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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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Effects of the growth conditions on expression of comX and cellular levels of MecA, ClpC and ClpP. A. Luciferase reporter activities (black circles and triangles) of the competence-specific promoter, PcomX, in S. mutans strain XT-Lx1 grown in THYE (open circles) or in CDM (open triangles) in response to CSP or XIP. B. Western blot analysis of cellular levels of MecA, ClpC or ClpP in S. mutans strains GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His) grown in THYE (+CSP) and in CDM (+XIP). The samples were taken from the cultures of these strains during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The MecA, ClpC and ClpP were detected by Western blotting using the anti-His antibody, while the protein loading controls were detected using the anti-S. mutans antibody.
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Figure 4: Effects of the growth conditions on expression of comX and cellular levels of MecA, ClpC and ClpP. A. Luciferase reporter activities (black circles and triangles) of the competence-specific promoter, PcomX, in S. mutans strain XT-Lx1 grown in THYE (open circles) or in CDM (open triangles) in response to CSP or XIP. B. Western blot analysis of cellular levels of MecA, ClpC or ClpP in S. mutans strains GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His) grown in THYE (+CSP) and in CDM (+XIP). The samples were taken from the cultures of these strains during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The MecA, ClpC and ClpP were detected by Western blotting using the anti-His antibody, while the protein loading controls were detected using the anti-S. mutans antibody.

Mentions: These findings led to two hypotheses: [1] that the stable levels of SigX in CDM might be due to a persistent expression of comX, which overcome degradation of SigX, or [2] that the growth conditions in CDM might negatively affect expression of MecA, ClpC or ClpP, resulting in insufficient levels of one or more of these proteins for SigX degradation. To test the first hypothesis, we examined kinetics of the luciferase reporter activities of the competence-specific promoter, PcomX, in S. mutans wild type background strain XT-Lx1 grown in CDM or in THYE in response to XIP or CSP. We found that the specific luciferase activities of the comX promoter, PcomX, still showed transient increases in response to XIP and then declined afterward, giving a pattern similar to that in THYE (Figure 4A). However, the luciferase reporter activities of this strain in CDM peaked around 200 min post XIP, which was about 1.5 hour later than the peak level in THYE post CSP. The pattern in the luciferase reporter activities appeared to be a reflection of the slower growth rate of this strain in CDM, although the reporter activities in CDM declined slightly later than those in THYE. We then began to test the second hypothesis by examining cellular levels of MecA, ClpC and ClpP in S. mutans strains, GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His), grown in both THYE (+CSP) and CDM (+XIP) by Western blot analysis. The results showed that considerable levels of MecA, ClpC and ClpP were detected during the (early, mid and late) exponential growth phases in both THYE and CDM (Figure 4B). No significant difference was observed in the cellular levels of these proteins between THYE and CDM. The results suggest that the stable levels of SigX detected in CDM may not necessarily result from variations in the cellular levels of MecA, ClpC or ClpP, but rather from an unknown mechanism that may negatively affect regulated proteolysis of SigX when S. mutans is grown in CDM.


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)

Effects of the growth conditions on expression of comX and cellular levels of MecA, ClpC and ClpP. A. Luciferase reporter activities (black circles and triangles) of the competence-specific promoter, PcomX, in S. mutans strain XT-Lx1 grown in THYE (open circles) or in CDM (open triangles) in response to CSP or XIP. B. Western blot analysis of cellular levels of MecA, ClpC or ClpP in S. mutans strains GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His) grown in THYE (+CSP) and in CDM (+XIP). The samples were taken from the cultures of these strains during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The MecA, ClpC and ClpP were detected by Western blotting using the anti-His antibody, while the protein loading controls were detected using the anti-S. mutans antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of the growth conditions on expression of comX and cellular levels of MecA, ClpC and ClpP. A. Luciferase reporter activities (black circles and triangles) of the competence-specific promoter, PcomX, in S. mutans strain XT-Lx1 grown in THYE (open circles) or in CDM (open triangles) in response to CSP or XIP. B. Western blot analysis of cellular levels of MecA, ClpC or ClpP in S. mutans strains GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His) grown in THYE (+CSP) and in CDM (+XIP). The samples were taken from the cultures of these strains during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The MecA, ClpC and ClpP were detected by Western blotting using the anti-His antibody, while the protein loading controls were detected using the anti-S. mutans antibody.
Mentions: These findings led to two hypotheses: [1] that the stable levels of SigX in CDM might be due to a persistent expression of comX, which overcome degradation of SigX, or [2] that the growth conditions in CDM might negatively affect expression of MecA, ClpC or ClpP, resulting in insufficient levels of one or more of these proteins for SigX degradation. To test the first hypothesis, we examined kinetics of the luciferase reporter activities of the competence-specific promoter, PcomX, in S. mutans wild type background strain XT-Lx1 grown in CDM or in THYE in response to XIP or CSP. We found that the specific luciferase activities of the comX promoter, PcomX, still showed transient increases in response to XIP and then declined afterward, giving a pattern similar to that in THYE (Figure 4A). However, the luciferase reporter activities of this strain in CDM peaked around 200 min post XIP, which was about 1.5 hour later than the peak level in THYE post CSP. The pattern in the luciferase reporter activities appeared to be a reflection of the slower growth rate of this strain in CDM, although the reporter activities in CDM declined slightly later than those in THYE. We then began to test the second hypothesis by examining cellular levels of MecA, ClpC and ClpP in S. mutans strains, GF-His2 (MecA-His), GF-His3 (ClpC-His) and GF-His4 (ClpP-His), grown in both THYE (+CSP) and CDM (+XIP) by Western blot analysis. The results showed that considerable levels of MecA, ClpC and ClpP were detected during the (early, mid and late) exponential growth phases in both THYE and CDM (Figure 4B). No significant difference was observed in the cellular levels of these proteins between THYE and CDM. The results suggest that the stable levels of SigX detected in CDM may not necessarily result from variations in the cellular levels of MecA, ClpC or ClpP, but rather from an unknown mechanism that may negatively affect regulated proteolysis of SigX when S. mutans is grown in CDM.

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