Limits...
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.

Show MeSH

Related in: MedlinePlus

The cellular levels of SigX in S. mutans during competence induction by CSP 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 S. mutans strain XT-His1 (wt) using the anti-His antibody. The total protein levels from the cell lysates of this strain are included as an example of protein loading controls detected by Western blotting using the anti-S. mutans antibody. B. Competence induction by CSP and the effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans UA159 (wt, black circle), XT-D1 (∆comX, open squares), XT-D4 (∆mecA, open diamonds), XT-D7 (∆clpC, open triangles), XT-D8 (∆clpP, black stars) and GF-Ox (MecA-Ox, open circles). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in S. mutans strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP). D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities were converted as 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
getmorefigures.php?uid=PMC4109385&req=5

Figure 1: The cellular levels of SigX in S. mutans during competence induction by CSP 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 S. mutans strain XT-His1 (wt) using the anti-His antibody. The total protein levels from the cell lysates of this strain are included as an example of protein loading controls detected by Western blotting using the anti-S. mutans antibody. B. Competence induction by CSP and the effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans UA159 (wt, black circle), XT-D1 (∆comX, open squares), XT-D4 (∆mecA, open diamonds), XT-D7 (∆clpC, open triangles), XT-D8 (∆clpP, black stars) and GF-Ox (MecA-Ox, open circles). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in S. mutans strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP). D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities were converted as relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.

Mentions: Previous studies showed that CSP induced a transient competence state or X state that allowed a subpopulation to take up transforming DNA when S. mutans was grown in a complex medium, the growth condition that is sub-permissive for competence development [17-22]. The X state normally maintained for 40–70 min and then shut off, suggesting that the SigX activity was unstable following transient competence activation. To confirm this observation, we examined the cellular level of SigX in a S. mutans wild type background strain XT-His1 (wt) grown in THYE for competence induction by CSP. This strain that carried a shuttle vector pSigX-His allowed detection of His-tagged SigX in the crude cell lysates by Western blotting. Without addition of CSP (T0), SigX was undetectable until about 60 min following addition of CSP a relatively high level of SigX was detected in the cell lysates (Figure 1A). This level of SigX remained for about 60 min and then rapidly declined to the levels nearly undetectable. However, the total protein levels in the cell lysates were relatively stable over times, particularly a 53-kDa protein, as indicated in the protein loading controls detected by Western blotting using the anti-S. mutans (serotype C) antibody. Consistent with the cellular levels of SigX, S. mutans UA159 (wt) showed relatively high but transient levels of transformation in response to CSP (Figure 1B). In contrast, the comX deletion mutant (ΔcomX; negative control) completely lost its transformability under the same condition. The results suggest that the synthesis of SigX is induced during competence induction by CSP, but SigX is quickly degraded during the escape from competence.


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 in S. mutans during competence induction by CSP 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 S. mutans strain XT-His1 (wt) using the anti-His antibody. The total protein levels from the cell lysates of this strain are included as an example of protein loading controls detected by Western blotting using the anti-S. mutans antibody. B. Competence induction by CSP and the effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans UA159 (wt, black circle), XT-D1 (∆comX, open squares), XT-D4 (∆mecA, open diamonds), XT-D7 (∆clpC, open triangles), XT-D8 (∆clpP, black stars) and GF-Ox (MecA-Ox, open circles). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in S. mutans strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP). D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities were converted as 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 1: The cellular levels of SigX in S. mutans during competence induction by CSP 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 S. mutans strain XT-His1 (wt) using the anti-His antibody. The total protein levels from the cell lysates of this strain are included as an example of protein loading controls detected by Western blotting using the anti-S. mutans antibody. B. Competence induction by CSP and the effects of mecA, clpC or clpP deletion on the transformation efficiency of S. mutans UA159 (wt, black circle), XT-D1 (∆comX, open squares), XT-D4 (∆mecA, open diamonds), XT-D7 (∆clpC, open triangles), XT-D8 (∆clpP, black stars) and GF-Ox (MecA-Ox, open circles). C. Western blot analysis of the effects of mecA, clpC or clpP deletion on the cellular levels of SigX in S. mutans strains XT-His1 (wt), XT-His2 (∆mecA), XT-His3 (∆clpC) and GF-His1 (∆clpP). D. The protein bands representing the cellular levels of SigX in these strains were scanned and the intensities were converted as relative integrated density values (RIDV), which were normalized to a maximum value of 1.0 for each strain.
Mentions: Previous studies showed that CSP induced a transient competence state or X state that allowed a subpopulation to take up transforming DNA when S. mutans was grown in a complex medium, the growth condition that is sub-permissive for competence development [17-22]. The X state normally maintained for 40–70 min and then shut off, suggesting that the SigX activity was unstable following transient competence activation. To confirm this observation, we examined the cellular level of SigX in a S. mutans wild type background strain XT-His1 (wt) grown in THYE for competence induction by CSP. This strain that carried a shuttle vector pSigX-His allowed detection of His-tagged SigX in the crude cell lysates by Western blotting. Without addition of CSP (T0), SigX was undetectable until about 60 min following addition of CSP a relatively high level of SigX was detected in the cell lysates (Figure 1A). This level of SigX remained for about 60 min and then rapidly declined to the levels nearly undetectable. However, the total protein levels in the cell lysates were relatively stable over times, particularly a 53-kDa protein, as indicated in the protein loading controls detected by Western blotting using the anti-S. mutans (serotype C) antibody. Consistent with the cellular levels of SigX, S. mutans UA159 (wt) showed relatively high but transient levels of transformation in response to CSP (Figure 1B). In contrast, the comX deletion mutant (ΔcomX; negative control) completely lost its transformability under the same condition. The results suggest that the synthesis of SigX is induced during competence induction by CSP, but SigX is quickly degraded during the escape from competence.

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