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. depicts a working model developed based on results presented in this communication.
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
(). 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 (). Similarly, mutations of ciaX
also do not affect the basal level transcription of the operon mediated via RRx (). However, in the absence of CiaH, none of these pathways can be activated and the operon transcription is severely diminished (). 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
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 b
rotein), 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 ). 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 (). 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 (). 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 (). In ASS medium, the operon turns on during lag phase, and turns off at early log phase (). 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 (). 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.