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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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In vitro degradation assays to determine MecA-mediated proteolysis of SigX. A. A colorimetrical assay of ATPase activity of ClpC. The reactions were initiated in a buffer by adding the following protein(s): (1) ClpC alone (black stars), (2) ClpC and MecA (open squires), (3) ClpC and SigX (black triangles), and (4) ClpC, MecA and SigX (black circles). These proteins were treated with PreSciession protease to remove GST-tag before used for the assay. B. The degradation reactions were initiated in a reaction buffer, including Group 1: ClpC-His, MecA-His, SigX-His, GST-ClpP and ATP (lane 1 and 2), Group 2: ClpC-His, MecA-His, SigX-His and GST-ClpP without addition of ATP, and Group 3: ClpC-His, SigX-His, GST-ClpP and ATP without MecA-His (lane 5 and 6). Aliquots of samples were taken from the reactions to assess degradation results by Western blot analysis of the interacting proteins using the anti-His or anti-GST antibody. C. Degradation assay of the reaction mixture containing ClpC, MecA, SigX and ClpP after removal of the GST-tag by PreScission protease cleavage. The SigX was detected by Western blotting using the anti-SigX antibody and the remaining SigX protein on the membrane was scanned and converted as RIDV values.
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Figure 6: In vitro degradation assays to determine MecA-mediated proteolysis of SigX. A. A colorimetrical assay of ATPase activity of ClpC. The reactions were initiated in a buffer by adding the following protein(s): (1) ClpC alone (black stars), (2) ClpC and MecA (open squires), (3) ClpC and SigX (black triangles), and (4) ClpC, MecA and SigX (black circles). These proteins were treated with PreSciession protease to remove GST-tag before used for the assay. B. The degradation reactions were initiated in a reaction buffer, including Group 1: ClpC-His, MecA-His, SigX-His, GST-ClpP and ATP (lane 1 and 2), Group 2: ClpC-His, MecA-His, SigX-His and GST-ClpP without addition of ATP, and Group 3: ClpC-His, SigX-His, GST-ClpP and ATP without MecA-His (lane 5 and 6). Aliquots of samples were taken from the reactions to assess degradation results by Western blot analysis of the interacting proteins using the anti-His or anti-GST antibody. C. Degradation assay of the reaction mixture containing ClpC, MecA, SigX and ClpP after removal of the GST-tag by PreScission protease cleavage. The SigX was detected by Western blotting using the anti-SigX antibody and the remaining SigX protein on the membrane was scanned and converted as RIDV values.

Mentions: We next examined whether MecA targeted SigX for degradation by the protease complex ClpC/ClpP. We first determined the ATPase activity of the ClpC protease by an ATPase activity assay [31,37]. The results showed that ClpC alone exhibited a very low ATPase activity, but the presence of MecA increased the ATPase activity of ClpC (Figure 6A). Interestingly, adding both MecA and SigX into the reaction increased the ATPase activity of ClpC by nearly 2-folds, although MecA or SigX alone exhibited little ATPases activity (data not shown). The results confirmed that the ATPase activity of ClpC depended on the presence of MecA and ATP, and the ATPase activity could be further enhanced by the presence of SigX. We then examined MecA-mediated proteolysis of SigX using a degradation assay. The reactions were mixed in the ATPase assay buffer by adding ClpC, MecA, SigX, ClpP and ATP. Aliquots of the samples were taken to assess degradation results by Western blot analysis of the interacting proteins. The results showed that degradation of SigX occurred in the presence of MecA, ClpC, ClpP and ATP in one-hour incubation (Figure 6B, lane 2). No detectable degradation of SigX was observed without MecA (lanes 5–6) or ATP (lane 3–4), suggesting that degradation of SigX required the presence of MecA and ATP. Interestingly, a similar level of degradation of MecA was also observed in the reaction (Figure 6B, lane 2), suggesting that MecA might serve as a degradation tag. To confirm the results, we also incubated these proteins for degradation after GST-tag was removed with PreScission protease. We then assessed degradation of SigX by Western blotting using the anti-SigX antibody. A similar result was observed, which showed that SigX degradation occurred as quickly as around 10 min (Figure 6C). Approximately 80% of SigX protein was degraded after 30-min incubation and over 90% of SigX was degraded after 60-min incubation, as indicated by the RIDV values of the remaining SigX in the reaction. In addition, we found that neither NTD1–82 nor CTD123–240 mediated degradation of SigX under the same condition (data not shown).


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)

In vitro degradation assays to determine MecA-mediated proteolysis of SigX. A. A colorimetrical assay of ATPase activity of ClpC. The reactions were initiated in a buffer by adding the following protein(s): (1) ClpC alone (black stars), (2) ClpC and MecA (open squires), (3) ClpC and SigX (black triangles), and (4) ClpC, MecA and SigX (black circles). These proteins were treated with PreSciession protease to remove GST-tag before used for the assay. B. The degradation reactions were initiated in a reaction buffer, including Group 1: ClpC-His, MecA-His, SigX-His, GST-ClpP and ATP (lane 1 and 2), Group 2: ClpC-His, MecA-His, SigX-His and GST-ClpP without addition of ATP, and Group 3: ClpC-His, SigX-His, GST-ClpP and ATP without MecA-His (lane 5 and 6). Aliquots of samples were taken from the reactions to assess degradation results by Western blot analysis of the interacting proteins using the anti-His or anti-GST antibody. C. Degradation assay of the reaction mixture containing ClpC, MecA, SigX and ClpP after removal of the GST-tag by PreScission protease cleavage. The SigX was detected by Western blotting using the anti-SigX antibody and the remaining SigX protein on the membrane was scanned and converted as RIDV values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: In vitro degradation assays to determine MecA-mediated proteolysis of SigX. A. A colorimetrical assay of ATPase activity of ClpC. The reactions were initiated in a buffer by adding the following protein(s): (1) ClpC alone (black stars), (2) ClpC and MecA (open squires), (3) ClpC and SigX (black triangles), and (4) ClpC, MecA and SigX (black circles). These proteins were treated with PreSciession protease to remove GST-tag before used for the assay. B. The degradation reactions were initiated in a reaction buffer, including Group 1: ClpC-His, MecA-His, SigX-His, GST-ClpP and ATP (lane 1 and 2), Group 2: ClpC-His, MecA-His, SigX-His and GST-ClpP without addition of ATP, and Group 3: ClpC-His, SigX-His, GST-ClpP and ATP without MecA-His (lane 5 and 6). Aliquots of samples were taken from the reactions to assess degradation results by Western blot analysis of the interacting proteins using the anti-His or anti-GST antibody. C. Degradation assay of the reaction mixture containing ClpC, MecA, SigX and ClpP after removal of the GST-tag by PreScission protease cleavage. The SigX was detected by Western blotting using the anti-SigX antibody and the remaining SigX protein on the membrane was scanned and converted as RIDV values.
Mentions: We next examined whether MecA targeted SigX for degradation by the protease complex ClpC/ClpP. We first determined the ATPase activity of the ClpC protease by an ATPase activity assay [31,37]. The results showed that ClpC alone exhibited a very low ATPase activity, but the presence of MecA increased the ATPase activity of ClpC (Figure 6A). Interestingly, adding both MecA and SigX into the reaction increased the ATPase activity of ClpC by nearly 2-folds, although MecA or SigX alone exhibited little ATPases activity (data not shown). The results confirmed that the ATPase activity of ClpC depended on the presence of MecA and ATP, and the ATPase activity could be further enhanced by the presence of SigX. We then examined MecA-mediated proteolysis of SigX using a degradation assay. The reactions were mixed in the ATPase assay buffer by adding ClpC, MecA, SigX, ClpP and ATP. Aliquots of the samples were taken to assess degradation results by Western blot analysis of the interacting proteins. The results showed that degradation of SigX occurred in the presence of MecA, ClpC, ClpP and ATP in one-hour incubation (Figure 6B, lane 2). No detectable degradation of SigX was observed without MecA (lanes 5–6) or ATP (lane 3–4), suggesting that degradation of SigX required the presence of MecA and ATP. Interestingly, a similar level of degradation of MecA was also observed in the reaction (Figure 6B, lane 2), suggesting that MecA might serve as a degradation tag. To confirm the results, we also incubated these proteins for degradation after GST-tag was removed with PreScission protease. We then assessed degradation of SigX by Western blotting using the anti-SigX antibody. A similar result was observed, which showed that SigX degradation occurred as quickly as around 10 min (Figure 6C). Approximately 80% of SigX protein was degraded after 30-min incubation and over 90% of SigX was degraded after 60-min incubation, as indicated by the RIDV values of the remaining SigX in the reaction. In addition, we found that neither NTD1–82 nor CTD123–240 mediated degradation of SigX under the same condition (data not shown).

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