The cia Operon of Streptococcus mutans Encodes a Unique Component Required for Calcium-Mediated Autoregulation

doi:10.1111/j.1365-2958.2008.06390.x

Mol Microbiol. Author manuscript; available in PMC 2010 October 15.

Published in final edited form as:

Mol Microbiol. 2008 October; 70(1): 112–126.

Published online 2008 August 4. doi: 10.1111/j.1365-2958.2008.06390.x

PMCID: PMC2955730

NIHMSID: NIHMS236274

The cia Operon of Streptococcus mutans Encodes a Unique Component Required for Calcium-Mediated Autoregulation

Xuesong He,²^ζ Chenggang Wu,¹^ζ Daniel Yarbrough,² Lucy Sim,² Guoqing Niu,¹ Justin Merritt,¹ Wenyuan Shi,² and Fengxia Qi¹^*

¹College of Dentistry, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73034

²UCLA School of Dentistry, Los Angeles, CA 90095

^* Corresponding author. University of Oklahoma Health Sciences Center, 975 NE 10^th Street, Oklahoma City, OK 73104. Phone: (405) 271-3668. FAX: (405) 271-3603. Email: Felicia-qi/at/ouhsc.edu

^ζThese two authors contributed equally to this work.

The publisher's final edited version of this article is available at Mol Microbiol

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Abstract

Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two-component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double-glycine-containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine-aspartate (SD) domain that is shared by calcium-binding proteins. A markerless in-frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD-domain resulted in the same phenotype as the in-frame deletion, indicating that the SD-domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in-frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real-time RT-PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three-component, self-regulatory system modulating cellular functions in response to calcium.

Introduction

Bacterial two-component signal transduction systems (TCS) play important roles in bacterial environmental adaptation, production of virulence factors, self-defense, and even biofilm formation. A typical TCS consists of a membrane-bound sensor kinase and a cytoplasmic response regulator. Upon receiving a signal, the sensor kinase undergoes autophosphorylation. The phosphorylated kinase then transfers the phosphate group to its cognate response regulator, which then activates or represses its target genes by binding to their promoter regions (Stock et al., 2000).

Streptococcus mutans is a primary pathogen causing dental caries in humans. S. mutans is also a member of the normal oral microbiota that colonize the tooth's surface in a biofilm commonly known as the dental plaque. The dental plaque is unique among known biofilms in that its cell density can reach as high as 10¹¹ cells/g wet weight (Hamilton, 2000). The oral cavity is also unique in that it experiences cycles of famine and feast from daily meals, and constant antagonistic insults from saliva, which contains saturated concentrations of calcium and phosphate as well as antimicrobial substances like isothiocyanate, peroxide, lysozyme, and secretory IgA. To survive, persist, and eventually become dominant, like in the caries lesion, S. mutans must be able to sense and respond to these various environmental signals as well as the presence of other competitors. Presumably, much of this ability is coordinated by the 14 pairs of TCS encoded in the S. mutans genome (Biswas et al., 2008).

The functions of these TCS in various aspects of cellular activities have been partially characterized. The ComED system is involved in competence regulation as well as bacteriocin production (Kreth et al., 2005; Li et al., 2002b), the LevSR system was found to be required for induction of the fruA and levD operons by fructose (Zeng et al., 2006), HK03/RR03 are involved in acid tolerance (Li et al., 2002a), and the VicRK system was found to be involved in regulation of sucrose-dependent adhesion, competence development, and biofilm formation (Senadheera et al., 2005; Senadheera et al., 2007).

Perhaps the best characterized TCS is the CiaRH system. In a previous study, we demonstrated that inactivation of ciaH, the membrane-bound histidine kinase sensor, in S. mutans strain UA140 diminished bacteriocin (mutacin) production, dramatically reduced natural competence, altered surface attachment and biofilm formation, and increased sensitivity to acidic pH (Qi et al., 2004). These findings were subsequently confirmed in another S. mutans strain, UA159 as well (Ahn et al., 2006; Biswas et al., 2008). While the phenotypic effect of cia operon mutations has been thoroughly investigated, the mechanism of how this TCS regulates such a diversity of cellular functions is still largely uncharacterized.

The CiaRH TCS is also one of the most studied two-component systems in the closely related streptococcal species, S. pneumoniae. It was first discovered in a laboratory mutant selected for resistance to the β-lactam antibiotic cefotaxime (Guenzi et al., 1994). This mutant was also found to be defective in competence. Further studies demonstrated that the phenotype is caused by a point mutation in the CiaH gene, rendering it constitutively active (Giammarinaro et al., 1999). Deletion of the response regulator, CiaR, increased competence as well as stationary phase lysis (Dagkessamanskaia et al., 2004). Solid phase DNA binding assays identified 26 potential CiaR target sites that include genes important for the synthesis and modification of cell wall polymers, peptide pheromones, bacteriocin production, and the htrA-spo0J region (Mascher et al., 2003; Mascher et al., 2006). A recent study identified a CiaR binding site consensus sequence TTTAAG-n5-TTTAAG that is ~10 bp upstream of the -10 region (Halfmann et al., 2007).

In this study, we further characterized the ciaRH operon in S. mutans. We demonstrate that unlike the ciaRH operon in S. pneumoniae, the ciaRH operon in S. mutans consists of three genes with the first gene encoding a small, double-glycine (GG) containing peptide, which acts as a calcium sensing signaling peptide that allows the CiaRH system to modulate its own operon expression in response to calcium. The significance of this finding in terms of how S. mutans adapts to the unique environment in the oral cavity is discussed.

Results

Phenotypic microarray screening for an environmental signal that regulates ciaRH operon expression

Previous studies in our laboratory demonstrated that the ciaH null mutation exhibits a pleiotropic phenotype: diminished mutacin production and competence development, reduced acid tolerance, and altered biofilm formation (Qi et al., 2004). Based upon these results, we were curious to determine how the CiaRH system is able to regulate such a diverse array of cellular functions and what the signal(s) for the system could be. We began by first investigating how the ciaRH operon itself is regulated. A luciferase (luc) reporter strain (UA140 ciaH-luc) was constructed, in which the promoter-less luciferase gene was transcriptionally fused with ciaH, the last gene of the operon, and the construct was integrated into the chromosome at its native location as a single copy. This reporter strain was used in a Phenotype Microarray assay (PM) (Biolog, Inc) to search for environmental signals that activate or repress ciaH-luc gene expression. A Phenotype microarray consists of a series of 96-well plates that contain unique chemicals and metabolites in each well. It was originally designed for screening mutations affecting carbon, nitrogen, phosphate, and sulfur utilization pathways, or growth stimulation or inhibition by amino acids, nutrients, osmolytes, metabolic inhibitors, and antibiotics (von Eiff et al., 2006; Zhou et al., 2003). We adapted PM analysis to screen for metabolic or environmental signals affecting ciaRH operon expression.

Overnight cultures of the reporter cells were diluted into fresh TH medium and added to the wells of the PM plates. Luciferase activity was measured during late-log phase, when ciaRH operon was at its peak expression level. As expected, the majority of the wells did not show altered luciferase activity as compared with the control. However, one of the wells (A2) in PM2A plate exhibited 3-fold higher luciferase activity (data not shown). The chemical affecting reporter activity was identified as chondroitin sulfate C, a member of the glycosaminoglycan family of heteropolysaccharides. Chondroitin sulfate C is found in humans in cartilage, bone, cornea, and skin, and binds to Ca⁺⁺ with high affinity (Tanaka, 1978). This implied that calcium might be an inhibiting factor for ciaRH operon expression.

To test this, different concentrations of EGTA were added to the TH medium, and the ciaH-luc gene expression was measured over the growth curve. As shown in Fig. 1A, adding 0.2 mM EGTA to the medium increased operon expression nearly 3-fold, although cell growth was not affected. Adding 5 mM CaCl₂ together with 0.2 mM EGTA completely reversed the stimulating effect. Adding the same concentration of EDTA did not have any effect, although higher concentrations of EDTA inhibited cell growth (data not shown). Since EDTA has lower chelating capacity for Ca⁺⁺ than EGTA, we suspected that the remaining free Ca⁺⁺ present in the TH medium was sufficient to repress ciaRH operon expression.

Fig. 1

Effect of calcium on ciaRH operon expression

To further test the effect of calcium on ciaRH operon expression, we developed a chemically defined artificial saliva solution (ASS) based on the formula of a chemically defined medium for streptococci and the electrolyte balance of saliva (see Materials and Methods). This medium supports a cell yield for S. mutans and other oral streptococci that is comparable to complex media such as BHI and TH (Merritt et al., unpublished). UA140ciaH-luc was grown in ASS medium with and without added calcium, and ciaH-luc gene expression was measured over time. As shown in Fig. 1B, ciaH-luc gene expression was reduced over 2-fold when cells were grown in ASS medium with 0.5 mM CaCl₂ compared with those grown in ASS medium without added calcium [higher concentrations of calcium further reduced operon expression, but also caused salt precipitation of the medium (data not shown)]. In contrast, MgCl₂ or MnCl₂ had no effect on operon expression (data not shown).

To further confirm that the calcium repression of ciaH-luc gene expression was not due to the fusion of the luc gene, real-time RT-PCR was performed directly with the RNA isolated from the original UA140 strain growing in ASS medium with and without added calcium. As shown in Fig. 1C, adding 0.5 mM calcium to the ASS medium reduced gene expression 2.5- to 3-fold for all three genes in the operon (see Fig. 2A for operon organization). Taken together, these results suggest that Ca⁺⁺ is perhaps the environmental signal repressing ciaRH operon expression.

Fig. 2

A. genomic organization of the ciaRH operon in sequenced genome of streptococci. B. Sequence of the CiaX prepeptide. The GG is the predicted cleavage site for the mature peptide. The SD domain sequence is underlined. C. Alignment of the S. aureus Bap (more ...)

Unique features of the ciaRH operon – identification of the ciaX gene

Data presented above demonstrated that Ca⁺⁺ is an environmental signal that represses ciaRH operon expression. We were next interested to determine how this signal was received and transduced to regulate ciaRH operon expression. The ciaRH operon is present in all sequenced streptococcal genomes, while it is not present in other gram-positive bacteria. An early study in S. pneumoniae demonstrated that the ciaRH operon was activated by calcium deprivation, and it was suggested that Ca⁺⁺ exerts its effect by either directly binding to CiaH or indirectly through an unknown mechanism (Giammarinaro et al., 1999). Comparison of the S. mutans CiaRH with that of S. pneumoniae revealed nearly identical sequences for CiaR (89% identities, 93% positives throughout the entire length), however, the similarity for CiaH is mainly at the C-terminal half of the protein where the His-kinase domain is located. A more striking difference was found in the genomic organization of the operon (Fig. 2A). While in S. pneumoniae and other non-oral streptococci the ciaRH operon contains only the ciaR and ciaH genes, the ciaRH operon in S. mutans actually contains three genes, the first encoding a hypothetical protein annotated as SMU.1131c. To see whether SMU1131c is indeed part of the ciaRH operon, real-time RT-PCR was performed on SMU1131c, ciaR, and ciaH. Similar transcript abundance was observed for all genes (Fig. S1), suggesting that SMU.1131c is indeed the first gene of the ciaRH operon.

SMU1131c encodes a small peptide of 87 aa with a double glycine (GG) at position 24-25, which is reminiscent of the leader peptide shared by most bacteriocin and pheromone prepeptides in gram-positive bacteria. InterProScan predicted a signal peptide sequence at the N-terminus from position 1-29, and thus designated the peptide as extracellular (http://www.oralgen.lanl.gov/). Hence, we assumed that a 62 aa peptide could be produced and secreted outside of the cell upon cleavage at the GG cleavage site. The most striking feature of this mature peptide is a C-terminal stretch of 28 aa that contains multiple serine + aspartate (SD) and serine + asparagine (SN) repeats. In addition, this segment is separated from the N-terminal portion by several proline residues (Fig. 2B). A literature search of proteins with SD repeats led us to a group of calcium binding proteins that are involved in a variety of cellular functions (Dominguez, 2004; Michiels et al., 2002; Rigden et al., 2003a; Rigden et al., 2003b). These proteins all contain an EF-hand motif with a SD-containing calcium binding loop. Sequence alignment of the SMU.1131c C-terminal domain with the EF4 calcium binding loop of Bap from Staphylococcus aureus (Arrizubieta et al., 2004) revealed high similarity. As shown in Fig. 2C, five out of the six conserved residues in the calcium-binding loop of EF4 are also conserved in the SD repeat region of SMU.1131c. Taken together, these findings suggest that SMU1131c could be a calcium binding peptide involved in calcium mediated signal transduction by the CiaRH TCS in S. mutans. Therefore, SMU1131c was renamed ciaX to reflect its affiliation with the ciaRH TCS, and the ciaRH operon was renamed ciaXRH.

CiaX is required for calcium repression of ciaXRH operon expression

To determine the function of CiaX, we first created an allelic replacement deletion of ciaX by inserting a terminator-less kanamycin (Kan) resistance gene. This mutation displayed a similar phenotype as the ciaH mutation described previously (Qi et al., 2004): reduced mutacin production (data not shown). However, a knock-in replacement of ciaX could not complement the phenotype (data not shown). This suggested that the observed phenotype could have resulted from altered expression of the downstream ciaRH gene, driven possibly by the Kan cassette promoter. Thus, we decided to create a markerless in-frame deletion using the recently developed galactose-mediated counterselection system for S. mutans (Merritt et al., 2007). For this deletion, ciaX was deleted from the start codon to the stop codon, with no antibiotic marker in between. Therefore, the operon was only under the control of its native promoter. This strain was transformed with the ciaH-luc construct to make a ciaH-luc reporter with a ciaX null mutation background, and named UA140ciaXIFD.

The effect of the ciaX null mutation on ciaXRH operon expression was measured in ASS medium with and without added Ca⁺⁺. As shown in Fig. 3A, deletion of ciaX reduced reporter activity over 2-fold in the absence of Ca⁺⁺ (compare the level of luciferase activity between the wt in Fig. 1B, and the mutant in Fig. 3A), and eliminated the calcium repressive effect on operon expression. When a complete copy of ciaX was integrated back to the chromosome of UA140ciaXIFD (UA140ciaXknock-in), a response similar to the wild-type was observed (Fig. 3B). These results suggest that ciaX is required for the Ca⁺⁺ repression of ciaXRH operon expression.

Fig. 3

Function of CiaX in calcium repression of ciaXRH operon expression

The SD domain is required for the function of CiaX

In silico analysis of the CiaX peptide suggested that the SD domain may be involved in Ca⁺⁺ binding and the function of the CiaX (Fig. 2C). To test this, we made point mutations in the ciaX gene, in which the SDSDSD repeats were changed to SASASA (Fig. 4A). This mutant gene was then integrated back onto the chromosome of UA140ciaXIFD to form UA140ciaXSD. The ciaH-luc expression of UA140ciaXSD was measured in ASS medium with and without Ca⁺⁺. As shown in Fig. 4B, the ciaX-SD mutation exhibited the same phenotype as the ciaX in-frame deletion (compare Fig. 4B with Fig. 3A), suggesting that the SD domain is essential for the function of CiaX.

Fig. 4

Effect of CiaX SD mutations on CiaX function

Chemically synthesized C-terminal CiaX peptide binds calcium ions in vitro

To test whether the C-terminal SD domain of CiaX indeed binds to calcium ions, a 26-aa C-terminal peptide (CiaX-SD) with the sequence DSSQSDSDSDSNSSNTNSNSSITNG was chemically synthesized and labeled with Fluorescein. Equilibrium dialysis experiments were then carried out against varying concentrations of CaCl₂ (see Materials and methods). The amount of calcium remaining in solution was calculated using the Calcium Green-2 fluorescent indicator system (Invitrogen), and binding of calcium to the peptide was inferred from reductions in the calcium concentration at equilibrium, relative to the expected equilibrium concentration in the absence of peptide. As shown in Fig. 5, the molar binding of Ca⁺⁺ bound to the peptide increases with increasing calcium concentrations. When a molar ratio of calcium/peptide of 1.6 was present in solution, a roughly 1:1 ratio of bound calcium to peptide was observed; when calcium concentration was increased to a 2.0 : 1 ratio, a roughly 3.5 : 1 of bound calcium to peptide was obtained. Further increase in calcium concentration did not seem to increase binding, indicating that the maximal binding capacity of the peptide is ~3-4 molecules of Ca⁺⁺ per molecule of peptide.

Fig. 5

Binding of Ca⁺⁺ to the CiaX-SD peptide

CiaX is involved in calcium mediated biofilm formation

Results presented above imply that CiaX may be involved in surface binding and biofilm formation on the tooth surface. To test this, a biofilm assay was performed with the wild-type, ciaX, ciaXknock-in, and ciaXSD strains. As shown in Fig. 6, the ciaX deletion and the ciaXSD mutation both diminished biofilm formation in the absence of added calcium in ASS medium supplemented with 1% sucrose (6C & 6G), while the ciaXknock-in rescued this phenotype (6F). Adding 0.3 mM calcium to the medium suppressed the ciaX defect in biofilm formation (6D & 6H), suggesting that CiaX is probably involved in calcium mediated biofilm formation in S. mutans.

Fig. 6

Effect of ciaX mutation on biofilm formation

The CiaXRH system is involved in autoregulation in response to Ca⁺⁺

Data presented so far demonstrate that the small peptide CiaX can bind to calcium and is required for Ca⁺⁺ mediated repression of ciaXRH operon expression as well as biofilm formation. It remained unclear whether the CiaX is a signaling peptide for the CiaRH TCS or it is part of a signaling system with other proteins. To test this, two strains, UA140ciaRIFD and UA140ciaH were constructed, which contained an in-frame deletion of ciaR and an insertional mutation of ciaH, respectively. UA140ciaRIFD contained the ciaH-luc fusion on the chromosome, which was constructed in the same manner as in UA140ciaXIFD. The UA140ciaH also contained a ciaH-luc fusion, which was constructed simultaneously with the insertional mutation (see Materials and Methods). The ciaH-luc gene expression in UA140ciaRIFD and UA140ciaH was measured in ASS medium with and without added Ca⁺⁺. As shown in Fig. 7, both ciaR and ciaH diminished calcium repression on operon expression, suggesting that the CiaRH TCS and CiaX are in the same pathway leading to ciaXRH operon regulation. It is also worth noting that deletion of ciaH appeared to have a much larger effect on operon expression than ciaX or ciaR mutations. While the ciaX and ciaR mutations reduced operon expression 2- to 2.5-fold in the absence of calcium, the ciaH mutation reduced operon expression ~10-fold (compare the RLU/OD₆₀₀ of Fig. 7A with Fig. 1B). This suggests that, in addition to controlling calcium modulation of operon expression together with CiaX and CiaR, CiaH also controls the basal level of ciaXRH operon expression, possibly through interacting with another response regulator.

Fig. 7

Effect of ciaR and ciaH mutations on ciaXRH operon expression in response to calcium

The ciaXRH promoter region contains a putative CiaR binding site

With a combination of in silico analysis and promoter probing studies, Halfman et al. identified a set of genes that are directly regulated by CiaR (Halfmann et al., 2007). Comparison of the promoter region of these genes identified a putative CiaR binding site with a consensus sequence TTTAAG-n5-TTTAAG that is located ~10 bp upstream of the -10 region. Mutagenesis and gel shift studies demonstrated that the core sequence TTAAG is absolutely required for CiaR binding. To see whether this sequence also exists in S. mutans, we scanned the intergenic regions (IGR) of the 427 contigs of the nearly completed genome sequence of UA140 for T/ATTAAG direct repeats located about 10 bp upstream of a putative -10 sequence. Six IGRs were identified, one of which is upstream of ciaX (IGR889 in UA159). Using a nested RT-PCR, we located the putative transcription start site and demonstrated that the putative CiaR binding site is indeed 10 bp upstream of the predicted -10 region (Fig. 8). In vitro gel shift assays will determine whether CiaR indeed binds to this putative CiaR binding site in S. mutans. It is also worth noting that another IGS with a putative CiaR binding site is upstream of htrA, which has been shown to be regulated by the CiaRH TCS (Ahn et al., 2006).

Fig. 8

The ciaXRH operon promoter region and the putative CiaR binding site

Discussion

In this study, we identified Ca⁺⁺ as the environmental signal that modulated the ciaXRH operon expression in S. mutans. Further studies demonstrated that the first gene of the operon, now renamed ciaX, encodes a small, secreted peptide with a calcium-binding (SD) domain. An in-frame deletion of ciaX as well as point mutations of the SD domain diminished calcium repression as well as reduced operon expression. An in-frame deletion of ciaR, the response regulator, resulted in the same phenotype as the ciaX mutations, while inactivation of ciaH, the histidine kinase sensor, further diminished operon expression. Based on these results, we concluded that the ciaXRH operon encodes a three-component signal transduction system, with the calcium-binding peptide CiaX mediating calcium repression of operon expression. In addition, the ciaXRH operon is subject to two tiers of regulation: the basal level, which only requires CiaH; and the activated level, which is modulated by calcium and dependent upon CiaR and CiaX. Furthermore, since CiaR is not required for the basal level expression, there exists a possibility of an as yet unknown response regulator (RRx) working with CiaH to regulate the basal level expression of the operon. Fig. 9 depicts a working model developed based on results presented in this communication.

Fig. 9

Model for CiaX mediated calcium modulation of ciaXRH operon expression

This model is reminiscent of the signal perception and transduction model proposed for the E. coli PhoQ (Bader et al., 2005). Early studies of the CiaH protein of S. pneumoniae suggested that it is distantly related to PhoQ (Giammarinaro et al., 1999), and a recent study suggests that they both belong to the same group of periplasm sensing HKs (Mascher et al. 2006), which contain 2 transmembrane (TM) domains separated by a 60-300 aa periplasmic loop. PhoQ is activated by low concentrations of cations like Mg⁺⁺ as well as by increasing concentrations of antimicrobial peptides, but inactivated by high concentrations of cations. The periplasmic domain of PhoQ forms an acidic flat surface at the membrane proximal side of the protein. It was proposed that Mg⁺⁺ binding at the acidic surface tethers the periplasmic domain to the membrane, and at this state, the PhoQ kinase is inactive. The binding of the antimicrobial peptide displaces the cation and disrupts the interaction between the periplasmic domain and the membrane. This disruption causes a structural distortion, which could be transmitted mechanically to the transmembrane (TM) helices, resulting in autophosphorylation of PhoQ (Bader et al., 2005).

In our model, CiaX is an unstructured peptide with the N-terminal half strongly positively charged (PI = 9.88) and the C-terminal half strongly negatively charged (PI = 3.32). The C-terminal half also contains the SD-domain. In the absence of calcium, CiaX probably folds into a hairpin structure through the ionic interaction between the N- and C-terminal halves. This folding neutralizes the charge enabling it to bind to the extracytoplasmic loop of CiaH (~140 aa). Binding of CiaX results in the autophosphorylation of CiaH, which then activates CiaR. Activated CiaR binds to the direct repeats upstream of the -10 region, enhancing transcription of the ciaXRH operon (Fig. 9A). In the presence of calcium, Ca⁺⁺ binds to the SD domain of CiaX, changing its configuration, thus preventing its binding to CiaH. In the absence of CiaX binding, CiaH is able to activate an unknown response regulator (RRx) via an unknown mechanism, leading to the basal level transcription of the ciaXRH operon (Fig. 9B). Similarly, mutations of ciaX or ciaR also do not affect the basal level transcription of the operon mediated via RRx (Fig. 9C). However, in the absence of CiaH, none of these pathways can be activated and the operon transcription is severely diminished (Fig. 9D). This model is consistent with the data presented so far. The assumption that CiaH may have a yet unknown partner in addition to CiaR is based on previous findings that a ciaR deletion did not have any effect on the cellular functions affected by the ciaH mutation (Qi et al., 2004). It is also supported by a recent transcriptome analysis of ciaH and ciaR mutations, in which we found that the majority of affected genes do not overlap between the two mutations (Wu et al. unpublished data). The possibility of a second response regulator for CiaH was suggested by other investigators as well (Ahn et al., 2006).

Calcium signaling is a very important cellular function in eukaryotic cells. It regulates a variety of cellular processes from cell cycle, metabolism, motility, to differentiation, stress response, and pathogenesis (Ikura et al., 2002; Mekalanos, 1992; Sanders et al., 1999; Whitaker & Larman, 2001). Different mechanisms are involved in calcium signaling, which include ion channels, calcium binding proteins, and ion condensation (Crivici & Ikura, 1995; Ripoll et al., 2004). Although evidence for the presence of calcium signaling in prokaryotes is still elusive, in silico analysis of proteins encoded by a large number of bacterial genomes identified a group of proteins that contain an EF-hand like domain similar to that of calmodulin, the prototype calcium binding protein in eukaryotic cells (Rigden et al., 2003a; Rigden et al., 2003b). The EF-hand domain contains a 12-aa calcium-binding loop flanked by two α-helices. Residues at position 1, 3, 5, 7, 9, and 12 of the loop provide ligands that chelate Ca⁺⁺ ions. Residues at these positions are highly conserved and are usually D, N, or E.

A common feature of these bacterial EF-hand containing proteins is that they are either surface bound or secreted. One of the proteins, calsymin from the gram-negative bacterium, Rhizobium etli, contains 6 EF-hand motifs, and has been shown to bind Ca⁺⁺, which is required for completion of the bacterial lifecycle (Xi et al., 2000). Another protein from the Gram-positive Staphylococcus aureus, Bap (for biofilm associated protein), contains 4 putative EF-hand domains, and two of them are shown to be required for calcium regulation of biofilm formation (Arrizubieta et al., 2004). CiaX is unique among these putative bacterial calcium-binding proteins in that it is extremely small (62 aa) and contains only the calcium binding loop (the SD-domain). Therefore, unlike the typical EF-hand domains, the SD loop in CiaX is not flanked by an α-helix; instead, the entire region appears to be in a non-structured configuration. This suggests that CiaX may not work as a typical calcium binding protein. Instead, it may work similarly like the cationic antimicrobial peptide that regulates the PhoQ activity in E. coli (Bader et al., 2005).

Calcium as a universal messenger plays a pivotal role in all aspects of a eukaryotic cell's life, but the role of calcium in a bacterial cell's life is less clear. However, unlike bacteria living in other environments, oral microbes are bathed in saliva, which is saturated with calcium [the average human saliva contains 1.2 mM Ca⁺⁺ (Agha-Hosseini et al., 2006)]. Salivary calcium is known to play an important role in the re-mineralization of the demineralized tooth enamel caused by acids produced by acid producing bacteria like S. mutans; however, the role of such a common salivary component in the growth of oral microbes is largely uncharacterized. A literature search found only one article, which reported that S. mutans growth was stimulated by low concentration of calcium (0.63 μM), but repressed by higher concentrations of calcium (1.3 -2.5 μM) (Aranha et al., 1986). Our data show that in addition to suppressing ciaXRH operon expression, 0.5 mM calcium also suppressed cell growth by increasing the length of the lag phase as well as reducing the growth rate (see Fig. 1B). Thus, it is conceivable that free-living S. mutans cells in saliva may not be able to grow as well as those in the dental plaque, in which a low-calcium microenvironment could exist as a result of consumption by other biofilm cells.

In addition to affecting cell growth, our results showed that calcium also affected biofilm formation, one of the most important virulence factors of S. mutans, and that CiaX and the SD-domain of CiaX were involved in this process (Fig. 6). Interestingly, the effect of calcium on biofilm formation appears to be opposite to that on cell growth. Here, in the absence of added calcium, CiaX is required for biofilm formation; however, in the presence of 0.3 mM calcium, biofilm forms in the absence of CiaX (Fig. 6). It is possible that even in the absence of added calcium, the ASS medium still contains trace amounts of calcium from contamination of the chemical ingredients. In this case, CiaX would act like a calcium scavenger to recruit calcium to the cell surface to assist biofilm formation; while in the presence of high concentrations of calcium, CiaX is no longer needed. This interesting paradox could be the result of co-evolution between the parasite (S. mutans) and the human host – saliva inhibits cell growth but promotes biofilm formation, an essential step for S. mutans to survive in the oral cavity. It will be very interesting to see whether other oral streptococcal species respond to calcium in similar fashions.

It is also worth noting that the ciaXRH operon exhibits a different expression pattern in complex medium such as TH than in chemically defined medium such as ASS. In TH, the operon expression follows the growth curve, turning on at early log and off at late log/early stationary phase (Fig. 1A). In ASS medium, the operon turns on during lag phase, and turns off at early log phase (Fig. 1B). The mechanism of this differential expression pattern is not understood at present, but promoter sequence analysis suggests that a second promoter-like sequence in the 50-bp intergenic region between ciaX and ciaR is probably utilized as a promoter to drive the transcription of ciaR and ciaH only when cells are grown in complex medium. This supposition is supported by our recent microarray study on cells grown in the complex medium BHI. We observed 3-5 times higher signal levels for ciaR and ciaH than for ciaX, which was also confirmed by real-time PCR (data not shown). In contrast, real-time PCR of the ciaXRH genes with cells grown in ASS medium with or without added calcium showed similar levels of gene expression for all three genes (see Fig. S1).

Finally, it is interesting to note that among the known ciaRH operons in the sequenced genomes of streptococci, the three oral streptococcal species (S. mutans, S. gordonii, S. sanguinis) all have an extra orf preceding ciaR (Fig. 2A). While the S. mutans ciaX has been shown in this study to be part of a TCS system involved in calcium mediated autoregulation, the function of the other orfs in S. gordonii and S. sanguinis is unknown. Interestingly, the other two orfs (SGO_1071 and SSA_0958) also encode peptides/small proteins (119 and 120 aa, respectively) with a predicted GG-containing leader peptide, similar to that of CiaX. However, while the mature S. mutans CiaX peptide is strongly positively charged in the N-terminal half and strongly negatively charged in the C-terminal half, the entire proteins of SGO_1071 and SSA_0958 are positively charged (PI=8.19 and 8.20, respectively). It will be interesting to see whether these two proteins are also involved in calcium-mediated autoregulation.

Materials and Methods

Bacterial strains and medium

S. mutans strain UA140 was used as the parent strain. UA140 derivatives constructed in this study are listed in Table S1 of Supplemental Materials. All S. mutans strains were grown in Brain Heart Infusion (BHI, Difco; Sparks, MD) agar plates supplemented with either spectinomycin (Spc; 800 μg/ml), kanamycin (Kan, 800 μg/ml), or erythromycin (Erm; 15 μg/ml). Todd-Hewitt broth (TH) was used in the phenotypic microarray (PM) assay and the EGTA assay for calcium. For luciferase reporter assays, all strains were grown in artificial saliva solution (ASS) with or without added calcium. The ASS medium was modified from the chemically defined medium by Socransky (Socransky et al., 1985), and contains the following ingredients:

Glu (2.54 g/L), Cys (0.2 g/L), Leu (0.15 g/L), Lys (0.15 g/L), Arg (0.21 g/L), Pro (0.006 g/L), Gly (0.004 g/L), adenine (0.009 g/L), K2HPO4 (11.9 mM), KH2PO4 (21.5 mM), NH4Cl (10 mM), Urea (5 mM), Sodium Pyruvate (5.5 mM), MgCl2 (5.9 mM), MnCl2 (0.1 mM), FeSO4 (0.1 mM), CaCl2 (1 mM), Riboflavin (1 mg/L), Thiamine HCl (0.5 mg/L), D-Biotin (0.1 mg/L), Nicotinic acid (1 mg/L), p-Amino-benzoic acid (0.1 mg/L), a-panthothenate (0.5 mg/L), Pyridoxine (1 mg/L), a-panthothenate (0.5 mg/L), Pyridoxine (1 mg/L), Folic acid (0.1 mg/L), and glucose (0.5 %). The amino acids, salts, and vitamins were made in stock mixtures separately, and filter-sterilized. Glucose was made as a stock, filter-sterilized, and added with other ingredient mixtures. Stock solutions were stored in 4° C and used within one month, and ASS solution was used within one week. All S. mutans strains were grown anaerobically (90% N₂ / 5% CO₂ / 5% H₂) at 37° C.

Phenotypic microarray assay

Overnight culture of S. mutans ciaH-luc reporter strain was diluted in fresh TH medium to an OD₆₀₀ of 0.005. 100 μl of the diluted culture was added to each well of the Phenotypic Microarrays plates (Biolog, Inc. Hayward, CA. USA). Plates were incubated at 37°C under anaerobic condition for 24 h before 25 μl 1 mM D-luciferin (Sigma) suspended in 100 mM citrate buffer was added to each well. Optical density at 600 nm and Luciferase activity of each well was measured using a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.).

Quantification of the ciaRH operon expression by Real-time RT-PCR

An overnight culture of S. mutans grown in ASS medium without calcium was diluted 1:20 in ASS medium with or without 0.5 mM calcium. The two sets of cultures were grown at 37°C in an anaerobic chamber for ~4 – 6 h, and cells were collected by centrifugation. The cell pellet was resuspended in 1 ml of ice cold Trizol and disrupted with 300 μl of 0.1 mm Zirconia Beads using Fastprep (MP Biomedicals). After centrifugation, the supernatant was collected and the RNA purified using the Ambion RiboPure™ –Bacteria Kit according to the manufacturer's protocol. Isolated RNA was treated with DNAse I (Ambion) to remove traces of chromosomal DNA. After the treatment, RNA samples were cleaned with the Qiagen RNeasy MinElute™ cleanup kit. 300 ng of total RNA was used for cDNA synthsis using Stratascipt RT (Stratagene) according to the manufacturer's protocol. Real-time PCR primers for ciaX, ciaR, ciaH, and 16S RNA are listed in Table 1 of the Supplemental Materials. For real-time PCR, SYBR green (Bio-Rad) was used for fluorescence detection with the 7300 real-time PCR system according to the manufacturer's protocol.

Construction of the ciaH-luc reporter gene fusion

The suicide vector for the construction of all reporter gene fusions was pFW5-luc, which contains a spectinomycin resistance marker (aad9) that works in both Gram-negative and Gram-positive bacteria (Podbielski et al., 1996). To construct a single-copy luciferase transcription fusion to the cia operon, the 3′ portion of ciaH was amplified by PCR from the chromosomal DNA of strain UA140 with primers ciaH3′F and ciaH3′R (Table S1). The PCR product was digested with XhoI and BamHI, and ligated into the BamHI and SalI site (compatible with Xho I) to form plasmid pFW5

3′ciaH-luc. Plasmid pFW5

3′ciaH-luc was transformed into UA140 and transformants were selected on BHI plates containing spectinomycin (800 μg/mL). Integration of the plasmid into the chromosome was confirmed by PCR.

Construction of in-frame deletion mutants of ciaX and ciaR

Previously, we have constructed a marker-less in-frame deletion system in S. mutans, which allows deletion of a gene within an operon without affecting the expression of the downstream gene (Merritt et al., 2007). To construct a ciaX in-frame deletion, a 1 kb fragment of upstream and downstream of ciaX was amplified by PCR with primers 1131upF and 1131upR, and 1131dnF and 1131dnR (Table S1), respectively. The upstream fragment was digested with Xba I and BamHI and cloned into pBluescript™ at the same restriction sites to generate pBS-1131up. The downstream fragment was then digested with BamHI and Kpn I, and cloned into pBS-1131up at the BamHI and Kpn I sites to generate pBS-Δ1131. The insert was removed by digestion with BssHII and ligated into the in-frame deletion vector pIFD-Sm (Merritt et al., 2007) digested with the same enzyme to create the plasmid pIFD-1131.

To create an in-frame deletion of ciaR, a 0.9 kb fragment from the upstream and downstream of ciaR was amplified by PCR using primers ciaRupF and ciaRupR, and ciaRdnF and ciaRdnR (Table S1), respectively. The upstream fragment was digested with Hind III and Sac I, the downstream fragment with Sac I and BamHI, and the in-frame deletion vector pIFD-sm with Bam HI and Hind III. The upstream fragment, downstream fragment, and the vector were ligated together in a single ligation step, and the correct plasmid, pIFD-ciaR, was confirmed by restriction enzyme digestion.

To construct the S. mutans strains with the ciaX and ciaR in-frame deletions, plasmid pIFD-1131 and pIFD-ciaR were transformed into the in-frame deletion recipient strain UA140IFD. Selections for in-frame deletion mutants were performed exactly as described previously (Merritt et al., 2007). Each positive isolate was confirmed first by spectinomycin-sensitivity assay, then by PCR to determine the genotype of each antibiotic-sensitive isolates. Three independent in-frame deletion clones were selected for each gene deletion (UA140 ciaXIFD, and UA140 ciaRIFD, respectively). UA140 ciaXIFD and UA140 ciaRIFD were further transformed with pFW5

3′ciaH-luc and used for luciferase assays.

Construction of a ciaH truncation mutant with a ciaH-luc transcriptional fusion

To study the cia operon expression in the ciaH mutant, a ciaH truncation mutation was constructed concomitantly with a luciferase reporter fusion. A 350 bp fragment from the 5′ portion of ciaH was amplified from the chromosomal DNA of strain UA140 with primers ciaH5′F and ciaH3′R (Table S1). The PCR product was digested with Nhe I and BamHI and ligated into pFW-luc at the same restriction enzyme sites to form plasmid pFW-5′ciaH-luc. Upon integration of this plasmid into the chromosome of UA140 via a single cross-over homologous recombination, the ciaH was truncated at 350 bp from the 5′-end and the luciferase reporter was fused at the same point.

Complementation of ciaX

To complement the in-frame deletion of ciaX, a 0.9 kb fragment encompassing the entire intergenic region (IGR) upstream of ciaX (IGR889) and the IGR upstream of the initiation codon for ciaR (IGR888) was amplified by PCR with primers IGR889-F and IGR888-R (Table S1). The PCR fragment was digested with Hind III and Sac I, and ligated into pFW-5 at the same restriction sites to form pFW-knock-in. The insert was released by digestion with Xho I and Hind III and cloned into pBS-Kan at the same restriction sites to generate pBS-knock-in. pBS-knock-in was transformed into UA140 ciaXIFD and the transformants were selected on BHI plus kanamycin plates (1000 mg/ml). Correct transformants were further confirmed by PCR.

Site-directed mutagenesis of the SD domain of CiaX

Mutagenesis of the SD domain was performed using an inverse PCR strategy with pBS-knock-in as the template. Two inverse PCR primers were designed, each with an engineered Nhe I site at the 5′ end. This site would not change the reading frame nor the amino acid that the sequence encodes, yet allows an easy enzyme digestion and religation of the inverse PCR product. The two primers SD1 (GCG GcTAGc GcT TCCGcTTCTA ATAGTAGCAA TAC) and SD2 (GGCG cCTAgC C GAtTGCGATG AATCATTTGA ATTG) also contain the mutations that would change the amino acid sequence SDSDSD to SASASA (see Fig. 4A). PCR was performed at 95°C/4 min for 1 cycle, then 30 cycles at 94°C/15 s, 55°C/20 s, 72°C/7min, and one cycle at 72°C/10 min. The PCR product was digested with Nhe I and self-ligated to generate pBS-SD. After the mutations were confirmed by sequencing, pBS-SD was transformed into strain UA140 ciaXIFD essentially as described for the ciaX complementation.

Luciferase assay

Luciferase assays were performed as previously described (Loimaranta et al., 1998). Briefly, 25 μl of 1 mM D-luciferin (Sigma; St. Louis, MO) suspended in 100 mM citrate buffer, pH6, was added to 100 μl of the cell culture. To ensure sufficient levels of intracellular ATP pool, cells were recharged with 1% glucose for 10 min prior to luciferin addition. Luciferase activity was measured by using a 20/20 luminometer (Turner Biosystems; Sunnyvale, CA). Usually, three parallel cultures were measured at each time point and the average value was taken. Each experiment was repeated at least three times.

Calcium binding assay

The Fluorescein labeled CiaX-SD peptide (sequence DSSQSDSDSDSNSSNTNSNSSITNG) was subject to equilibrium dialysis as follows: Samples containing varying concentrations of CaCl₂ (16-128 μM) were dialysed against samples containing 10μM CiaX-SD peptide (Micro-equilibrium dialyser, Harvard Apparatus). Sample volume was 100 μl/chamber and all samples contained 50 mM Tris, pH 7.2; dialysis membranes with a molecular weight cutoff of 1,000 Daltons were used, and equilibrium was achieved after 1.5 hours. Samples were recovered and mixed with Calcium Green-2 (Invitrogen) to a final concentration of 2 μM in a total volume of 300 μl, followed by measurement of full fluorescence spectra using a Varian Eclipse Fluorescence Spectrophotometer with Microplate reader attachment. Excitation was at 505nm, spectra were recorded between 515 and 650 nm, and integrated between 520 and 600 nm. The amount of calcium bound to peptide was calculated by comparing the fluorescence of the samples to a standard calibration curve.

Biofilm assay

UA140 and the mutant derivatives were grown overnight in ASS medium at 37°C anaerobically. The culture was diluted 1:20 into fresh ASS and further incubated until the culture reached an OD ₆₀₀ nm of 0.5. The culture was then diluted 1:1,000 into ASS supplemented with 1% sucrose, and 0.4 ml of the cell suspension was added to each well of an eight-well Lab-Tek II chamber slide system (Nalge Nunc International, Naperville, Ill.). The chamber was incubated at 37°C for 18 hours as a static culture to allow for biofilm formation. The supernatant was removed, and biofilm was gently washed with PBS, and a labeling solution containing PBS plus 0.2 mM Cyto 59 (Invitrogen) was added to the biofilm. The labeling proceeded for 1 h to allow the dye to stain cells' nucleic acid. Biofilms were then analyzed by confocal laser scanning microscopy.

Acknowledgments

We would like to thank Dr. Jian He for synthesizing the CiaXSD peptide. This work was supported in part by NIH grants R01-DE014757 to F. Q., a COBRE P20 grant to J. M., and a Delta Dental grant WDS78956 to W. S.

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