmental Microbiology 2017 Nagasawa E00869 17.full
Applied and Environmental Microbiology 2017 Nagasawa E00869 17.full
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August 2017 Volume 83 Issue 15 e00869-17 Applied and Environmental Microbiology aem.asm.org 1
Nagasawa et al. Applied and Environmental Microbiology
RESULTS
The stimulatory effect of raffinose on biofilm formation. We tested whether
biofilm formation was induced by the presence of raffinose using several media. In this
study, we used sucrose and glucose as the biofilm-inducing and non-biofilm-inducing
substrates, respectively. As shown in Fig. 1B and in Fig. S1 in the supplemental material,
biofilm formation was significantly increased in the tryptic soy broth with raffinose
(TSBr) medium compared to TSB with glucose (TSBg), which served as a negative
control for biofilm formation. In contrast, the biomass of the biofilm was significantly
lower in TSBr than in TSB with sucrose (TSBs), which was used as a positive control. This
result suggested that raffinose had the ability to induce biofilm formation by S. mutans.
Conversely, biofilm formation was not induced when the base medium was changed to
semidefined medium (SDM), brain heart infusion (BHI) agar, or Todd Hewitt broth (THB)
(Fig. 1B and S2A and B). These results suggested that some components of TSB were
necessary for the induction of the biofilm in the presence of raffinose. To identify the
reason why a biofilm was induced in TSBr, we focused on the presence of sucrose in
TSB. TSB consists of the components derived from tryptone and soy and contains
approximately 0.0075% (wt/vol) sucrose, which is not sufficient for biofilm induction
(data not shown). To evaluate the effect of raffinose at the low sucrose concentration,
a biofilm formation assay was conducted in SDM supplemented with 0.002% (wt/vol)
sucrose, which was a more severe condition in terms of sucrose availability, and with
various raffinose concentrations. As shown in Fig. 1C, the biofilm biomass was signif-
icantly increased when a raffinose concentration greater than 0.125% was added to
SDM supplemented with 0.002% (wt/vol) sucrose as the base substrate. As a control,
the addition of glucose did not induce biofilm formation. This result suggested that
raffinose had a stimulatory effect on biofilm formation. Therefore, raffinose was not
only a nutrient.
Raffinose incorporated by S. mutans is degraded to galactose and sucrose under the
influence of ␣-galactosidase as a first step of metabolism (23). We tested whether the
sucrose formed by ␣-galactosidase during this process induced biofilm formation in
TSBr. Then, we constructed an isogenic deletion mutant of the agaL gene (ΔagaL
mutant), which encodes ␣-galactosidase. The biofilm formed by the ΔagaL mutant was
quantified to examine the effect of ␣-galactosidase on the biofilm in TSBr. The biofilm
biomass produced by the ΔagaL mutant was almost equal to the biofilm biomass of the
wild-type (WT) strain (Fig. 1D). This result suggested that the sucrose derived from
endogenous (intracellular) raffinose did not contribute to biofilm formation. Collec-
tively, these results showed that extracellular sucrose was important for biofilm forma-
tion in TSBr.
Quantitative analysis and contribution of eDNA to biofilm formation. eDNA is
a major component of biofilms in many bacteria, including S. mutans. We determined
the amount of eDNA in the biofilm by isolating and calculating the eDNA weight per
unit of dry weight of the biofilm. The amount of eDNA in the biofilm formed in TSBr was
similar to the amount in the biofilm formed in TSBs but significantly greater than the
amount in the biofilm formed in TSBg (Fig. 2A). The eDNA was confirmed to be derived
from the genomic DNA of S. mutans UA159 using the random amplified polymorphic
DNA (RAPD) method (Fig. S3). In contrast, the total eDNA included in the biofilm and
culture supernatant did not change in any of the media (Fig. 2B). These results indicated
that raffinose and sucrose, but not glucose, might induce the biofilm formation
associated with eDNA. Next, to examine the contribution of eDNA to biofilm formation,
we assessed the inhibitory effect of DNase I on biofilm formation. Biofilm formation was
significantly but not completely inhibited by DNase I, whereas inhibition was not
observed by heat-inactivated DNase I (Fig. 2C). These results indicated that eDNA was
an important factor for biofilm formation in TSBr.
Quantitative analysis of polysaccharides. Polysaccharides are important factors
for the structural and functional stability of biofilms. In particular, insoluble polysac-
sucrose, which was contained in TSB. When an amount equal (0.0075% [wt/vol]) to the
amount of sucrose included in TSB was added to SDM, the bacterial cell surface
hydrophobicity was significantly increased compared to SDM, but the level of hydro-
phobicity was lower than the level in TSB. These results suggested that components of
TSB other than sucrose also contributed to this process. After upregulation of hydro-
phobicity on the cell surface, other responses associated with eDNA and fructan might
have induced cell aggregation and biofilm formation. Therefore, gtfB contributed to cell
aggregation and biofilm formation in TSBr by utilizing sucrose, and other components
included in TSB increased the bacterial cell surface hydrophobicity and other responses
associated with eDNA and fructan.
Biofilm formation on the sHA surface. To confirm whether the biofilm formation
in TSBr occurred on the tooth surface, biofilm formation by S. mutans was examined on
the human saliva-coated hydroxyapatite disk (sHA) as an in vitro model of a smooth
surface. We examined the cells in biofilms stained with the LIVE/DEAD BacLight
bacterial viability kit by CLSM. Biofilm formation was observed on the sHA surface in
TSBs and TSBr but not in TSBg. Small cell aggregates were scattered in TSBr on the sHA
surface compared to those in TSBs (Fig. 6). The same result was obtained by examining
safranin-stained biofilms (Fig. S5). In TSBr, colocalization of live cells (green) and dead
cells (red) was observed in some small aggregates in the biofilm, whereas in other
aggregates, dead cells were mainly observed on the entire sHA surface. In TSBs,
colocalization of live and dead cells was observed in large aggregates in the biofilm.
These results showed that the biofilm formed in TSBr was not simply a sediment on the
bottom of the culture plate.
DISCUSSION
Our study revealed that biofilm formation by S. mutans was induced in TSBr but not
in SDMr. Accordingly, the mechanism underlying biofilm formation in TSBr is divided
into the participation of TSB and raffinose. Concerning the role of TSB, we propose that
sucrose contained in TSB is important for increasing the hydrophobicity of the cell
surface, which is mediated by the reaction of GtfI encoded by gtfB. Cell surface
hydrophobicity has been reported to be important for microbial attachment and
adhesion (25, 26). Indeed, the deletion of gtfB resulted in the reduction of hydropho-
bicity of the cell surface and decreased biofilm formation. Although the bacterial cell
surface hydrophobicity of the ΔgtfB mutant was reduced by half compared to the WT
level, the biofilm formation was dramatically decreased. According to these results,
other effects were also predicted to be involved in the biofilm formation via the gtfB
deletion. The importance of gtfB for biofilm formation was also suggested by RT-qPCR
and the two-compartment biofilm formation assay results. Nonetheless, the effects of
the gtfB deletion other than the decrease in hydrophobicity were not determined in
this study.
The biofilm formation mechanism in the TSBr medium was different from the
(34). Thus, the role of fructan in biofilm formation is complicated and unclear. In this
study, we identified some of the functions of fructan in biofilm formation. During
quantification of the biofilm, we found that deletion of the sacB gene did not appar-
ently affect the biofilm biomass stained with safranin in TSBr. However, during quali-
tative observation of the biofilm, a flat form was represented in the biofilm of the ΔsacB
mutant stained with the LIVE/DEAD BacLight bacterial viability kit. In contrast, aggre-
gates were largely observed in the WT biofilm. These results indicated that the
participation of fructan was associated with cell aggregates in the WT strain. Then, we
focused on the role of fructan in the biofilm features. Levan, which is a type of fructan,
was reported to enhance the viscosity of DNA solutions and was necessary for biofilm
formation by Bacillus subtilis (24). Here, we showed that fructan from S. mutans
facilitated cell aggregation when it interacted with eDNA. We believe that bacterial cells
Plasmid
pUC19 Cloning vector Ampr 54
pResEmMCS10 Streptococcal integration plasmid Ermr 42
pDL276 E. coli-Streptococcus shuttle vector; Kanr 55
pDL278 E. coli-Streptococcus shuttle vector; Spcr 56
pSBB pUC19 containing gtfB fragment; Ampr Kanr This study
pSBC pUC19 containing gtfC fragment; Ampr Ermr This study
pSBD pUC19 containing gtfD fragment; Ampr Kanr This study
pSMsacB pDL278::pldh, sacB; Spcr This study
aErms,
erythromycin susceptibility; Kans, kanamycin susceptibility; Ermr, erythromycin resistance; Kanr,
kanamycin resistance; Spcr, spectinomycin resistance; Ampr, ampicillin resistance.
was grown in Luria-Bertani (LB) broth (Affymetrix, Inc., Santa Clara, CA) or 1.5% agar at 37°C to be used
for cloning and plasmid amplification.
Construction of mutants. The primers used for the construction of bacterial mutants in this study
are listed in Table 2. Sequence information was obtained from the KEGG (http://www.genome.jp/kegg/)
and NCBI (http://www.ncbi.nlm.nih.gov/) databases. The ΔgtfB, ΔgtfC, and ΔgtfD mutants were created
cells. The eDNA was isolated using the CTAB method. The eDNA concentration was measured by
absorptiometry using a NanoDrop lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA).
RAPD method. A random amplified polymorphic DNA (RAPD) method was performed as described
elsewhere, with slight modifications (48). Genomic DNA (gDNA) was isolated from S. mutans UA159 using
the DNeasy blood and tissue kit. eDNA was isolated from the biofilm by the same procedure as in the
quantification of eDNA. RAPD was carried out using the random primers that were published previously:
OPA02 (5=-TGCCGAGCTG-3=) and OPA18 (5=-AGGTGACCGT-3=) (49, 50). The PCR was conducted using the
PrimeSTAR premix (TaKaRa Bio, Inc., Shiga, Japan) under the following cycling conditions: 94°C for 1 min,
32°C for 1 min, and 72°C for 2 min for 40 cycles. RAPD products were separated by gel electrophoresis
in 1% agarose and visualized with 0.2 g/ml ethidium bromide.
Measurement of the inhibitory effect of DNase I on biofilm formation. Fifty units per milliliter of
DNase I (Roche Applied Science, Mannheim, Germany) was added to the medium prior to the biofilm
formation assay. The preparation of the bacterial cell suspensions and the quantitation procedure were
the same as those described for the biofilm formation assay. The incubation time was changed to 8 h,
because eDNA was assumed to be an important contributor to the initial attachment, which was the
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AEM
.00869-17.
SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.
ACKNOWLEDGMENTS
We thank Ryoma Nakao, Nozomu Obana, and Satoru Hirayama for their advice and
valuable discussions. We also thank American Journal Experts for English language
editing.
This work was supported in part by a grant-in-aid for the development of scientific
research (grants 21390506, 24659821, and 16K11537) from the Ministry of Education,
Science, and Culture of Japan.
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