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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Lett Appl Microbiol. Author manuscript; available in PMC Apr 1, 2008.
Published in final edited form as:
PMCID: PMC2062497

Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties



To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties.

Methods and Results

For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development.


Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis.

Significance and Impact of the Study

Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.

Keywords: acid resistance, biofilm formation, gene inactivation, oral streptococci, stress tolerance, two-component system


Bacteria monitor and adapt to changing environmental conditions via environmental signal transduction. Two-component systems (TCS) are an important mode of signal transduction in bacteria as well as some archaea, protozoa, fungi, and plants. TCS allow cells to respond to a wide variety of environmental stimuli, including nutrient starvation, osmotic shock, pH variations, host–pathogen interactions, and other stress conditions, by modulating gene expression (Beier and Gross 2006). TCS are typically composed of a membrane-bound sensor histidine kinase (HK) that has an extracellular input domain and a cytosolic response regulator (RR) that binds to promoter operator regions of genes. Environmental signals are sensed by the HK resulting in autophosphorylation at a specific histidine residue creating a high-energy phosphoryl group that is subsequently transferred to a specific aspartate residue within the N-terminal half of the cognate RR. Phosphorylation induces a conformational change in the regulatory domain resulting in activation of the RR. The activated RR then regulates gene expression by acting as a DNA-binding transcriptional regulator, which activates or represses genes whose products are specifically utilized to respond to the given input signal (Stock et al. 2000).

Streptococcus mutans, a primary agent involved in the initiation and progression of human dental caries, must contend with a highly dynamic environment and has evolved several virulence factors that allow it to accumulate within the dental plaque biofilm, and produce and tolerate acids generated from bacterial sugar metabolism. Of the 13 putative TCS found in the S. mutans UA159 genome (Ajdic et al. 2002), the ComDE TCS (TCS-13) is typical in that it consists of a membrane-associated HK, ComD, and a cognate RR, ComE. This TCS has been shown to be involved in the development of competence, biofilm formation, and bacteriocin production (Bhagwat et al. 2001; Cvitkovitch et al. 2003; Kreth et al. 2005; Ahn et al. 2006). TCS-11, comprised of the hk11 and rr11 genes, has also been described, and found to be involved in S. mutans survival at an acidic pH. Deletion of hk11 or rr11 also resulted in biofilms with sponge-like architecture that were composed of cells organized in very long chains, suggesting a role for TCS-11 in cell segregation and biofilm architecture (Cvitkovitch et al. 2003). In addition, the VicRK TCS (TCS-1) has been shown to be involved in the regulation of sucrose-dependent adhesion, biofilm formation, and competence development in S. mutans (Senadheera et al. 2005a).

The aim of the present study was to target each of the HK-encoding genes identified in UA159 for insertion mutagenesis, and assess their role in the expression of several S. mutans virulence properties.

Materials and methods

Bacterial strains and growth conditions

Streptococcus mutans wild-type strain UA159 was used in this study (Ajdic et al. 2002). The S. mutans hk defective mutants were constructed by polymerase chain reaction (PCR) ligation mutagenesis (Lévesque et al. 2005). Streptococcus mutans strains were grown in THYE (Todd-Hewitt yeast extract) broth at pH of 7·5 and 5·5 (pH adjusted with HCl), and incubated statically at 37 °C in air with 5% CO2 (Li et al. 2001).

Growth kinetics

All strains were grown in THYE broth without antibiotics for 16 h to assay their growth kinetics using a Bioscreen microbiology reader (Bioscreen C Labsystems, Helsinki, Finland) (Hasona et al. 2005). Doubling time (Td) of bacterial growth was calculated by measuring the slope of the logarithmic growth phase using the formula: Td = (t2 t1) ln(2)/ ln(OD2) – ln(OD1) (Khalichi et al. 2004). Results represent an average of five replicates with standard deviation of the mean. Each experiment was performed two to four times. Statistical significance was determined by using Student's t-test and a P value < 0·05.

In vitro model for growing biofilms

Static biofilms were developed in 96-well polystyrene microtitre plates using semi-defined minimal medium (SDM) supplemented with 20 mmol l–1 of glucose or 10 mmol l–1 of sucrose and quantified as described previously (Lévesque et al. 2005). A biomass of ≥ ±20% of the wild-type level was considered significant. Scanning electron microscopy (SEM) was performed on 16-h biofilms grown in SDM-glucose on the surface of glass discs deposited in 12-well polystyrene microtitre plates to examine their structure and cell morphology (Li et al. 2001).

Transformation experiments

Overnight cultures were diluted (1 : 20) with prewarmed THYE and incubated at 37 °C until an OD600 of c. 0·1 was reached at which time 1 μg of plasmid pDL277, conferring spectinomycin resistance was added. Cultures were spread on THYE plates and transformation efficiency (TE) was expressed as the percentage of spectinomycin-resistant transformants over the total number of recipient cells. To test the effect of S. mutans competence-stimulating peptide (CSP), experiments were also conducted with synthetic CSP dissolved in sterile double-distilled water and added to a final concentration of 1 μg ml–1.


Classification of HK and RR

Bioinformatic analysis of the S. mutans UA159 genome sequence identified 13 putative TCS (TCS-1 to TCS-13) and one independent RR (GcrR). All of the TCS are arranged in pairs, either with the HK-encoding gene first followed by the RR-encoding gene, or vice versa. The criteria used to identify the TCS were the conserved ATP-binding site characteristic of sensor HK in conjunction with a conserved histidine motif and the overall similarity of the phosphorylated aspartate domains of the RR. Every ORF shared significant similarities to HK or RR from several Streptococcus sp., Lactobacillus sp., Staphylococcus sp., and Clostridium sp. strains (Table 1).

Table 1
Two-component systems (TCS) of Streptococcus mutans UA159

All of the HK in UA159 are predicted to be membrane localized, consistent with the observation that localization of the sensor kinase to the membrane of the bacterial cells appears to be a general feature of most TCS (Stock et al. 2000). The S. mutans HK were grouped based on the residues on either side of the phosphorylated histidine (Table 2). According to the classification of Fabret et al. (1999), the histidine motifs fell into five homology groups (I, II, IIIA, IIIB, and IV). Based on that classification scheme, S. mutans possesses eight HK (HK1, HK2, HK3, HK4, HK5, HK6, HK7, and HK8) that are members of the group IIIA. This group appears to be the most abundant class of kinases in the gram-positive pathogens whose genomes have been sequenced to date (Kiil et al. 2005). A single sensor kinase, HK10, was found to belong to group I; while HK9, HK11, and HK12 were classified into group II. HK13, the ComD sensor of the CSP quorum-sensing signaling system (Li et al. 2001), does not appear to fit into the current classification scheme based on the residues near the conserved histidine.

Table 2
Classification of Streptococcus mutans UA159 two-component systems (TCS)

Subsequent BLAST (Basic Local Alignment Search Tool) searches and PFAM (Protein domain Families) database analysis were performed to classify the S. mutans RR. They display sequence similarities to the following families of DNA-binding proteins: OmpR (RR1, RR2, RR3, RR4, RR5, RR6, RR7, and RR8), NarL (RR9, RR11, and RR12), and LytR (RR10 and RR13) (Table 2) (Galperin 2006).

Construction of insertion mutants

To assess the role of the S. mutans TCS, each of the HK-encoding genes were inactivated [except VicK (TCS-1), HK11 (TCS-11), and ComD (TCS-13)] as these systems have already been studied by our group. None of the hk-deletion mutations were lethal for S. mutans as viable strains were obtained for all interrupted HK-encoding genes. Growth kinetics showed that all mutants but one, SMHK9 (Td = 54·7 ± 0·8 min), grew similarly in THYE (pH 7·5) compared with UA159 (Td = 48·6 ± 0·7 min). Although modest, this increase in Td was statistically significant (P < 0·05). However, SMHK9 showed a similar growth curve and final growth yield after 16 h of unstressed growth compared with UA159 (data not shown).

Growth during acid challenge

The effect of the HK mutations on S. mutans acid tolerance was determined by measurements of growth rates in THYE broth adjusted to pH 5·5. The SMHK3 mutant displayed a significantly slower Td compared with the wild-type strain; SMHK3 had a Td of 74·2 ± 2·2 min, while UA159 doubled every 67·7 ± 1·0 min. However, the final growth yield of SMHK3 after 16 h was the same as that of UA159 (Fig. 1a). The SMHK9 mutant also had a significantly slower Td in liquid culture at pH 5·5 compared with UA159. SMHK9 doubled every 77·1 ± 1·8 min compared with 67·7 ± 1·0 min for UA159.

Figure 1
Growth kinetics of UA159 (unstressed [diamond], stressed ■), SMHK2 (▲), SMHK3 (•), and SMHK9 (×). (a) THYE (pH 5·5); (b) THYE (pH 7·5) with 0·5 mmol l–1 of hydrogen peroxide; (c) THYE (pH ...

Tolerance to oxidative and osmotic stress

The effect of hk gene disruption on tolerance to other environmental stressors including osmotic and oxidative shock was evaluated for all mutants. The Td of all mutants but one, SMHK2, was not significantly different during growth in the presence of 2% (w/v) NaCl or 0·5 mmol l–1 of H2O2 relative to the wild-type strain. Surprisingly, SMHK2 displayed a shorter Td both in the presence of NaCl or H2O2 relative to UA159; SMHK2 had a Td of 130·3 ± 3·3 min and 109·1 ± 2·7 min following osmotic and oxidative stress, respectively, while UA159 doubled every 196·8 ± 5·3 min and 131·5 ± 3·1 min. In fact, while the growth kinetics of UA159 and other hk-defective mutants was altered by growth in presence of 0.5 mmol l–1 of H2O2, the growth curve of SMHK2 resembled that of UA159 under unstressed conditions (Fig. 1b). A similar trend was observed for this mutant during growth under high salinity, where hk2 deletion partially restored growth rates to the wild-type unstressed level (Fig. 1c). All other mutants grew similarly to UA159 under both oxidative and osmotic stress (data not shown). The final growth yield of all mutants was the same after 16 h growth in the presence of NaCl or H2O2, with the exception of SMHK2 and SMHK9, which showed significantly higher growth yields than the wild-type under osmotic stress (Fig. 1c).

Competence development

The involvement of TCS in the development of natural genetic competence was tested by assaying hk-defective mutants for their ability to be transformed with plasmid DNA. The SMHK9 mutant (%TE: 7·0 ± 1·0 × 10–3) had an eightfold significant reduction in TE relative to UA159 (%TE: 5·5 ± 1·7 × 10–2). Although the addition of CSP greatly increased the number of SMHK9 transformants, the TE did not reach the wild-type level. The TE of SMHK9 (%TE: 0·3 ± 0·1) with addition of CSP was approximately sixfold lower compared with UA159 (%TE: 1·9 ± 0·4) with exogenously added CSP. In S. mutans and other naturally competent streptococci, genetic competence is primarily regulated by the ComDE TCS (Senadheera et al. 2005b). As an effector molecule, ComE is involved in the regulation of specific early competence genes, including comX, encoding an alternative sigma factor that initiates transcription of late competence genes required for DNA uptake and recombination. In Streptococcus pneumoniae, mutations in ciaRH de-repress competence by modulating comCDE transcription (Echenique et al. 2000). To assess whether changes in comD, comE, and comX expression were responsible for the difference in SMHK9 TEs, we measured gene expression of these early competence genes in SMHK9 and wild-type cells by real-time reverse transcriptase (RT)-PCR (Pfaffl 2001). The results showed that the expression of comD, comE, and comX was not significantly altered in SMHK9 in comparison with UA159 (data not shown).

Biofilm formation

Assessment of the biofilm-forming capacity of the hk-defective mutants relative to the wild-type strain showed that all hk-defective mutants formed stable and reproducible biofilms typical of the parent strain in SDM-sucrose and SDM-glucose. No significant difference in the total biomass of the hk-defective mutant biofilms compared with the wild-type biofilm was noted in sucrose-supplemented medium. However, the SMHK2 mutant cells supplemented with glucose as the sugar source generated thinner biofilms relative to UA159. The SMHK2 biofilms had a biomass of 20·2 ± 2·8% less than the wild-type. SEM examination of the glucose-grown biofilms revealed that SMHK2 biofilm cells formed very short chains in comparison with UA159 (Fig. 2).

Figure 2
Scanning electron micrographs of Streptococcus mutans UA159 (a–c) and SMHK2 (d–f) biofilms. Magnifications, ×1000 (a and d), ×2500 (b and e), and ×5000 (c and f).


The human oral cavity is a complex biological system in which bacteria must overcome a wide range of environmental conditions in order to survive. The pH levels in dental plaque are highly variable and frequently shift from neutral pH to as low as 3·0 during the ingestion of dietary carbohydrates by the host. Despite the fact that the mouth is highly aerobic and oxygen is abundant at mucosal surfaces, oxygen gradients exist in the dental biofilm. Solutes also accumulate from different sources, including exogenous nutrients from the host diet and salts from tooth demineralization. Thus, the ability of S. mutans to withstand extreme fluctuations allows it to compete with other micro-organisms within the dental plaque. Monitoring and adapting to changing environmental conditions is the key function of bacterial signal transduction, which is generally carried out by the TCS. Although the basic biochemistry of TCS is well understood, the role of TCS in the pathogenicity of bacteria remains to be determined in many cases. In an effort to gain a more comprehensive view of the role of each TCS in the biology of S. mutans, whose virulence is linked to diet and environment, we targeted the signal transduction pathways for gene inactivation and phenotypic assessment.

We identified a novel TCS (TCS-3) involved in S. mutans's ability to tolerate acid, which exhibits significant identity with the Streptococcus pyogenes ScnRK system. In S. pyogenes, the ScnRK system has been shown to be essential for the production of the bacteriocin SA-FF22 (McLaughlin et al. 1999). The bacteriocins produced by S. mutans are small ribosomally synthesized antimicrobial peptides, called mutacins. The SA-FF22 bacteriocin of S. pyogenes is known to share a high degree of similarity with mutacin II produced by S. mutans. Interestingly, bacteriocin production has been reported to be stimulated by acidic pH in lactic acid bacteria (Guerra and Pastrana 2003). We may thus hypothesize that by sensing pH fluctuations, S. mutans may also regulate the expression of the mutacin-encoding genes to allow its own population to compete with other acidogenic bacteria within the human dental biofilm.

We demonstrated that inactivation of the CiaH-encoding gene (hk2) resulted in greater tolerance to oxidative stress. Qi et al. (2004) revealed a role for CiaH in stress tolerance of S. mutans UA140, a clinical strain isolated from a severe caries lesion. Recently, a link between CiaH signal transduction and the surface-anchored serine-protease HtrA has been suggested, which may connect ciaH to stress response pathways in S. mutans (Ahn et al. 2006). Deletion of CiaH also resulted in mutant biofilms with reduced biomass and very short chains, suggesting a role for this TCS in regulating S. mutans cell growth and/or cell division. In fact, growth defective phenotypes have been described in S. pneumoniae cia defective mutants indicating a possible role for CiaRH system in the maintenance of the overall integrity of the cell wall (Mascher et al. 2003).

We also identified a novel system, TCS-9, that controls the development of streptococcal genetic competence. The development of genetic competence enables many streptococci to uptake and integrate exogenous free DNA from their environment, leading to the acquisition of novel genes, the emergence of antibiotic resistance, and the evolution of virulence factors. Consequently, this process enhances the bacterium's ability to survive and adapt in its natural environment. It has been proposed that development of streptococcal competence may be controlled at multiple levels, including more than one TCS sensing different environmental signals (Senadheera et al. 2005b). Our results reinforce the hypothesis that S. mutans may have evolved multiple TCS linked in a complex network to govern genetic competence. Ahn et al. (2006) proposed a model for competence development in S. mutans in which ComDE is the primary TCS responding to CSP while CiaRH integrates signals detected in serum components. In this model, the regulatory network is separated into circuits that are CSP-dependent and -independent. Our results suggests that TCS-9 may also be involved in regulating competence in a CSP-independent manner, albeit outside the comDE circuit, as TE was almost completely restored with the addition of CSP.

It is clear from this study that multiple TCS govern important biological parameters of S. mutans. It will be of particular interest to determine the genes that are controlled by these TCS and whether their function is consistent with the speculative cellular role proposed for each system. TCS are widespread systems used for sensing of extracellular signals. Because of the absence of TCS in human cells, these systems might be relevant targets in a search for more effective ways to treat microbial infections.


This research was supported by NIDRC grant DE013230 and by CIHR operating grant MT-15431. D.G. Cvitkovitch is supported by a Canada Research Chair.


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