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J Bacteriol. Dec 2009; 191(23): 7363–7366.
Published online Sep 25, 2009. doi:  10.1128/JB.01054-09
PMCID: PMC2786547

Multiple Two-Component Systems of Streptococcus mutans Regulate Agmatine Deiminase Gene Expression and Stress Tolerance[down-pointing small open triangle]


Induction of the agmatine deiminase system (AgDS) of Streptococcus mutans requires agmatine and is optimal at low pH. We show here that the VicRK, ComDE, and CiaRH two-component systems influence AgDS gene expression in response to acidic and thermal stresses.

The agmatine deiminase system (AgDS) of Streptococcus mutans catalyzes the conversion of agmatine, a decarboxylated derivative of arginine, to putrescine, CO2, and ammonia, with the concomitant generation of ATP (8). The pathway augments acid tolerance in S. mutans by neutralizing the cytoplasm, and the ATP generated can be used for growth and maintenance (8, 15). Agmatine, which is present in oral biofilms, is inhibitory to the growth of S. mutans, and the AgDS plays a role in eliminating this compound from the environment (8). In S. mutans, the AgDS (aguBDAC) genes are organized in an operon and could be induced by agmatine and further activated by environmental stresses, including acidic and thermal stresses (2). Immediately 5′ to the AgDS genes, but transcribed in the opposite orientation, is the aguR gene, which encodes a transcriptional regulator that activates aguBDAC expression and has its DNA-binding activity controlled by the agmatine-putrescine antiporter (AguD) (15). AgDS gene expression is also sensitive to catabolite repression, affected in part by CcpA (8). Oxygen does not have a substantial impact on AgDS expression. Notably, only a partial Flp protein, which is an activator of the arginine deiminase (AD) genes of Streptococcus gordonii, is encoded in S. mutans, but it appears to be nonfunctional and has no similarity to AguR (A. R. Griswold and R. A. Burne, unpublished data).

Bacterial adaptation to changing environmental conditions is often accomplished by two-component systems (TCS), which modulate gene expression in response to a wide variety of stimuli (3). In many streptococci and some other gram-positive bacteria, the VicRK, ComDE, and CiaRH TCS have been shown to play central roles in stress tolerance. In a companion paper, we demonstrate that the expression of the AD system (ADS), which is highly similar to the AgDS, is influenced by the Cia, Com, and Vic TCS in response to acid and oxidative stresses (14a). We investigated here the involvement of Cia, Com, and Vic in the regulation of the AgDS of S. mutans in response to environmental signals known to activate aguBDAC gene expression.

Strains of S. mutans UA159 carrying mutations in the cia, com, and vic genes and harboring a gene fusion to the aguB promoter (PaguB) fused to the lacZ gene (S. mutans/PaguB-lacZ) (15) were constructed. To make deletions of com (comD and comE), cia (ciaH and ciaR), and vicK genes in S. mutans, 5′ and 3′ flanking regions of each gene were amplified from chromosomal DNA from S. mutans UA159 by using primers described elsewhere (2). The PCR products were ligated together by using BamHI sites designed into each primer set and cloned into the pGEM-T Easy vector (Promega, Madison, WI). These plasmids were digested with BamHI and a tetracycline cassette (Tet) from pLN2 (6) was inserted (Table (Table1)1) . The desired mutagenic plasmids were identified and used to transform S. mutans/PaguB-lacZ (15). Double-crossover mutants of each gene were confirmed by PCR and DNA sequencing. The nonpolar Tet insertion was confirmed to allow efficient readthrough to the downstream genes by real-time PCR (data not shown). To evaluate effects of replacement of the entire ciaRH and comDE operons, both ciaR and ciaH were deleted in strain SmciaRH, and comD and comE were deleted in SmcomDE. Previous studies have indicated that VicR is an essential gene in S. mutans (1, 19), so only a vicK mutant was utilized in these studies. Cells were grown to mid-exponential phase in TY medium, which had been acidified to pH 5.5 by using HCl or buffered at pH 7.0 using 50 mM potassium phosphate buffer. Cells were harvested, and the LacZ and agmatine deiminase (AgD) activities were measured, as previously detailed (15). LacZ activity was expressed in Miller units, and AgD activity was expressed as nmol of N-carbamoylputrescine produced (min × mg of protein)−1 (15). In the SmWT background, cells expressed twofold-higher LacZ activity from PaguB at pH 5.5 than at pH 7.0. A similar phenotype was observed in the SmciaR and SmcomE mutants (Fig. (Fig.1A),1A), whereas no induction of PaguB activity by low pH could be detected in the SmciaH, SmciaRH, SmcomD, SmcomDE, and SmvicK mutants (Fig. (Fig.1A).1A). Plasmid-borne copies of the ciaRH, vicK, or comDE genes of S. mutans on the shuttle plasmid pDL278 (11) were used to complement the respective deletions strains (SmciaRH/ciaRH, SmcomDE/comDE, and SmvicK/vicK). The complementing plasmid constructs were generated by using methods described elsewhere (Y. Liu and R. A. Burne, unpublished data). In all cases, complementation of the mutations restored the ability of transcription from the aguB promoter to be induced at low pH at a level comparable to that observed in the SmWT strain (Fig. (Fig.1).1). In addition to using promoter fusion activity to monitor AgD gene expression activity, AgD enzyme activity was also monitored in cells growing under inducing conditions in acidic or neutral pH (Fig. (Fig.1).1). The results confirmed the involvement of the TCS in low-pH induction and the complementation results. Thus, the Com, Cia, and Vic systems affect the expression of AgDS at the transcriptional level by low pH, but only the histidine kinases CiaH, ComD, and VicK were required for this induction.

FIG. 1.
LacZ (A) and AgD (B) activities of various S. mutans UA159 derivatives carrying a PaguB-lacZ gene fusion (Table (Table1)1) in response to growth under different pH values. All strains were cultured in TY broth containing 25 mM galactose and 10 ...
S. mutans strains used in this study

A previous study from our laboratory demonstrated that the AgDS of S. mutans could be induced by heat stress (8) but not by exposure to H2O2. We recently determined that, unlike the ADS of S. gordonii, there was no difference in AgDS gene expression in S. mutans when cells were cultivated under aerobic or anaerobic conditions (data not shown). To examine a possible involvement of these TCS in heat stress activation of the AgDS in S. mutans, the various mutants were cultured to an optical density at 600 nm (OD600) of 0.3 in TV medium containing 25 mM galactose and 10 mM agmatine. Subsequently, the cultures were incubated at 42°C for 1 h, and the LacZ and AgD activities were measured. In the SmWT background, cells expressed 3.5-fold-higher LacZ activity from PaguB at 42°C than cells maintained at 37°C (Fig. (Fig.2A).2A). A similar phenotype was retained in the com mutants and the SmvicK and SmciaR strains, whereas only 1.5-fold higher PaguB expression at 42°C was noted in the SmciaH and SmciaRH strains compared to the same strains growing at 37°C (Fig. (Fig.2A).2A). Complementation of the ciaRH deletion with plasmid-borne copies of ciaRH restored full responsiveness to thermal stress (Fig. (Fig.2A).2A). AgD activity was consistent with the gene fusion results (Fig. (Fig.2B),2B), indicating a defect in heat induction of the AgDS associated with loss of CiaH.

FIG. 2.
LacZ (A) and AgD (B) activities of various S. mutans UA159 derivatives carrying a PaguB-lacZ gene fusion (Table (Table1)1) with or without heat shock. The strains were cultured in TV medium supplemented with 25 mM galactose and 10 mM agmatine ...

A number of studies have connected acid tolerance in S. mutans with expression of various TCS, including CovR, CiaRH, VicRK, and ComDE (2, 5, 10, 19), although an impaired capacity of cells to grow at pH 5.0 was only observed in com mutants of NG8 (14) and not UA159 (2). In the present study, acid killing experiments were used to evaluate the ability of the cia, com, and vicK mutants to survive a lethal pH. After 45 min of exposure at pH 2.8, the survival rate of cia, comDE, and comD mutants was ~2 logs lower than that of SmWT (Fig. (Fig.3A),3A), whereas a 1-log-lower survival rate of the SmcomE and SmvicK strains was observed (Fig. (Fig.3A).3A). S. mutans is known to adapt to a low pH environment and mount an acid tolerance response (ATR) (4, 9, 17, 18), as manifested by an increased resistance to acid killing and an elevated capacity to grow and metabolize carbohydrates at lower pH values after initial exposure to mildly acidic conditions. After 2 h of preincubation in BHI medium that had been adjusted to pH 5.0 with HCl to allow adaptation, the survival rates of cia mutants and the SmvicK strain, which showed severely compromised acid tolerance, were found to be restored to a level comparable to that of the SmWT strain that had been cultivated at pH 5.0 before challenge at pH 2.8 (Fig. (Fig.3B).3B). The survival rate of com mutants was elevated in a proportion similar to that of strain SmWT, but again the survival rate was lower than that of the acid-adapted SmWT by 1 to 2 logs (Fig. (Fig.3B).3B). Thus, although ComDE were not required for growth of S. mutans strain UA159 at pH 5.0, our studies support that ComDE does contribute to acid resistance in non-acid-adapted S. mutans UA159 in a way that enhances the resistance to acid killing. However, in contrast to what has been previously reported for strain BM71, that the lack of comC, -D, or -E resulted in an attenuated ATR (12), the loss of ComDE in the UA159 background did not diminish the ability of the organisms to mount an ATR. These results confirm an essential role for vicK and ciaRH in acid resistance (2, 5, 10, 13, 19) but also show that these latter TCS are not needed for cells to show enhanced acid tolerance after preexposure to mildly acidic conditions.

FIG. 3.
Acid tolerance assay. (A) S. mutans UA159 derivatives carrying a PaguB-lacZ gene fusion (Table (Table1)1) were grown in BHI medium adjusted to pH 7.0 to an OD600 of 0.3, washed with 0.1 M glycine buffer (pH 7.0), and subjected to acid killing ...

In summary, the present study is the first to disclose a requirement of TCS in activation of the AgD genes by low pH. Further, our results add support to the idea that CiaRH may participate in the general stress response of S. mutans (2, 16), as evidenced by the poor induction of the AgDS by thermal stress in CiaRH-deficient strains. The different behaviors of certain kinase and response regulator mutants also add to the growing body of evidence that there may be substantial “cross-regulation” (2, 7) between sensor kinases and response regulators of the TCS of S. mutans. The AgDS conveys bioenergetic advantages to S. mutans through enhancement of ΔpH and generation of ATP (8) and benefits the organisms by the elimination of agmatine, which antagonizes the growth of this organism, from the environment. Thus, it is by these mechanisms that the AgDS is thought to enhance the persistence and virulence of this organism. Clearly, an important question arising from the present study is how ComE, CiaR, or VicK exert their effects on PaguB-driven transcription. Direct binding of all three response regulators to PaguB seems unlikely, whereas cross-regulation of a single response regulator by different histidine kinases or alterations in expression of a separate regulatory protein by all of the TCS seem more plausible. Whether there is direct binding of one or more of the response regulators to the aguB promoter region is currently under investigation. Deciphering the molecular mechanism(s) that underlie Cia, Com, and Vic signaling in AgDS regulation should foster a better understanding of virulence gene regulation in S. mutans. Furthermore, comparing the signals for the Vic, Com, and Cia and the scope of the regulons of these TCS in the oral pathogen S. mutans and the oral commensal S. gordonii (Liu and Burne, unpublished) may highlight differences that could guide the development of therapeutics that compromise the cariogenic potential of S. mutans without adversely affecting beneficial commensals.


This study was supported by Public Health Service grant DE10362 and DE13239 from the National Institute of Dental and Craniofacial Research.


[down-pointing small open triangle]Published ahead of print on 25 September 2009.


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