Subtilisin NAT, a subtilisin-like serine protease present in fermented soybean “natto” extract, inhibits Streptococcus mutans biofilm formation

Abstract

Our previous study indicated that the water-soluble extract obtained from commercially available natto products inhibited the sucrose-dependent cariogenic biofilm formation of Streptococcus mutans. This inhibitory effect correlated with the protease activity levels of the extract. Subtilisin NAT (nattokinase), a subtilisin-like serine protease, is a dominant protease found in natto products. In this study, we investigated the effect of natto products produced by a subtilisin NAT-deficient mutant (aprN⁻) of the Bacillus subtilis strain NBRC16449 on S. mutans biofilm formation. The mutation decreased the γ-poly glutamic acid (γ-PGA) levels and reduced protease activity in natto products. A significant reduction of the inhibitory effect was observed in the extract from natto products prepared with the aprN⁻ strain. The γ-PGA obtained from the natto products did not affect biofilm formation. We propose that subtilisin NAT plays a major role in biofilm inhibition induced by natto extract.

Introduction

Streptococcus mutans    is a Gram-positive bacterium and is the principal microbial etiologic agent of dental caries (Kuramitsu et al., 2007). One of the important virulence properties of this strain is its biofilm-forming ability. S. mutans utilizes glucosyltransferases (GTFs) to convert dietary sucrose to water-insoluble glucans which enhance the adherence and accumulation of cariogenic streptococci on the surfaces of teeth (Chen et al., 2016).

In our previous study, we found that commercially available proteases such as subtilisin NAT (nattokinase), trypsin, and subtilisin interfered with sucrose-dependent S. mutans biofilm formation (Narisawa et al., 2014). In contrast, we found that papain and bromelain did not inhibit the biofilm formation. These results suggest that different proteases have different impacts on S. mutans biofilm formation. Although the detailed inhibitory mechanism of these enzymes was not elucidated, we considered that the proteases might inhibit S. mutans biofilm formation by interfering with GTFs.

Natto, a fermented food product made from soybeans using Bacillus subtilis natto as a starter strain, is currently one of the most widely consumed fermented foods in Japan. In our previous study, a water-soluble fraction from a commercially available natto product interfered with the sucrose-dependent biofilm formation of S. mutans without reducing viable cell numbers (Iwamoto et al., 2018). Our results indicated that natto could be used as a safe functional food with anti-caries activity. The inhibitory activity is thought to be related to proteases, but this has not yet been confirmed.

Subtilisin NAT (E.C. 3.4.21.62), produced by B. subtilis natto, is a profibrinolytic serine protease with potent fibrin-degrading activity. This enzyme is the major protease in natto products. Subtilisin NAT is encoded by the aprN gene in B. subtilis natto (Nakamura et al., 1992). In the present study, to determine the effect of subtilisin NAT in natto on biofilm formation of S. mutans, we prepared natto with a subtilisin NAT-deficient mutant (aprN⁻) strain and investigated its effects. In addition, because subtilisin NAT during natto fermentation are involved in production of γ-poly glutamic acid (γ-PGA) that is considered to be responsible for the high viscosity of natto product (Kada et al., 2013), we investigated the effect of γ-PGA on biofilm biomass.

Materials and Methods

Bacterial strains and culture conditions S. mutans    UA159 (Ajdic et al., 2002) was cultured at 37 °C for 20 h in an atmosphere containing 5 % CO₂. Brain-heart infusion medium (BHI; Becton Dickinson and Company, San Jose, CA) was used to maintain the bacterial culture. SPII medium (Tran et al., 2000) was used for B. subtilis transformation. The fermentation starter B. subtilis NBRC16449 and its aprN⁻ derivative (see below) were cultured at 37 °C for 20 h with shaking under aerobic conditions.

Mutant construction    The aprN gene, which encodes subtilisin NAT, an extracellular serine protease (Nakamura et al., 1992), was disrupted by inserting the spectinomycin-resistance gene (Spc^R) into aprN via homologous recombination (Tran et al., 2000). The nucleotide sequence of aprN was obtained from the B. subtilis 168 genome (https://www.ncbi.nlm.nih.gov/genomes). Upstream and downstream DNA regions of aprN with 5′- and 3′-flanks were amplified by polymerase chain reaction (PCR) with oligonucleotide primers (Table 1). They were then sub-cloned into the EcoRI–KpnI and XbaI–HindIII sites of the pUC19 plasmid (Vieira and Messing, 1987), respectively, using a kit (Ligation High ver. 2; Toyobo, Osaka, Japan). The resultant plasmid was digested with BamHI, and an Spc^R fragment from pFW5 (Podbielski et al., 1996) was inserted. The resultant plasmid pAPR was linearized with EcoRI and HindIII digestion to transform B. subtilis cells. Spectinomycinresistant cells were selected on BHI agar plates containing 100 µg/mL spectinomycin. DNA from bacterial cells was extracted using DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). The isolated DNA was used as the template for the PCR and nucleotide sequencing.

Soybean fermentation    Soybeans (cultivar Suzumaru) were soaked in distilled water for 20 h at 4 °C. The soaked soybeans were steamed for 15 min at 121 °C. After steaming, a 40-g sample (wet weight) of soybeans was inoculated with approximately 2.5 × 10⁹ colony forming units (CFU)/mL starter vegetative cells suspended in 0.5 mL sterile water and packed in a polystyrene box (90 × 90 × 35 mm) with small pores. Fermentation was performed at 37 °C for 40 h under 85 % to 90 % relative humidity. Samples were stored at −30 °C until use.

Extract preparation    Extracts were prepared as follows: a 40-g sample (wet weight) of natto was homogenized in 360 mL cold distilled water in a Stomacher laboratory blender (BagMixer; Interscience, St. Nom, France) for 3 min. Soybean extracts were prepared in a manner similar to that used for the natto extracts by using soybeans soaked in distilled water for 20 h at 4 °C. The cell-free extracts were prepared using an Aspirator filter (0.2 µm; Millipore, Bedford, MA).

Crude protein, protease activity, and sucrose quantification    The crude protein content of the soybean extracts was determined using a protein assay kit (Thermo Fisher Scientific; Waltham, MA) with bovine serum albumin as the standard. Proteolytic activity was measured based on azo-casein hydrolysis (Narisawa et al., 2014). One unit of proteolytic activity was defined as the amount of enzyme required for an increase of absorbance at 440 nm (Δ A₄₄₀/min/mL). The sucrose content of the extracts was measured using a Saccharose/D-Glucose/D-Fructose F-kit (JK International, Tokyo).

Viable cell numbers    S. mutans culture tubes were prepared as described above. Aliquots (50 µL) of the cultures and 2.5 mL of 2 × tryptic soy broth without dextrose (TSB; Becton Dickinson and Company) supplemented with 0.5 % glucose were added to the culture tubes, followed by addition of cell-free soybean extract, natto extract and/or sterile distilled water to a total volume of 5.0 mL. The viable cell count of S. mutans was evaluated in terms of CFUs using appropriate decimal dilutions of the culture in sterile distilled water, which were plated on BHI agar and incubated at 37 °C for two days.

γ-PGA extraction and quantification    γ-PGA was extracted as follows. A 25-g aliquot (wet weight) of the natto product was incubated with 75 mL of 2.5 % trichloroacetic acid (TCA) solution for 3 min at 50 °C. After incubation, the solution was filtered using a metal mesh and adjusted to 100 mL with 2.5 % TCA. The pH of the supernatant aliquot (25 mL) after centrifugation (9 200 × g for 10 min at 4 °C) was adjusted to pH 7.0 with 1.0 M NaOH. Aliquots (300 µL) of the samples were added to 1.2 mL ethanol, incubated on ice for 5 min, and centrifuged at 9 200 × g for 10 min at 4 °C. The precipitate was dissolved in 1.2 mL distilled water. The γ-PGA amount was determined by the cetyltrimethylammonium bromide method (Kanno and Takamatsu, 1995) and calculated as the glutamic acid equivalent (Kiuchi, 2010).

Biofilm formation assay on 96-well microtiter plates    Biofilm formation was assayed by measuring the ability of cells to adhere to and grow on the wells of 96-well polystyrene microtiter plates (Sumitomo Bakelite, Tokyo). Aliquots (2 µl) of S. mutans cultures and 40 µL of TSB supplemented with 0.5 % sucrose were added to 96-well polystyrene microtiter plates, followed by addition of cell-free soybean extract, natto extract, and/or sterile distilled water to a total volume of 200 µl. The plates were incubated at 37 °C in a 5 % CO₂ atmosphere for 20 h. After incubation, the plates were washed with distilled water, and adherent cells were stained with safranin solution (Becton Dickinson and Company). The dye was solubilized in 70 % ethanol, and the absorbance at 492 nm was determined using a microplate reader (Colona Electric, Ibaraki, Japan). When required, purified γ-PGA, D- and L-glutamic acid (Wako Pure Chemicals, Osaka, Japan) and commercially available nattokinase (Wako Pure Chemicals) were added to the growth medium.

Biofilm formation assay on hydroxyapatite discs    Biofilm formation was assayed by measuring the ability of cells to adhere to and grow on discs of hydroxyapatite (HA). HA discs (5 × 1.6 mm; 3D Biotek, Hillsborough, NJ) were placed on 48-well polystyrene microtiter plates (Sumitomo Bakelite). Aliquots (10 µL) of S. mutans cultures and 400 µL TSB supplemented with 0.5 % sucrose were then poured into the wells, and cell-free natto extract, soybean extract, and/or sterile distilled water were added to fill the wells to 1 mL. The plates with HA discs were incubated under 5 % CO₂ atmospheric conditions at 37 °C for 20 h. The biofilms on HA discs that remained after washing with sterile distilled water were stained using safranin solution. When required, nattokinase was added to the growth medium (0.1U/mL medium).

Statistical analysis    All data are expressed as the mean±standard deviation. Data were analyzed using Student's t-test, and values of p < 0.05 were considered statistically significant.

Results and Discussion

As B. subtilis natto has a lower transformation efficiency than B. subtilis Marburg 168, it is difficult to create a mutant strain using B. subtilis natto. We selected B. subtilis natto NBRC16449 as a host for constructing the mutant in this study because it possesses natto fermentation ability and transformation competence (Urushibata et al., 2002). Subtilisin NAT is encoded by the aprN gene in B. subtilis natto (Nakamura et al., 1992). Insertion–inactivation of aprN in B. subtilis NBRC16449 was performed with Spc^R insertion using homologous recombination with a transformation efficiency of 8.42 × 10⁻⁸. PCR amplification showed that the lengths of the wild type and transformant aprN genes with 5′- and 3′-flanks were approximately 1.0 and 1.5 Kbp, respectively (data not shown). The nucleotide sequence indicated that the spectinomycin-resistance gene was appropriately located in the aprN region of the transformant (data not shown). The constructed aprN⁻ strain had a growth ability similar to that of the wild type in BHI liquid medium under aerobic conditions (data not shown).

To characterize the natto products produced by the B. subtilis wild type and aprN⁻ strains, we investigated the protease-specific activity and quantified γ-PGA. The extract from the natto product made by the aprN⁻ strain showed a significant decrease in protease activity compared with that produced by the parental strain (p < 0.05; Fig. 1A). The amount of γ-PGA in the natto product made by the aprN⁻ strain was below the detection limit (< 5mg/ml) (Fig. 1B). These results were in agreement with the findings of Kada et al. (2013) and were caused by the lack of glutamic acid due to insufficient hydrolysis of soy proteins (Kada et al., 2013).

Fig. 1.

Protease-specific activity (A) and γ-PGA quantity (B) in the natto product. “Wild type” and “aprN⁻” on the x-axis indicate the natto product made with the B. subtilis natto wild type and aprN⁻ strains, respectively. Data represent average values from three independent experiments (*p < 0.05). N.D., not detected.

To determine the impact of subtilisin NAT in the extract from natto product on S. mutans biofilm formation, we investigated the effect of the extract from natto products with wild type B. subtilis and aprN⁻ strain. The presence of more than 25 % (vol/vol) cell-free extract from the natto products made with wild type B. subtilis (protease activity: < 0.08 U/mL medium) significantly decreased the safranin-stained S. mutans biofilm biomass (Fig. 2A). The presence of 25 % (vol/vol) natto extract made with the aprN⁻ strain (protease activity: 0.01 U/mL medium) had little effect on biofilm formation (Fig. 2A). The presence of 50 % (vol/vol) natto extract made with the aprN⁻ strain (protease activity: 0.02 U/mL medium) decreased the biofilm formation (Fig. 2A). This result was consistent with the inhibition of biofilm formation by commercially available subtilisin NAT: the presence of approximately 0.05 and 0.02 U/mL medium of commercially available subtilisin NAT reduced the safranin-stained biofilm by about 80 % and 20 %, respectively (data not shown). These results strongly support our previous conclusion that subtilisin NAT is the biofilm inhibitor in natto extract (Iwamoto et al., 2018; Narisawa et al., 2014). Kitagawa et al. (2017) showed that the natto peptide, which consists of 45 amino acid residues, has a bactericidal effect against Gram-positive bacteria, such as S. pneumoniae and some Bacillus species; however, in our study, the natto extracts did not affect S. mutans growth in a liquid medium (Fig. 2B). HA is a main component of teeth and is used as a model material for investigating dental biofilm formation (Kawarai et al., 2016). Our previous conclusion (Iwamoto et al., 2018; Narisawa et al., 2014) was also supported by the results of biofilm formation assay on HA discs: biofilm formation on the HA disc surfaces was also inhibited by the natto extracts made with the wild type strain (protease activity: 0.14 U/mL medium) and commercially available subtilisin NAT (0.10 U/mL medium), but not by the natto extracts made with the aprN⁻ strain (protease activity: 0.02 U/mL medium) (Fig. 3).

Fig. 2.

The effect of natto and soybean extract on biofilm formation (A) and viable cell number (B). Black bars, dark-gray bars, and whitish-gray bars represent soybean extract, the extract from natto made with the wild type B. subtilis natto strain, and the extract from natto made with the B. subtilis natto aprN⁻ strain, respectively. The circles represent the protease activity of the cell-free extract (U/mL medium). Dotted lines indicate the value in the control. (A) Biofilm formation: A₄₉₂ = 0.52 ± 0.02. (B) Number of viable cells: 8.96 ± 0.66 log CFU/mL. Data represent average values from three independent experiments (*p < 0.05).

Fig. 3.

The effects of natto and soybean extracts on the formation of biofilm by cariogenic streptococci on discs of HA. Natto and soybean extracts (50 %, vol/vol) were added to the biofilm medium of S. mutans UA159 together with HA discs. The control indicates the biofilm formation of S. mutans without natto extract. The negative control indicates no inoculation of S. mutans.

Next, we determined the effect of γ-PGA on S. mutans biofilm formation. Crude γ-PGA obtained from natto made with the wild-type strain was tested in this study. The presence of γ-PGA in the culture medium with sucrose did not affect biofilm formation (data not shown), and the biofilm formation in the medium containing γ-PGA was the same as that in the culture medium with D- and L-glutamic acid, two components of γ-PGA (data not shown).

The crude protease fraction obtained from commercially available natto inhibited water-insoluble glucan production (Iwamoto et al., 2018). S. mutans has at least three GTFs that are involved in glucan production as follows. GtfB (165.8 kDa) primarily synthesizes insoluble glucans with α(1–3) glycosidic linkages, GtfD (163.4 kDa) primarily synthesizes soluble glucans with α(1–6) linkages, and GtfC (163.4 kDa) synthesizes a mixture of insoluble and soluble glucans (Chen et al., 2016). We investigated the effect of subtilisin NAT on GTFs obtained from S. mutans culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography indicated that subtilisin NAT degraded GTFs, resulting in the loss of GTF activity (unpublished data). We propose that subtilisin NAT inhibits biofilm formation by interfering with GTFs. However, it is difficult to confidently determine the impact of subtilisin NAT on biofilm formation because numerous factors are involved in S. mutans biofilm formation, e.g., the transport of virulence factors through membrane vesicles (Liao et al., 2014), the genetics and physiology of the competence cascade through extracellular peptides (Li et al., 2002), and amyloid fibers on the cell surfaces (Besingi et al., 2017). Further studies are required to determine the target of subtilisin NAT for the inhibitory action.

The extract obtained from soybeans before fermentation significantly promoted biofilm formation (Fig. 2). A similar result was obtained for other soybean extracts in our previous study (Iwamoto et al., 2018). The soybean extract, even after low molecular weight compounds (<Mw. 14000) including sucrose, were eliminated by dialysis, promoted biofilm production (unpublished data). Initial bacterial attachment to abiotic and/or biotic surfaces is an important step in biofilm development (Wen and Burne, 2002). It has been reported that inducing aggregation in S. mutans interferes with the initial attachment to teeth following biofilm development (Lee and Boran, 2003), and several plant agglutinins interfere with S. mutans adherence to the tooth surface (Yang et al., 2015). A recent study indicated that genistein, an isoflavone in soybeans, induces S. mutans aggregation, and thereby inhibits biofilm formation (Lee et al., 2014). Although we were unable to induce S. mutans aggregation with soybean extract in the present study (data not shown), a recent study indicated that the genistein content in soybeans is affected by soaking and steaming (Kasuga et al., 2006). Future studies should examine the behavior of genistein in soybeans during natto fermentation and its effects on biofilm formation.

In this study, we revealed that the biofilm inhibitor in the fermented soybean product known as “natto” is subtilisin NAT, encoded by the aprN gene. The subtilisin NAT content in natto is impacted by fermentation conditions, starter strains, and bean cultivars (Akimoto et al., 1993; Matsumoto et al., 1995; Kubo et al., 2011). These are subjects of future study. In addition, clinical studies are required to confirm the anticarious effect of natto.

Acknowledgements    We thank Hidenobu Senpuku (National Institute of Infectious Diseases) for advice and valuable discussions. This work was supported in part by a Nihon University College of Bioresource Science Research Grant for 2020.

References

•  Ajdic,  D.,  McShan,  W.M.,  McLaughlin,  R.E.,  Savic,  G.,  Chang,  J.,  Carson,  M.B.,  Primeaux,  C.,  Tian,  R.,  Kenton,  S.,  Jian,  H.,  Lin,  S.,  Qian,  Y.,  Li,  S.,  Zhu,  H.,  Najar,  F.,  Lai,  H.,  White,  J.,  Roe,  B.A., and  Ferretti,  J.J. (2002). Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA, 99, 14434-14439.
•  Akimoto,  T.,  Matsumoto,  I., and  Imai,  S. (1993). Effect of temperature and inoculum size on Natto's enzyme activities during fermentation. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kyogo Gakkaishi). 40, 83-90. (in Japanese)
•  Besingi,  R.N.,  Wenderska,  I.B.,  Senadheera,  D.B.,  Cvitkovitch,  D.G.,  Long,  J.R.,  Wen,  Z.T., and  Brady,  L.J. (2017). Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c. Microbiology, 163, 488-501.
•  Chen,  L.,  Ren,  Z.,  Zhou,  X.,  Zeng,  J.,  Zou,  J., and  Li,  Y. (2016). Inhibition of Streptococcus mutans biofilm formation, extracellular polysaccharide production, and virulence by an oxazole derivative. Appl. Microbiol. Biotechnol., 100, 857-867.
•  Iwamoto,  A.,  Nakamura,  T.,  Narisawa,  N.,  Kawasaki,  Y.,  Abe,  S.,  Torii,  Y.,  Senpuku,  H., and  Takenaga,  F. (2018). The Japanese fermented food natto inhibits sucrose-dependent biofilm formation by cariogenic streptococci. Food Sci. Tech. Res., 24, 129-137.
•  Kada,  S.,  Ishikawa,  A.,  Ohshima,  Y., and  Yoshida,  K. (2013). Alkaline serine protease AprE plays an essential role in poly-γ-glutamate production during natto fermentation. Biosci. Biotechnol. Biochem., 77, 802-809.
•  Kanno,  A. and  Takamatsu,  H. (1995). Determination of γ-polyglutamic acid in “natto” using cetyltrimethylammonium bromide. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kogyo Gakkai-Shi). 42, 878-886. (in Japanese)
•  Kasuga,  A.,  Ogiwara,  E.,  Aoyagi,  Y., and  Kimura,  H. (2006). Changes in isoflavone content of soybeans during heating process. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin Kogyo Gakkai-Shi). 53, 365-372. (in Japanese)
•  Kawarai,  T.,  Narisawa,  N.,  Yoneda,  S.,  Tsutsumi,  Y.,  Ishikawa,  J.,  Hoshino,  Y., and  Senpuku,  H. (2016). Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea. Arch. Oral Biol., 68, 73-82.
•  Kitagawa,  M.,  Shiraishi,  T.,  Yamamoto,  S.,  Kutomi,  R.,  Ohkoshi,  Y.,  Sato,  T.,  Wakui,  H.,  Itoh,  H.,  Miyamoto,  A., and  Yokota,  S. (2017). Novel antimicrobial activities of a peptide derived from a Japanese soybean fermented food, Natto, against Streptococcus pneumoniae and Bacillus subtilis group strains. AMB Express, 7, 10.1186/s13568-017-0430-1.
•  Kiuchi,  K. (2010). Investigative approach to NATTO. In “Functional component analysis,” ed. by  K.  Akishige Koseisha Koseikaku Inc., Tokyo, pp. 133-135.
•  Kubo,  Y.,  Rooney,  A.P.,  Tsukakoshi,  Y.,  Nakagawa,  R.,  Hasegawa,  H., and  Kimura,  K. (2011). Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production. Appl. Environ. Microbiol., 77, 6463-6469.
•  Kuramitsu,  H.K.,  He,  X.,  Lux,  R.,  Anderson,  M.H., and  Shi,  W. (2007). Interspecies interactions within oral microbial communities. Microbiol. Mol. Biol. Rev., 71, 653-670.
•  Lee,  S.F. and  Boran,  T.L. (2003). Roles of sortase in surface express ion of the major protein adhesin P1, saliva-induced aggregation and adherence, and cariogenicity of Streptococcus mutans. Infect. Immun., 71, 676-681.
•  Lee,  H.J.,  Kwon,  T.Y.,  Kim,  K.H., and  Hong,  S.H. (2014). Soybean extracts facilitate bacterial agglutination and prevent biofilm formation on orthodontic wire. J. Med. Food, 17, 135-141.
•  Liao,  S.,  Klein,  M.I.,  Heim,  K.P.,  Fan,  Y.,  Bitoun,  J.P.,  Ahn,  S.J.,  Burne,  R.A.,  Koo,  H.,  Brady,  L.J., and  Wen,  Z.T., (2014). Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J. Bacteriol., 196, 2355-2366.
•  Li,  Y.H.,  Tang,  N.,  Aspiras,  M.B.,  Lau,  P.C.,  Lee,  J.H.,  Ellen,  R.P., and  Cvitkovitch,  D.G. (2002). A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J. Bacteriol., 184, 2699-2708.
•  Matsumoto,  I.,  Akimoto,  T., and  Imai,  S. (1995). Effect of soybean cooking condition on the quality of natto. Nippon Shokuhin Kagaku Kogaku Kaishi, 42, 338-343. (in Japanese)
•  Nakamura,  T.,  Yamagata,  Y., and  Ichishima,  E. (1992). Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem., 56, 1869-1871.
•  Narisawa,  N.,  Kawasaki,  Y.,  Nakashima,  K.,  Abe,  S.,  Torii,  Y., and  Takenaga,  F. (2014). Interference effects of proteolytic nattokinase on biofilm formation of cariogenic streptococci. Food Preserv. Sci., 40, 273-278.
•  Podbielski,  A.,  Spellerberg,  B.,  Woischnik,  M.,  Pohl,  B., and  Liitticken,  R. (1996). Novel series of plasmid vectors for gene inactivation and expression analysis in group A streptococci (GAS). Gene, 177, 137-147.
•  Tran,  L.S.,  Nagai,  T., and  Itoh,  Y. (2000). Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis. Mol. Microbiol., 37, 1159-1171.
•  Urushibata,  Y.,  Tokuyama,  S., and  Tahara,  Y. (2002). Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production. J. Bacteriol., 184, 337-343.
•  Vieira,  J. and  Messing,  J. (1987). Production of singlestranded plasmid DNA. Methods Enzymol., 153, 3-11.
•  Wen,  Z.T. and  Burne,  R.A. (2002). Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl. Environ. Microbiol., 68, 1196-203.
•  Yang,  W.Y.,  Won,  T.H.,  Ahn,  C.H.,  Lee,  S.H.,  Yang,  H.C.,  Shin,  J., and  Oh,  K.B. (2015). Streptococcus mutans sortase A inhibitory metabolites from the flowers of Sophora japonica. Bioorg. Med. Chem. Lett., 25, 1394-1397.