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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 protein-protein interactions of MecA, SigX and ClpC by co-elution experiments. A. Protein-protein interactions were initiated by incubating the protein mixtures as follows: (1) MecA-His and GST (control), (2) MecA-His and GST-SigX, (3) MecA-His and GST-ClpC and (4) all three proteins. The reactions were incubated at 37°C for 1 hour before applied to Ni-charged resin for co-elution. The elution samples were then analyzed by Western blotting using the anti-His or anti-GST antibody, respectively. B. Effects of deletion of the N-terminal domain or C-terminal domain of MecA on its interaction with SigX or ClpC. Lane 1 and 4 indicate the interactions of the full-length MecA with both SigX and ClpC. Lane 2 indicates an interaction of the N-terminal domain (NTD1–82) with SigX, but not with ClpC (Lane 5). Lane 6 indicates an interaction of the C-terminal domain (CTD123–240) with ClpC but not with SigX (Lane 3).
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Figure 5: In vitro protein-protein interactions of MecA, SigX and ClpC by co-elution experiments. A. Protein-protein interactions were initiated by incubating the protein mixtures as follows: (1) MecA-His and GST (control), (2) MecA-His and GST-SigX, (3) MecA-His and GST-ClpC and (4) all three proteins. The reactions were incubated at 37°C for 1 hour before applied to Ni-charged resin for co-elution. The elution samples were then analyzed by Western blotting using the anti-His or anti-GST antibody, respectively. B. Effects of deletion of the N-terminal domain or C-terminal domain of MecA on its interaction with SigX or ClpC. Lane 1 and 4 indicate the interactions of the full-length MecA with both SigX and ClpC. Lane 2 indicates an interaction of the N-terminal domain (NTD1–82) with SigX, but not with ClpC (Lane 5). Lane 6 indicates an interaction of the C-terminal domain (CTD123–240) with ClpC but not with SigX (Lane 3).

Mentions: Recent study using bacterial two-hybrid (B2H) system shows that MecA interacts with SigX and ClpC, mediating the formation of a ternary complex [34]. To obtain direct evidence of the interaction among these proteins, we cloned, expressed and produced recombinant proteins of SigX, MecA, ClpC and ClpP using protein expression technology. Each of the resulting proteins carried a His-tag or GST-tag that facilitated subsequent purification and identification of these proteins. SDS-PAGE analysis of these proteins showed successful production of sufficient amounts of all fusion proteins. Their identities and predicted molecular sizes following purification are described in Additional file 1: Figure S1, Additional file 1: Figure S2 and Additional file 1: Figure S3. The fusion proteins were further confirmed by Western blotting using the anti-His or anti-GST antibody. With these purified proteins, we next examined MecA-mediated protein-protein interactions by co-elution experiments using Ni-charged resin. After extensive washings, the proteins were eluted in a buffer and the elution samples were analyzed by Western blotting. As shown in Figure 5A, MecA did not interact with GST-tag (lane 1), but interacted with SigX (lane 2) or ClpC (lane 3) or both (lane 4). The results clearly indicate that MecA protein interacts simultaneously with both SigX and ClpC, likely mediating the formation of a ternary SigX-MecA-ClpC complex.


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 protein-protein interactions of MecA, SigX and ClpC by co-elution experiments. A. Protein-protein interactions were initiated by incubating the protein mixtures as follows: (1) MecA-His and GST (control), (2) MecA-His and GST-SigX, (3) MecA-His and GST-ClpC and (4) all three proteins. The reactions were incubated at 37°C for 1 hour before applied to Ni-charged resin for co-elution. The elution samples were then analyzed by Western blotting using the anti-His or anti-GST antibody, respectively. B. Effects of deletion of the N-terminal domain or C-terminal domain of MecA on its interaction with SigX or ClpC. Lane 1 and 4 indicate the interactions of the full-length MecA with both SigX and ClpC. Lane 2 indicates an interaction of the N-terminal domain (NTD1–82) with SigX, but not with ClpC (Lane 5). Lane 6 indicates an interaction of the C-terminal domain (CTD123–240) with ClpC but not with SigX (Lane 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 5: In vitro protein-protein interactions of MecA, SigX and ClpC by co-elution experiments. A. Protein-protein interactions were initiated by incubating the protein mixtures as follows: (1) MecA-His and GST (control), (2) MecA-His and GST-SigX, (3) MecA-His and GST-ClpC and (4) all three proteins. The reactions were incubated at 37°C for 1 hour before applied to Ni-charged resin for co-elution. The elution samples were then analyzed by Western blotting using the anti-His or anti-GST antibody, respectively. B. Effects of deletion of the N-terminal domain or C-terminal domain of MecA on its interaction with SigX or ClpC. Lane 1 and 4 indicate the interactions of the full-length MecA with both SigX and ClpC. Lane 2 indicates an interaction of the N-terminal domain (NTD1–82) with SigX, but not with ClpC (Lane 5). Lane 6 indicates an interaction of the C-terminal domain (CTD123–240) with ClpC but not with SigX (Lane 3).
Mentions: Recent study using bacterial two-hybrid (B2H) system shows that MecA interacts with SigX and ClpC, mediating the formation of a ternary complex [34]. To obtain direct evidence of the interaction among these proteins, we cloned, expressed and produced recombinant proteins of SigX, MecA, ClpC and ClpP using protein expression technology. Each of the resulting proteins carried a His-tag or GST-tag that facilitated subsequent purification and identification of these proteins. SDS-PAGE analysis of these proteins showed successful production of sufficient amounts of all fusion proteins. Their identities and predicted molecular sizes following purification are described in Additional file 1: Figure S1, Additional file 1: Figure S2 and Additional file 1: Figure S3. The fusion proteins were further confirmed by Western blotting using the anti-His or anti-GST antibody. With these purified proteins, we next examined MecA-mediated protein-protein interactions by co-elution experiments using Ni-charged resin. After extensive washings, the proteins were eluted in a buffer and the elution samples were analyzed by Western blotting. As shown in Figure 5A, MecA did not interact with GST-tag (lane 1), but interacted with SigX (lane 2) or ClpC (lane 3) or both (lane 4). The results clearly indicate that MecA protein interacts simultaneously with both SigX and ClpC, likely mediating the formation of a ternary SigX-MecA-ClpC complex.

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