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. 2008 Oct;70(1):112-26.
doi: 10.1111/j.1365-2958.2008.06390.x. Epub 2008 Aug 4.

The cia operon of Streptococcus mutans encodes a unique component required for calcium-mediated autoregulation

Xuesong He  1 , Chenggang Wu, Daniel Yarbrough, Lucy Sim, Guoqing Niu, Justin Merritt, Wenyuan Shi, Fengxia Qi
Affiliations

The cia operon of Streptococcus mutans encodes a unique component required for calcium-mediated autoregulation

Xuesong He et al. Mol Microbiol. 2008 Oct.

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.

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Figures

Fig. 1
Fig. 1. Effect of calcium on ciaRH operon expression
S. mutans UA140 ciaH-luc reporter strain was grown to stationary phase, centrifuged, and diluted to OD600 of 0.01 in fresh medium. Samples were taken at designated time points and optical density at 600 nm and luciferase activities were measured. Usually a triplicate sample was measured at each time point, and the average was taken for analysis. The experiment was repeated at least 3 times. Presented here are representative results. A. Effect of EGTA on ciaRH operon expression in TH medium. Diamond: TH medium; Circle: TH medium with 0.2 mM EGTA; Triangle: TH medium with 0.2 mM EGTA and 5 mM Ca++. Broken line: Growth curve; Solid line: ciaH-luc operon expression B. Effect of calcium on ciaRH operon expression in chemically defined (ASS) medium. Diamond: ASS medium without added Ca++; Square: ASS medium with 0.5 mM Ca++; Open symbol: Growth curve; Closed symbol: ciaH-luc operon expression. C. Effect of calcium on ciaX, ciaR, and ciaH gene expression as measured by real-time RT-PCR. The experimental design was the same as in (B), except that the samples were taken at 6-h post-inoculation when the operon was at its peak level. Transcript levels of samples without calcium were arbitrarily set as 1. Open bars: without calcium; filled bars: with calcium.
Fig. 1
Fig. 1. Effect of calcium on ciaRH operon expression
S. mutans UA140 ciaH-luc reporter strain was grown to stationary phase, centrifuged, and diluted to OD600 of 0.01 in fresh medium. Samples were taken at designated time points and optical density at 600 nm and luciferase activities were measured. Usually a triplicate sample was measured at each time point, and the average was taken for analysis. The experiment was repeated at least 3 times. Presented here are representative results. A. Effect of EGTA on ciaRH operon expression in TH medium. Diamond: TH medium; Circle: TH medium with 0.2 mM EGTA; Triangle: TH medium with 0.2 mM EGTA and 5 mM Ca++. Broken line: Growth curve; Solid line: ciaH-luc operon expression B. Effect of calcium on ciaRH operon expression in chemically defined (ASS) medium. Diamond: ASS medium without added Ca++; Square: ASS medium with 0.5 mM Ca++; Open symbol: Growth curve; Closed symbol: ciaH-luc operon expression. C. Effect of calcium on ciaX, ciaR, and ciaH gene expression as measured by real-time RT-PCR. The experimental design was the same as in (B), except that the samples were taken at 6-h post-inoculation when the operon was at its peak level. Transcript levels of samples without calcium were arbitrarily set as 1. Open bars: without calcium; filled bars: with calcium.
Fig. 1
Fig. 1. Effect of calcium on ciaRH operon expression
S. mutans UA140 ciaH-luc reporter strain was grown to stationary phase, centrifuged, and diluted to OD600 of 0.01 in fresh medium. Samples were taken at designated time points and optical density at 600 nm and luciferase activities were measured. Usually a triplicate sample was measured at each time point, and the average was taken for analysis. The experiment was repeated at least 3 times. Presented here are representative results. A. Effect of EGTA on ciaRH operon expression in TH medium. Diamond: TH medium; Circle: TH medium with 0.2 mM EGTA; Triangle: TH medium with 0.2 mM EGTA and 5 mM Ca++. Broken line: Growth curve; Solid line: ciaH-luc operon expression B. Effect of calcium on ciaRH operon expression in chemically defined (ASS) medium. Diamond: ASS medium without added Ca++; Square: ASS medium with 0.5 mM Ca++; Open symbol: Growth curve; Closed symbol: ciaH-luc operon expression. C. Effect of calcium on ciaX, ciaR, and ciaH gene expression as measured by real-time RT-PCR. The experimental design was the same as in (B), except that the samples were taken at 6-h post-inoculation when the operon was at its peak level. Transcript levels of samples without calcium were arbitrarily set as 1. Open bars: without calcium; filled bars: with calcium.
Fig. 2
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 EF-4 calcium binding loop with CiaX. Bold letters are the conserved residues for calcium binding.
Fig. 2
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 EF-4 calcium binding loop with CiaX. Bold letters are the conserved residues for calcium binding.
Fig. 3
Fig. 3. Function of CiaX in calcium repression of ciaXRH operon expression
A. Effect of ciaX in-frame deletion on calcium repression of operon expression. B. Effect of ciaX complementation on calcium repression of operon expression. Experiments were performed exactly as described in Fig. 1B. Diamond: without Ca++; square: with 0.5 mM Ca++. Open symbols: growth curve; closed symbols: ciaH-luc operon expression
Fig. 3
Fig. 3. Function of CiaX in calcium repression of ciaXRH operon expression
A. Effect of ciaX in-frame deletion on calcium repression of operon expression. B. Effect of ciaX complementation on calcium repression of operon expression. Experiments were performed exactly as described in Fig. 1B. Diamond: without Ca++; square: with 0.5 mM Ca++. Open symbols: growth curve; closed symbols: ciaH-luc operon expression
Fig. 4
Fig. 4. Effect of CiaX SD mutations on CiaX function
A. construction of the SD mutation by inverse PCR. The coding sequence of ciaX and the primers used for the mutagenesis (SD1 and SD2) are presented. B. Effect of the SD mutation on calcium repression of operon expression. Experiments were performed as described in Fig. 1B. Diamond: without Ca++; square: with 0.5 mM Ca++. Open symbols: growth curve; closed symbols: ciaH-luc operon expression
Fig. 4
Fig. 4. Effect of CiaX SD mutations on CiaX function
A. construction of the SD mutation by inverse PCR. The coding sequence of ciaX and the primers used for the mutagenesis (SD1 and SD2) are presented. B. Effect of the SD mutation on calcium repression of operon expression. Experiments were performed as described in Fig. 1B. Diamond: without Ca++; square: with 0.5 mM Ca++. Open symbols: growth curve; closed symbols: ciaH-luc operon expression
Fig. 5
Fig. 5. Binding of Ca++ to the CiaX-SD peptide
Samples containing varying concentrations of CaCl2 were dialysed against samples containing 10μM CiaX-SD peptide in dialysis tubing with a molecular weight cutoff of 1,000 Daltons. After 1.5 h, unbound calcium was measured by using the Calcium Green-2 kit. The amount of calcium bound to peptide was calculated by comparing the fluorescence of the samples to a standard calibration curve.
Fig. 6
Fig. 6. Effect of ciaX mutation on biofilm formation
Mid-log cultures of the WT and mutant strains were diluted 1:1,000 into ASS supplemented with 1% sucrose, and incubated in an 8-well Lab-Tek II chamber slide system for 18 hours as a static culture. After the biofilm was gently washed with PBS, Cyto 59 was used to label the biofilm. Biofilms were then analyzed by confocal laser scanning microscopy. A & B: wild-type without and with 0.3 mM calcium, respectively; C & D: ciaX without and with 0.3 mM calcium, respectively; E & F: ciaX-knock-in without and with 0.3 mM calcium, respectively; G & H: ciaXSD without and with 0.3 mM calcium, respectively.
Fig. 7
Fig. 7. Effect of ciaR and ciaH mutations on ciaXRH operon expression in response to calcium
Experiments were performed essentially as described in Fig. 1B. A. ciaH-luc gene expression in ciaH background. B. ciaH-luc gene expression in ciaR background. Diamond : without calcium; square: with calcium. Open symbols: growth curve; closed symbols: luciferase activity.
Fig. 7
Fig. 7. Effect of ciaR and ciaH mutations on ciaXRH operon expression in response to calcium
Experiments were performed essentially as described in Fig. 1B. A. ciaH-luc gene expression in ciaH background. B. ciaH-luc gene expression in ciaR background. Diamond : without calcium; square: with calcium. Open symbols: growth curve; closed symbols: luciferase activity.
Fig. 8
Fig. 8. The ciaXRH operon promoter region and the putative CiaR binding site
DR1 and DR2 are the two putative direct repeats bound by CiaR. The -10 and +1 sites are labeled. The 3 primers used in the nested RT-PCR are also labeled. Primer 1311PB-F and 1311R did not yield a PCR product, but primer 1311PA-F and 1311R yielded a PCR product of expected size (data not shown).
Fig. 9
Fig. 9. Model for CiaX mediated calcium modulation of ciaXRH operon expression
A: wild-type without calcium, high level ciaXRH operon expression; B: wild-type with calcium, basal level ciaXRH operon expression; C: ciaR or ciaX mutant, basal level ciaXRH operon expression regardless of calcium; D: ciaH mutant, severely diminished operon expression regardless of calcium. Detailed description of the model is provided in Discussion.
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