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  • Research article
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  • Open Peer Review

Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans

BMC Oral HealthBMC series – open, inclusive and trusted201616:47

  • Received: 26 August 2015
  • Accepted: 1 April 2016
  • Published:
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Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.


The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation.


SrtAN40(UA159) and the mutant enzymes, SrtAN40(D56E) and SrtAN40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtAN40(D56E) and SrtAN40(R157H) remained approximately equal to the affinity of SrtAN40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtAN40(D56E) were reduced compared with those of SrtAN40(R157H) and SrtAN40(UA159), whereas the k cat and k cat /K m values of SrtAN40(R157H) were slightly lower than those of SrtAN40(UA159).


The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.


  • Caries
  • Missense mutation
  • srtA
  • Streptococcus mutans
  • Enzyme activity


Dental caries is an infective transmittable bacterial disease characterized by a multi-factorial pathology, and Streptococcus mutans (S. mutans) is considered as the primary aetiological agent of dental caries [1, 2]. Adhesion to a tooth surface and biofilm formation by S. mutans are the initial steps in caries development [2]. Pac (also called P1 and SpaP) is a multi-functional adhesive and is considered the primary factor that mediates the early attachment to tooth enamel [3]. Glucan binding protein C (GbpC), wall-associated protein A (wapA) and dextranase have been demonstrated to be closely related to adherence and biofilm properties [46]. The aforementioned proteins all contain a conserved LPXTG motif [7, 8]. The sortase A (SrtA) enzyme has been demonstrated as an essential transpeptidase that recognizes the LPXTG motif and responsible for sorting and anchoring those proteins to the cell wall of S. mutans [9]. Inactivation of the srtA gene could result in defective pathogenesis [10]. For example, Pac from S. mutans srtA inactivated strain could not attach to cell wall, which inhibits the ability of the mutant strain to colonize teeth and form a biofilm, and consequently reduces the occurrence of caries [11, 12]. Therefore, SrtA is thought to take a critical role in pathogenesis of S. mutans.

The various genotypes of S. mutans are involved in the susceptibility to dental decay [13, 14], and the distribution of genotypes of S. mutans differs by population. In our previous studies, we compared the srtA gene of S. mutans strains isolated from caries-free children and children with high-severity caries. Chromosomal DNA of S. mutans strains were extracted and amplified by PCR (polymerase chain reaction) to obtain the srtA gene. Then the purified PCR products were sequenced. The srtA gene sequence of S. mutans UA159 was selected as a reference sequence. The srtA gene sequences of S. mutans clinical isolates were compared with that of S. mutans UA159 using Variant Reporter™ Software (Applied Biosystems, CA, USA) (accession numbers: KP301259 - KP301500). The distributions of missense mutations were compared between the groups [15, 16]. A total of 17 missense mutation sites were found and remarkably, the prevalence of the point mutations T168G and G470A significantly differed between the two groups [16]. The total length of the srtA gene in S. mutans UA159 is 741 bp. T168G is a point mutation at the 168th base in the srtA gene; this base was T in S. mutans UA159, while some clinical isolates had a G base substitution at that site. Additionally, G470A denotes a G base at the 470th base in the srtA gene of S. mutans UA159, while an A base is substituted in the srtA gene of some clinical isolates. The frequency of mutations at the 168 locus was significantly higher in the caries-free group than in the high-severity caries group. Moreover, strains with the locus 470 polymorphism exhibited a significantly higher mutation frequency in the high-severity caries group.

Since SrtA is closely associated with adherence and biofilm formation, we hypothesized that the missense mutations T168G and G470A in the srtA gene might affect the function of the SrtA enzyme and consequently lead to the changes in the cariogenicity of S.mutans. Based on our previous study, we constructed T168G and G470A missense mutations using the srtA gene of S. mutans UA159 as a template, and investigated the effects of the two missense mutations on SrtA activity in S. mutans.


Bacterial strains, plasmids, and culture conditions

S. mutans UA159 (ATCC700610) (Guangdong Culture Collection Centre of Microbiology, Guangzhou, China) was used as the source of chromosomal DNA for the PCR. The Escherichia coli (E. coli) BL21 (TaKaRa, Kyoto, Japan) as a host of gene operation and expression vector pET32a (Novagen, Madison, WI, USA) were used for gene expression. E. coli BL21 strains were grown in Luria-Bertani (LB) broth and plated onto LB medium containing 1.5 % (w/v) agar at 37 °C. Ampicillin was added when needed at 100 μg/mL (final concentration).

Construction of srtA N120(UA159) and mutant expression vectors

SrtA is a membrane-anchoring protein containing an N-terminal signal peptide that can decrease its hydrophilicity. Therefore, full-length SrtA is difficult to purify and is unstable [17]. However, the transpeptidase activity of the truncated SrtA enzyme is not influenced by the absence of the N-terminal signal peptide because the deleted hydrophobic N-terminal region of SrtA functions as a signal peptide for secretion and a stop-transfer signal for membrane anchoring [18, 19]. Thus, to decrease the hydrophobicity of SrtA, the truncated SrtA lacking the N-terminal 40 amino acids was expressed in this study according to previous studies [2022].

This study protocol was approved by the Ethics Committee of Guanghua School of Stomatology, Sun Yat-sen University (ERC-[2012]-13). Based on our previous epidemiological investigation [16], srtA N120(T168G) and srtA N120 (G470A) were constructed using the srtA gene of S. mutans UA159 as a template. The chromosomal DNA of S. mutans UA159 was extracted and amplified DNA fragment which contains truncated SrtA coding gene according to previously described methods [20, 21] with modifications. In brief, the primers 5′-CGGGATCC GCTTGGAATACCAATAGATATCAG-3′ (BamHI site is italic) and 5′-CCGCTCGAG TTAAAATGATATTTGATTATAGGACTGC-3′ (XhoI site is italic) were used to amplify the truncated srtA fragment (621 bp) from S. mutans UA159 chromosomal DNA by PCR. The srtA fragment was cloned into linearized pET32a vector by digested with BamHI and XhoI to generate the X6 HIS tagged recombinant plasmid pET32a-srtA N120(UA159) . The site-directed mutagenesis of T168G and G470A was performed using the MutanBEST Kit (TaKaRa, Kyoto, Japan) with pET32a-srtA N120(UA159) as a template plasmid to construct pET32a-srtA N120(T168G) and pET32a-srtA N120(G470A) by following the manufacturer’s instructions. The primers 5′-GCAAGAAAGAGG ATTGAACACAACAAGGC-3′ (mutated base is italic) and 5′-TAACATTAGAAACCTG ATATCTATTGGTATTCCAAG-3′ were used to generate the T168G mutation, and the primers 5′-CCTTTAGAACATGCAAAAGAAGGCATGG-3′ (mutated base is italic) and 5′-TGAAAAGAGCATCTGTGAAGATCCGGTC-3′ were used to generate the G470A mutation. As expressed products of these gene mutations, the D56E and R157H mutants of SrtA were generated. All primers used in this study were synthesised by Shanghai Sangon Company (Shanghai, China). The plasmids were sequenced by Shanghai Sangon Company to verify that the expected sites were mutated.

Expression and purification of SrtAN40(UA159) and mutant enzymes

The pET32a-srtA N120(UA159) and mutant constructs were introduced into E. coli BL21 by chemical transformation by the manufacturer’s protocol. The transformed cells were grown in Luria broth medium containing 50 μg/mL ampicillin at 37 °C until the OD600 reached 0.6. The expression of truncated SrtA in E. coli BL21 was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cells were grown for another 6 h at 15 °C. The supernatant was then collected and centrifuged at 16,000 × g for 15 min. All soluble recombinant SrtA enzymes were purified on a Ni Sepharose 6 Fast Flow column (GE healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions. The purity and specificity of the SrtAN40(UA159) and mutant enzyme preparations were verified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using a rabbit anti-His tag monoclonal antibody (Abgent, San Diego, CA, USA).

Non-denaturing polyacrylamide Gel analysis

The soluble recombinant SrtA enzymes were subjected to non-denaturing polyacrylamide gel electrophoresis (native PAGE) described previously [23, 24] with modifications. The proteins were loaded onto 4–16 % Bis-Tris gels and resolved by electrophoresis at 4 °C. The gels were stained with Coomassie Brilliant Blue R250 (Bio-Rad, Hercules, CA, USA) and the protein bands were visualized. The ratios of dimers/monomers of SrtA proteins were evaluated by comparing the densities of dimer bands and monomer bands using ImageJ software (National Institutes of Health, Bethesda, MD, USA). All the reported ratios are the means of triplicate assays.

SrtA activity assay

The activities of the purified SrtAN40(UA159) and the two point mutated enzymes were monitored as described previously [21, 22] with modifications. The synthetic peptide 4-(4-dimethylamino phenylazo) benzoic acid (Dabcyl)-QALPETGEE-5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid (Edans) (Dabcyl-QALPNTGEE-Edans) (Jiershenghua, Shanghai, China) was used as the substrate to determine SrtA activity. The substrate Dabcyl-QALPNTGEE-Edans contains a fluorescent luminophore and a fluorescence quencher. When Dabcyl-QALPNTGEE-Edans is cleaved by SrtA, the fluorophore Edans group is separated from the quencher Dabcyl group, which enhances the fluorescence signal. Dabcyl-QALPNTGEE-Edans was added to the kinetic reaction at a final concentration from 0.2 μM to 12.8 μM. Reactions were performed in 400 μL of reaction buffer (50 mM Tris–HCl, 5 mM CaCl2, 150 mM NaCl, pH 7.5) containing varying concentrations of fluorescent peptide substrate Dabcyl-QALPNTGEE-Edans (0.2-12.8 μM), 1.2 μM purified SrtA, and 0.2 M NH2OH. The experiments were performed for 30 min at 37 °C at an excitation wavelength at 350 nm and an emission wavelength at 495 nm. SrtA activity was assessed by quantifying increases in fluorescence intensity using a Victor3 1420 multilabel counter (PerkinElmer, Waltham, MA, USA). The maximum velocity (V max ) and Michaelis constant (K m ) were calculated based on the curve fit for the Michaelis-Menten equation using Origin 8 software (OriginLab, Northampton, MA, USA):
$$ v= Vmax\left[S\right]/\left({K}_m+\left[S\right]\right) $$

where v is the slope during the linear phase of cleavage and [S] is the substrate concentration. The catalytic rate constant (k cat ) was calculated based on the ratio of V max to the enzyme concentration, and the catalytic efficiency was determined based on the k cat /K m ratio. All reported values are the means of triplicate assays.


Site-specific mutation of srtA N120(UA159)

The sortase coding gene srtA N120(T168G) and srtA N120(G470A) were generated by point mutation procedure from srtA gene of S. mutans UA159. The mutated nucleotide positions in each srtA genes were indicated in Fig. 1.
Fig. 1
Fig. 1

Point mutations in mutants compared with the srtA gene of S. mutans UA159. Detailed legend: srtA N120(T168G) contains a point mutation at base 168, and srtA N120 (G470A) contains a point mutation at base 470 (labelled in green)

Expression and purification of SrtAN40 (UA159) and mutants

The SrtAN40(UA159) and point mutated sortase SrtAN40(D56E) and SrtAN40(R157H) were expressed as the recombinant protein that coded by srtA N120(UA159) , srtA N120(T168G) and srtA N120 (G470A) , respectively. Compared with the amino acid sequence of SrtAN40(UA159), the mutant enzyme SrtAN40(D56E) contains a single amino acid substitution from aspartate (D) to glutamate (E) at the 56th amino acid residue, while SrtAN40(R157H) contains a single amino acid substitution from arginine (R) to histidine (H) at the 157th amino acid residue. The amino acid sequences are shown in Fig. 2. SDS-PAGE analysis of expressed and purified SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) is shown in Fig. 3.
Fig. 2
Fig. 2

Point mutations in mutant enzymes compared with the SrtA enzyme of S. mutans UA159. Detailed legend: SrtAN40(D56E) contains a single mutation at the 56th amino acid residue, and SrtAN40(R157H) contains a single mutation at the 157th amino acid residue (labelled in green). The putative catalytic domain of SrtA is shown in red

Fig. 3
Fig. 3

SDS-PAGE analysis of SrtA enzyme expression. Detailed legend: Expression of non-purified and purified SrtA analysed by SDS-PAGE. Lane M: Protein marker. Lane 1: Supernatant from E. coli BL21 transfected with pET32a. Lanes 2, 4, 6: Supernatants from E. coli BL21 transfected with pET32a-srtA N120(UA159) , pET32a-srtA N120(T168G) and pET32a-srtA N120(G470A) , respectively, before purification. Lanes 3, 5, 7: Supernatants from E. coli BL21 transfected with pET32a-srtA N120(UA159) , pET32a-srtA N120(T168G) and pET32a-srtA N120(G470A) , respectively, after purification using Ni sepharose 6 Fast Flow columns. New bands appeared in the supernatants from E. coli BL21 containing pET32a-srtA N120(UA159) , pET32a-srtA N120(T168G) and pET32a-srtA N120(G470A) compared with the supernatant from E. coli BL21 containing pET32a. The estimated molecular weight of SrtAN40(UA159) and the mutant enzymes was approximately 42 kDa as indicated by SDS-PAGE

As shown in Fig. 3, the estimated molecular weight of the purified enzymes was approximately 42 kDa, which was consistent with the theoretical molecular weight. Western blot analysis for SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) probed with a rabbit anti-His tag monoclonal antibody revealed anti-His antibody-reactive bands (Fig. 4).
Fig. 4
Fig. 4

Western blot of SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) probed with rabbit anti-His tag monoclonal antibody. Detailed legend: Lane M: Marker. Lane 1: Supernatant from E. coli BL21 transfected with pET32a. Lanes 2, 3, 4: Supernatants from E. coli BL21 transfected with pET32a-srtA N120(UA159) , pET32a-srtA N120(T168G) and pET32a-srtA N120(G470A) , respectively

Native PAGE analysis

Native PAGE was used to evaluate the native status of purified SrtAN40(UA159) and mutants. The dimer/monomer ratios of recombinant SrtA enzymes were estimated by comparing the densities of dimer bands and monomer bands. As shown in Fig. 5, the recombinant SrtA enzymes primarily existed as monomers and dimers. The dimer/monomer ratios of SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) were 3.25 ± 0.16, 3.28 ± 0.25 and 3.21 ± 0.32, respectively. The dimer/monomer ratios of SrtAN40(D56E) and SrtAN40(R157H) were close to that of SrtAN40(UA159) on native gel.
Fig. 5
Fig. 5

Native PAGE of SrtAN40(UA159) and mutant proteins. Detailed legend: Lane 1: Dimer and monomer of SrtAN40(UA159). Lane 2: Dimer and monomer of SrtAN40(D56E). Lane 3: Dimer and monomer of SrtAN40(R157H). The band labelled with D indicates the band of dimeric protein; the band labelled with M indicates the band of monomeric protein

SrtA activity assay

To measure the rate of cleavage by SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H), the substrate Dabcyl-QALPNTGEE-Edans was incubated with the purified enzymes, and the kinetic constants were calculated for the hydrolysis catalysed by SrtAN40(UA159) and the mutant enzymes.

The kinetic parameters for SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) are compared in Table 1. The cleavage activity of the mutant enzymes was reduced compared with SrtAN40(UA159), and the cleavage activity of SrtAN40(R157H) was more similar to SrtAN40(UA159) than SrtAN40(D56E).
Table 1

Comparison of the enzymatic activities of SrtAN40(UA159) and the mutant enzymes


V max (μM · s-1) (×10-3)

k cat (s-1) (×10-3)

K m (μM)

k cat /K m (μM-1 · s-1) (×10-4)


3.75 ± 0.15

3.04 ± 0.12

15.50 ± 0.25

1.96 ± 0.08


0.91 ± 0.13

0.74 ± 0. 11

15.34 ± 0.32

0.48 ± 0.06


2.78 ± 0.26

2.25 ± 0.21

15.35 ± 0.07

1.47 ± 0.14

The k cat values of SrtAN40(UA159) and SrtAN40(R157H) were approximately 3.1-fold and 2.0-fold higher than the k cat of SrtAN40(D56E), respectively, whereas the k cat of SrtAN40(UA159) was only 0.3-fold higher than the k cat of SrtAN40(R157H).

The K m values of SrtAN40(D56E) and SrtAN40(R157H) showed negligible decreases compared with the K m of SrtAN40(UA159), suggesting that the affinities of SrtAN40(D56E) and SrtAN40(R157H) for substrate Dabcyl-QALPNTGEE-Edans were approximately equal to those of SrtAN40(UA159).

SrtAN40(UA159) and SrtAN40(R157H) catalysed the sorting reaction more efficiently than SrtAN40(D56E), as indicated by the k cat /K m ratios. The most apparent effect on SrtA catalysis was produced by the mutation D56E. The k cat /K m values of SrtAN40(UA159) and SrtAN40(R157H) were approximately 4.1 and 3.0 times the k cat /K m of SrtAN40(D56E), respectively. The k cat /K m of SrtAN40(UA159) was slightly higher than the k cat /K m of SrtAN40(R157H).


S. mutans is the primary pathogen of dental caries, and because of a variety of different genetic events, S. mutans strains exhibit considerable phenotypic variation and differ in cariogenicity [13]. The protease SrtA is an important virulence factor that catalyses the cell wall anchoring of surface proteins containing an LPXTG motif [7] and the polymorphisms of the srtA gene could lead to variations in cariogenic capacity [1].

Previously, we performed two epidemiological investigations to explore and compare the genetic polymorphisms of the srtA gene among clinical strains of S. mutans that were isolated from children with distinct caries status [15, 16]. The results indicated that almost all clinical isolates harboured point mutations when S. mutans UA159 served as the template strain. Although the locations and periods of the two epidemiological investigations differed, the two epidemiological investigations yielded similar results. The T168G mutation was primarily observed in the caries-free group, whereas the G470A mutation was mainly detected in the caries-active group [16]. Based on these results, this study was conducted to assess the effects of missense mutations T168G and G470A in the srtA gene on the activity of the SrtA enzyme, which has not been previously reported.

A single point mutation in srtA gene was demonstrated to be able to completely change the enzyme activity. The srtA genes in S. mutans Ingbritt and S. mutans NG5 both contain nonsense mutations that cause premature termination and result in the production of incomplete SrtA enzymes and defective cell wall sorting activity [14, 25]. The srtA gene of S. mutans NG5 contains a stop codon arising from a single base substitution from G to T at a GAA codon that is 70 amino acids upstream of the putative active site of the enzyme [14]. The generation of a new termination codon in the srtA gene of S. mutans Ingbritt arises from a deletion of 11 bp [25]. In Staphylococcus aureus, mutations at H120, C184 and R197, the catalytic triad of SrtA, could affect the enzymatic activity. For example, the point mutated sortases SrtAN24(H120Q), SrtAN24(C184S) and SrtAN24(R197A) were expressed as the recombinant protein that coded by srtA N72(C360A) , srtA N72(T550A) , and srtA N72(C589G/G590C) , respectively. Compared with the wild-type SrtA, the enzymatic activities of these point mutated sortases decreased dramatically [26]. However, the mutation that is not at the active site of SrtA could also influence the enzymatic activity. For instance, the average activity of point mutated sortase SrtAN59(I123G), which was expressed as the recombinant protein that coded by srtA N177(A367G/T368G) , was also lower than the average activity of wild-type SrtA [23].

Our study showed that missense mutations arising from single base substitutions of T168G and G470A in the srtA gene in S. mutans could also result in changes in enzyme activity. Compared with the amino acid sequence of SrtAN40(UA159), the mutant enzymes SrtAN40(D56E) and SrtAN40(R157H) contained single amino acid substitutions from D to G at the 56th amino acid residue and from R to H at the 157th amino acid residue, respectively. Although the D56E and R157H mutations in the amino acid sequence did not lead to marked changes in the affinity of SrtA for the Dabcyl-QALPNTGEE-Edans substrate, the catalytic efficiency of SrtAN40(D56E) was decreased compared with those of SrtAN40(UA159) and SrtAN40(R157H). The D56E mutation significantly affected SrtA catalysis. And the enzymatic activity of SrtA(D56E), which is primarily expressed in the caries-free group, was notably decreased compared with the enzymatic activity of SrtAN40(UA159) and SrtAN40(R157H), which is primarily expressed in the caries-active group (Table 1). This difference may be responsible for the significantly lower incidence of caries in the caries-free group than in the caries-active group. Nevertheless, dental caries is an infective bacterial disease characterized by a multi-factorial pathology, and many factors other than the srtA gene of S. mutans contribute to dental caries.

The mutation data may be explained through correlation with the physical-chemical characteristics of amino acids [27]; the physical-chemical characteristics such as charge and size more or less differ among D, E, R and H. However, the mechanisms by which amino acid mutations affect protein function are complex and related to many factors [23, 2730], including protein dimerization and structure, among others. Additionally, some cases could not be easily explained or identified by structure alone. Multiple amino acid sequence alignments of sortase enzymes with determined structures in closely related species indicated that the catalytic triad of SrtA in S. mutans was composed of H139, C205 and R213 [22]. Thus, amino acid residues D56 and R157 do not belong to the catalytic triad, which is important for enzyme activity. To preliminarily explore the possible mechanism of the effects of the D56E and R157H amino acid mutations on SrtA enzyme activities, we analysed the generated SrtAN40(UA159) and mutants for dimerization using native PAGE. The results showed that the recombinant SrtA enzymes existed primarily in both dimeric and monomeric forms, which was consistent with previous study [23]. No apparent differences in the ratios of dimeric/monomeric SrtAN40(UA159), SrtAN40(D56E) and SrtAN40(R157H) were found, indicating that SrtAN40(D56E) and SrtAN40(R157H) were similar to SrtAN40(UA159) in terms of monomer-dimer equilibrium and that the effects of the D56E and R157H mutations on the enzyme activities may not be related to SrtA dimerization. Further in-depth studies are needed to investigate the exact mechanism by which the D56E and R157H amino acid mutations affect the enzyme activities.

This study was subject to certain limitations. Research limitations precluded us from investigating the effects of D56E and R157H mutations on the structure or conformation of SrtA. Mutant strains of S. mutans should be constructed in future studies to observe the effects of the D56E and R157H mutations in SrtA on strain phenotypes, such as adhesion and biofilm formation. However, the selection of mutation sites and mutation types in our study were based on two previous clinical epidemiological investigations whereas traditional studies of random mutations and specific studies of enzyme active sites or domains did not rely on such epidemiological investigations [15, 16]. Therefore, the results of this study were an approximation of the clinical condition and manifestation of caries, which is significant for the guidance of clinical preventive services. To the best of our knowledge, the effects of the D56E and R157H mutations on the catalytic activity of SrtA have not yet been studied in the context of missense mutations of srtA in S. mutans.


The reasons for the differences in cariogenicity among clinical isolates of S. mutans are complex. The results of the present study suggest that the diversity of the srtA gene can lead to the differences in enzyme activity in clinical isolates of S. mutans. Specifically, the T168G mutation in the srtA gene of S. mutans can decrease the enzyme activity.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its Additional file 1.



4-(4-dimethylamino phenylazo)benzoic acid (Dabcyl)-QALPETGEE -5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid (Edans)


deoxyribonucleic acid

E. coli

Escherichia coli


glucan binding protein C


isopropyl β-D-1-thiogalactopyranoside

k cat

catalytic rate constant

k cat /K m

catalytic efficiency

K m

michaelis constant

native PAGE: 

non-denaturing polyacrylamide gel electrophoresis


polymerase chain reaction

S. mutans

Streptococcus mutans


sodium dodecyl sulphate–polyacrylamide gel electrophoresis


sortase A


maximum velocity


wall-associated protein A



Funding for this research was provided by the National Natural Science Foundation of China (Grant No. 81271123).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-Sen University, 56 Ling Yuan Road West, Guangzhou, China
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, Guangzhou, China
Department of Stomatology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Road West, Guangzhou, China


  1. Werneck RI, Mira MT, Trevilatto PC. A critical review: an overview of genetic influence on dental caries. Oral Dis. 2010;16(7):613–23. doi:10.1111/j.1601-0825.2010.01675.x.View ArticlePubMedGoogle Scholar
  2. Krzyściak W, Jurczak A, Kościelniak D, Bystrowska B, Skalniak A. The virulence of Streptococcus mutans and the ability to form biofilms. Eur J Clin Microbiol Infect Dis. 2014;33(4):499–515. doi:10.1007/s10096-013-1993-7.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Sato Y, Okamoto-Shibayama K, Azuma T. A mechanism for extremely weak SpaP-expression in Streptococcus mutans strain Z1. J Oral Microbiol. 2011;3. doi:10.3402/jom.v3i0.5495.
  4. Tamura H, Yamada A, Kato H. Molecular characterization of the dextran-binding lectin B gene dblB of streptococcus criceti in Streptococcus mutans strain GS-5 with mutations in both gbpC and spaP genes. Genes Genet Syst. 2014;89(2):41–50. doi:10.1266/ggs.89.41.View ArticlePubMedGoogle Scholar
  5. Li Y, Liu Z, Zhang Y, Su QP, Xue B, Shao S, et al. Live-cell and super-resolution imaging reveal that the distribution of wall-associated protein A is correlated with the cell chain integrity of Streptococcus mutans. Mol Oral Microbiol. 2015; [Epub ahead of print]. doi:10.1111/omi.12100.
  6. Otsuka R, Imai S, Murata T, Nomura Y, Okamoto M, Tsumori H, et al. Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation. Microbiol Immunol. 2015;59(1):28–36. doi:10.1111/1348-0421.12214.View ArticlePubMedGoogle Scholar
  7. Marraffini LA, Dedent AC, Schneewind O. Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol Mol Biol Rev. 2006;70(1):192–221. doi:10.1128/MMBR.70.1.192-221.2006.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009;73(3):407–50. doi:10.1128/MMBR.00014-09.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Spirig T, Weiner EM, Clubb RT. Sortase enzymes in Gram-positive bacteria. Mol Microbiol. 2011;82(5):1044–59. doi:10.1111/j.1365-2958.2011.07887.x.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Yamaguchi M, Terao Y, Ogawa T, Takahashi T, Hamada S, Kawabata S. Role of streptococcus sanguinis sortase A in bacterial colonization. Microbes Infect. 2006;8(12-13):2791–6. doi:10.1016/j.micinf.2006.08.010.View ArticlePubMedGoogle Scholar
  11. Lee SF, Boran TL. Roles of sortase in surface expression of the major protein adhesin P1, saliva-induced aggregation and adherence, and cariogenicity of Streptococcus mutans. Infect Immun. 2003;71(2):676–81. doi:10.1128/IAI.71.2.676-681.2003.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Lévesque CM, Voronejskaia E, Huang YC, Mair RW, Ellen RP, Cvitkovitch DG. Involvement of sortase anchoring of cell wall proteins in biofilm formation by Streptococcus mutans. Infect Immun. 2005;73(6):3773–7. doi:10.1128/IAI.73.6.3773-3777.2005.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Lembo FL, Longo PL, Ota-Tsuzuki C, Rodrigues CR, Mayer MP. Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries-free and caries-active children. Oral Microbiol Immunol. 2007;22(5):313–9. doi:10.1111/j.1399-302X.2007.00361.x.View ArticlePubMedGoogle Scholar
  14. Lee SF, McGavin MK. Identification of a point mutation resulting in loss of cell wall anchoring activity of SrtA of Streptococcus mutans NG5. Infect Immun. 2004;72(7):4314–7. doi:10.1128/IAI.72.7.4314-4317.2004.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Zhang XH, Zhou Y, Zhi QH, Tao Y, Lin HC. Genetic polymorphisms of the sortase A gene and early childhood caries in two-year-old children. Arch Oral Biol. 2012;57(7):948–53. doi:10.1016/j.archoralbio.2012.02.002.View ArticlePubMedGoogle Scholar
  16. Yu LX, Tao Y, Qiu RM, Zhou Y, Zhi QH, Lin HC. Genetic polymorphisms of the sortase A gene and social-behavioural factors associated with caries in children: a case-control study. BMC Oral Health. 2015;15:54. doi:10.1186/s12903-015-0039-1.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Lu C, Zhu J, Wang Y, Umeda A, Cowmeadow RB, Lai E, et al. Staphylococcus aureus sortase A exists as a dimeric protein in vitro. Biochemistry. 2007;46(32):9346–54. doi:10.1021/bi700519w.View ArticlePubMedGoogle Scholar
  18. Ilangovan U, Ton-That H, Iwahara J, Schneewind O, Clubb RT. Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc Natl Acad Sci U S A. 2001;98(11):6056–61. doi:10.1073/pnas.101064198.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Deng FK, Zhang L, Wang YT, Schneewind O, Kent SB. Total chemical synthesis of the enzyme sortase A(ΔN59) with full catalytic activity. Angew Chem Int Ed Engl. 2014;53(18):4662–6. doi:10.1002/anie.201310900.View ArticlePubMedGoogle Scholar
  20. Hu P, Huang P, Chen WM. Curcumin inhibits the Sortase A activity of the Streptococcus mutans UA159. Appl Biochem Biotechnol. 2013;171(2):396–402. doi:10.1007/s12010-013-0378-9.View ArticlePubMedGoogle Scholar
  21. Huang P, Hu P, Zhou SY, Li Q, Chen WM. Morin inhibits sortase A and subsequent biofilm formation in Streptococcus mutans. Curr Microbiol. 2014;68(1):47–52. doi:10.1007/s00284-013-0439-x.View ArticlePubMedGoogle Scholar
  22. Wallock-Richards DJ, Marles-Wright J, Clarke DJ, et al. Molecular basis of Streptococcus mutans sortase A inhibition by the flavonoid natural product trans-chalcone. Chem Commun (Camb). 2015;51(52):10483–5. doi:10.1039/c5cc01816a.View ArticleGoogle Scholar
  23. Zhu J, Lu C, Standland M, Lai E, Moreno GN, Umeda A, et al. Single mutation on the surface of Staphylococcus aureus Sortase A can disrupt its dimerization. Biochemistry. 2008;47(6):1667–74. doi:10.1021/bi7014597.View ArticlePubMedGoogle Scholar
  24. Swamy M, Siegers GM, Minguet S, et al. Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes. Sci STKE. 2006;2006(345):pl4.PubMedGoogle Scholar
  25. Igarashi T. Deletion in sortase gene of Streptococcus mutans Ingbritt. Oral Microbiol Immunol. 2004;19(3):210–3. doi:10.1111/j.0902-0055.2004.00136.x.View ArticlePubMedGoogle Scholar
  26. Frankel BA, Tong Y, Bentley ML, Fitzgerald MC, McCafferty DG. Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA. Biochemistry. 2007;46(24):7269–78. doi:10.1021/bi700448e.View ArticlePubMedGoogle Scholar
  27. Taylor WR. The classification of amino acid conservation. J Theor Biol. 1986;119:205–18.View ArticlePubMedGoogle Scholar
  28. Studer R, Dessailly B, Orengo C. Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochem J. 2013;449:581–94. doi:10.1042/BJ20121221.View ArticlePubMedGoogle Scholar
  29. Teng S, Srivastava AK, Schwartz CE, Alexov E, Wang L. Structural assessment of the effects of amino acid substitutions on protein stability and protein-protein interaction. Int J Comput Biol Drug Des. 2010;3:334–49. doi:10.1504/IJCBDD.2010.038396.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Yates CM, Sternberg MJ. The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions. J Mol Biol. 2013;425:3949–63. doi:10.1016/j.jmb.2013.07.012.View ArticlePubMedGoogle Scholar


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