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Regulated proteolysis of the alternative sigma factor SigX in Streptococcus mutans: implication in the escape from competence.

Dong G, Tian XL, Gomez ZA, Li YH - BMC Microbiol. (2014)

Bottom Line: A deletion of the N-terminal or C-terminal domain of MecA abolishes its binding to SigX or ClpC.Adaptor protein MecA in S. mutans plays a crucial role in recognizing and targeting SigX for degradation by the protease ClpC/ClpP.Thus, MecA actually acts as an anti-sigma factor to regulate the stability of SigX during competence development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Oral Sciences, Faculty of Dentistry, Dalhousie University, 5981 University Avenue, Halifax, Nova Scotia B3H 1 W2, Canada. yung-hua.li@dal.ca.

ABSTRACT

Background: SigX (σX), the alternative sigma factor of Streptococcus mutans, is the key regulator for transcriptional activation of late competence genes essential for taking up exogenous DNA. Recent studies reveal that adaptor protein MecA and the protease ClpC act as negative regulators of competence by a mechanism that involves MecA-mediated proteolysis of SigX by the ClpC in S. mutans. However, the molecular detail how MecA and ClpC negatively regulate competence in this species remains to be determined. Here, we provide evidence that adaptor protein MecA targets SigX for degradation by the protease complex ClpC/ClpP when S. mutans is grown in a complex medium.

Results: By analyzing the cellular levels of SigX, we demonstrate that the synthesis of SigX is transiently induced by competence-stimulating peptide (CSP), but the SigX is rapidly degraded during the escape from competence. A deletion of MecA, ClpC or ClpP results in the cellular accumulation of SigX and a prolonged competence state, while an overexpression of MecA enhances proteolysis of SigX and accelerates the escape from competence. In vitro protein-protein interaction assays confirm that MecA interacts with SigX via its N-terminal domain (NTD1-82) and with ClpC via its C-terminal domain (CTD123-240). Such an interaction mediates the formation of a ternary SigX-MecA-ClpC complex, triggering the ATP-dependent degradation of SigX in the presence of ClpP. A deletion of the N-terminal or C-terminal domain of MecA abolishes its binding to SigX or ClpC. We have also found that MecA-regulated proteolysis of SigX appears to be ineffective when S. mutans is grown in a chemically defined medium (CDM), suggesting the possibility that an unknown mechanism may be involved in negative regulation of MecA-mediated proteolysis of SigX under this condition.

Conclusion: Adaptor protein MecA in S. mutans plays a crucial role in recognizing and targeting SigX for degradation by the protease ClpC/ClpP. Thus, MecA actually acts as an anti-sigma factor to regulate the stability of SigX during competence development.

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The cellular levels of SigX during competence induction by XIP in CDM and the effects of mecA, clpC or clpP deletion on the stability of SigX and competence. A. Western blot analysis of the cellular levels of SigX in strain XT-His1 (wt) by the anti-His antibody. The protein loading controls this strain were detected by Western blotting using the anti-S. mutans antibody. B. The effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans strains UA159 (wt), XT-D1 (∆comX), XT-D4 (∆mecA), XT-D7 (∆clpC) and XT-D8 (∆clpP). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP) using the anti-His antibody. D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities of the bands were converted as the relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.
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Figure 3: The cellular levels of SigX during competence induction by XIP in CDM and the effects of mecA, clpC or clpP deletion on the stability of SigX and competence. A. Western blot analysis of the cellular levels of SigX in strain XT-His1 (wt) by the anti-His antibody. The protein loading controls this strain were detected by Western blotting using the anti-S. mutans antibody. B. The effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans strains UA159 (wt), XT-D1 (∆comX), XT-D4 (∆mecA), XT-D7 (∆clpC) and XT-D8 (∆clpP). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP) using the anti-His antibody. D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities of the bands were converted as the relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.

Mentions: Recent studies show that when S. mutans is grown in a chemically defined medium (CDM), competence induction through the ComRS signaling system is active for hours in response to XIP [18,25,26]. These studies suggest that S. mutans activates competence differently through the ComRS signaling system in CDM. We therefore examined the stability of SigX and the effects of inactivated MecA, ClpC or ClpP on the cellular levels of SigX by Western blot analysis of strain XT-His1 (wt) and three mutant background strains, XT-His2 (ΔmecA), XT-His3 (ΔclpC) and GF-His1 (ΔclpP) grown in CDM in response to XIP. The results showed that XIP induced a rapid synthesis of SigX in strain XT-His1 (wt) grown in CDM (Figure 3A). Surprisingly, an increasing level of SigX was detected in this strain throughout the experiment, although relatively stable levels of the protein samples were loaded on the gel, as indicated by the protein loading controls. The results suggested a cellular accumulation of the SigX after its synthesis in CDM. Consistent with these levels of SigX, a prolonged competent state was observed in S. mutans UA159 grown under the same condition (Figure 3B). We then examined the effects of deletion of mecA (ΔmecA), clpC (ΔclpC) or clpP (ΔclpP) on the cellular levels of SigX in CDM. The results clearly showed the cellular accumulation of SigX in all three mutant strains grown in CDM (Figure 3C). As indicated by the relative integrated density values (RIDVs) (Figure 3D), the cellular levels of SigX in these mutant strains remained high or even higher than the wild type control strain throughout the experiments. In agreement with such stable levels of SigX, all the strains showed a prolonged competence state for taking up transforming DNA in CDM (Figure 3B). The results clearly show that SigX is relatively stable after the synthesis during competence induction by XIP in CDM, resulting in a prolonged competent state for taking up exogenous DNA.


Regulated proteolysis of the alternative sigma factor SigX in Streptococcus mutans: implication in the escape from competence.

Dong G, Tian XL, Gomez ZA, Li YH - BMC Microbiol. (2014)

The cellular levels of SigX during competence induction by XIP in CDM and the effects of mecA, clpC or clpP deletion on the stability of SigX and competence. A. Western blot analysis of the cellular levels of SigX in strain XT-His1 (wt) by the anti-His antibody. The protein loading controls this strain were detected by Western blotting using the anti-S. mutans antibody. B. The effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans strains UA159 (wt), XT-D1 (∆comX), XT-D4 (∆mecA), XT-D7 (∆clpC) and XT-D8 (∆clpP). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP) using the anti-His antibody. D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities of the bands were converted as the relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109385&req=5

Figure 3: The cellular levels of SigX during competence induction by XIP in CDM and the effects of mecA, clpC or clpP deletion on the stability of SigX and competence. A. Western blot analysis of the cellular levels of SigX in strain XT-His1 (wt) by the anti-His antibody. The protein loading controls this strain were detected by Western blotting using the anti-S. mutans antibody. B. The effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans strains UA159 (wt), XT-D1 (∆comX), XT-D4 (∆mecA), XT-D7 (∆clpC) and XT-D8 (∆clpP). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP) using the anti-His antibody. D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities of the bands were converted as the relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.
Mentions: Recent studies show that when S. mutans is grown in a chemically defined medium (CDM), competence induction through the ComRS signaling system is active for hours in response to XIP [18,25,26]. These studies suggest that S. mutans activates competence differently through the ComRS signaling system in CDM. We therefore examined the stability of SigX and the effects of inactivated MecA, ClpC or ClpP on the cellular levels of SigX by Western blot analysis of strain XT-His1 (wt) and three mutant background strains, XT-His2 (ΔmecA), XT-His3 (ΔclpC) and GF-His1 (ΔclpP) grown in CDM in response to XIP. The results showed that XIP induced a rapid synthesis of SigX in strain XT-His1 (wt) grown in CDM (Figure 3A). Surprisingly, an increasing level of SigX was detected in this strain throughout the experiment, although relatively stable levels of the protein samples were loaded on the gel, as indicated by the protein loading controls. The results suggested a cellular accumulation of the SigX after its synthesis in CDM. Consistent with these levels of SigX, a prolonged competent state was observed in S. mutans UA159 grown under the same condition (Figure 3B). We then examined the effects of deletion of mecA (ΔmecA), clpC (ΔclpC) or clpP (ΔclpP) on the cellular levels of SigX in CDM. The results clearly showed the cellular accumulation of SigX in all three mutant strains grown in CDM (Figure 3C). As indicated by the relative integrated density values (RIDVs) (Figure 3D), the cellular levels of SigX in these mutant strains remained high or even higher than the wild type control strain throughout the experiments. In agreement with such stable levels of SigX, all the strains showed a prolonged competence state for taking up transforming DNA in CDM (Figure 3B). The results clearly show that SigX is relatively stable after the synthesis during competence induction by XIP in CDM, resulting in a prolonged competent state for taking up exogenous DNA.

Bottom Line: A deletion of the N-terminal or C-terminal domain of MecA abolishes its binding to SigX or ClpC.Adaptor protein MecA in S. mutans plays a crucial role in recognizing and targeting SigX for degradation by the protease ClpC/ClpP.Thus, MecA actually acts as an anti-sigma factor to regulate the stability of SigX during competence development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Oral Sciences, Faculty of Dentistry, Dalhousie University, 5981 University Avenue, Halifax, Nova Scotia B3H 1 W2, Canada. yung-hua.li@dal.ca.

ABSTRACT

Background: SigX (σX), the alternative sigma factor of Streptococcus mutans, is the key regulator for transcriptional activation of late competence genes essential for taking up exogenous DNA. Recent studies reveal that adaptor protein MecA and the protease ClpC act as negative regulators of competence by a mechanism that involves MecA-mediated proteolysis of SigX by the ClpC in S. mutans. However, the molecular detail how MecA and ClpC negatively regulate competence in this species remains to be determined. Here, we provide evidence that adaptor protein MecA targets SigX for degradation by the protease complex ClpC/ClpP when S. mutans is grown in a complex medium.

Results: By analyzing the cellular levels of SigX, we demonstrate that the synthesis of SigX is transiently induced by competence-stimulating peptide (CSP), but the SigX is rapidly degraded during the escape from competence. A deletion of MecA, ClpC or ClpP results in the cellular accumulation of SigX and a prolonged competence state, while an overexpression of MecA enhances proteolysis of SigX and accelerates the escape from competence. In vitro protein-protein interaction assays confirm that MecA interacts with SigX via its N-terminal domain (NTD1-82) and with ClpC via its C-terminal domain (CTD123-240). Such an interaction mediates the formation of a ternary SigX-MecA-ClpC complex, triggering the ATP-dependent degradation of SigX in the presence of ClpP. A deletion of the N-terminal or C-terminal domain of MecA abolishes its binding to SigX or ClpC. We have also found that MecA-regulated proteolysis of SigX appears to be ineffective when S. mutans is grown in a chemically defined medium (CDM), suggesting the possibility that an unknown mechanism may be involved in negative regulation of MecA-mediated proteolysis of SigX under this condition.

Conclusion: Adaptor protein MecA in S. mutans plays a crucial role in recognizing and targeting SigX for degradation by the protease ClpC/ClpP. Thus, MecA actually acts as an anti-sigma factor to regulate the stability of SigX during competence development.

Show MeSH
Related in: MedlinePlus