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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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Constitutive expression of MecA and its effect on the cellular levels of SigX. A. Western blot analysis of cellular levels of MecA in S. mutans strain GF-His2 (MecA-His) and strain GF-Ox (MecA-Ox) that constitutively expressed MecA. The samples were taken from the cultures of these strains grown during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The cellular MecA of these strains were detected by Western blotting using the anti-His antibody. The intensities of the bands were scanned and converted as relative integrated density values (RIDV) for comparison. The total protein loading controls from the cell lysates of these strains were detected using the anti-S. mutans antibody. B. The effect of constitutively expressed MecA on the cellular levels of SigX in GF-Ox (MecA-Ox) grown in THYE in response to CSP. The cellular MecA was detected by Western blotting using the anti-His antibody, while the cellular SigX in the same strain was detected using the anti-SigX antibody.
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Figure 2: Constitutive expression of MecA and its effect on the cellular levels of SigX. A. Western blot analysis of cellular levels of MecA in S. mutans strain GF-His2 (MecA-His) and strain GF-Ox (MecA-Ox) that constitutively expressed MecA. The samples were taken from the cultures of these strains grown during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The cellular MecA of these strains were detected by Western blotting using the anti-His antibody. The intensities of the bands were scanned and converted as relative integrated density values (RIDV) for comparison. The total protein loading controls from the cell lysates of these strains were detected using the anti-S. mutans antibody. B. The effect of constitutively expressed MecA on the cellular levels of SigX in GF-Ox (MecA-Ox) grown in THYE in response to CSP. The cellular MecA was detected by Western blotting using the anti-His antibody, while the cellular SigX in the same strain was detected using the anti-SigX antibody.

Mentions: To further investigate the effect of MecA on the stability of SigX, we constructed a constitutively expressed MecA strain, GF-Ox (MecA-Ox), which expressed MecA under the control of a constitutively expressed promoter of ldh, the gene encoding lactate dehydrogenase in S. mutans. In addition, this strain allowed detection of MecA protein by Western blotting using the anti-His antibody, since a His-tag was added to the end of mecA-coding sequence just before the stop codon. Our results confirmed that strain MecA-Ox constitutively expressed MecA when grown in THYE, independently on the growth phases. Based on the relative integrated density values (RIDV), this strain (MecA-Ox) produced as much as two-fold MecA protein when compared with that of strain GF-His2 (MecA-His) grown under the same condition (Figure 2A), suggesting that strain MecA-Ox overexpressed MecA under the conditions tested. It was noted from the Western blot that MecA-Ox showed an extra band that slightly bigger than the MecA. The feature of this extra band was unclear. One possibility was that over-expressed MecA might also bind to a small, unknown protein or peptide in S. mutans. Alternatively, this band might be a non-specific reaction. We then began to determine the effect of an overexpressed MecA on the cellular levels of SigX in response to CSP. The results revealed that the overexpression of MecA negatively affected the cellular levels of SigX in this strain, since addition of CSP induced very low levels of SigX, as detected by Western blotting using the anti-SigX antibody (Figure 2B). Consistent with this result, the constitutive expression of MecA resulted in hundred-fold reduction in transformation efficiency (Figure 1B). The results suggest that the overexpressed MecA enhances MecA-mediated proteolysis of SigX, accelerating 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)

Constitutive expression of MecA and its effect on the cellular levels of SigX. A. Western blot analysis of cellular levels of MecA in S. mutans strain GF-His2 (MecA-His) and strain GF-Ox (MecA-Ox) that constitutively expressed MecA. The samples were taken from the cultures of these strains grown during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The cellular MecA of these strains were detected by Western blotting using the anti-His antibody. The intensities of the bands were scanned and converted as relative integrated density values (RIDV) for comparison. The total protein loading controls from the cell lysates of these strains were detected using the anti-S. mutans antibody. B. The effect of constitutively expressed MecA on the cellular levels of SigX in GF-Ox (MecA-Ox) grown in THYE in response to CSP. The cellular MecA was detected by Western blotting using the anti-His antibody, while the cellular SigX in the same strain was detected using the anti-SigX antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Constitutive expression of MecA and its effect on the cellular levels of SigX. A. Western blot analysis of cellular levels of MecA in S. mutans strain GF-His2 (MecA-His) and strain GF-Ox (MecA-Ox) that constitutively expressed MecA. The samples were taken from the cultures of these strains grown during the early- (E), mid- (M) and late- (L) exponential phases to prepare the crude cell lysates. The cellular MecA of these strains were detected by Western blotting using the anti-His antibody. The intensities of the bands were scanned and converted as relative integrated density values (RIDV) for comparison. The total protein loading controls from the cell lysates of these strains were detected using the anti-S. mutans antibody. B. The effect of constitutively expressed MecA on the cellular levels of SigX in GF-Ox (MecA-Ox) grown in THYE in response to CSP. The cellular MecA was detected by Western blotting using the anti-His antibody, while the cellular SigX in the same strain was detected using the anti-SigX antibody.
Mentions: To further investigate the effect of MecA on the stability of SigX, we constructed a constitutively expressed MecA strain, GF-Ox (MecA-Ox), which expressed MecA under the control of a constitutively expressed promoter of ldh, the gene encoding lactate dehydrogenase in S. mutans. In addition, this strain allowed detection of MecA protein by Western blotting using the anti-His antibody, since a His-tag was added to the end of mecA-coding sequence just before the stop codon. Our results confirmed that strain MecA-Ox constitutively expressed MecA when grown in THYE, independently on the growth phases. Based on the relative integrated density values (RIDV), this strain (MecA-Ox) produced as much as two-fold MecA protein when compared with that of strain GF-His2 (MecA-His) grown under the same condition (Figure 2A), suggesting that strain MecA-Ox overexpressed MecA under the conditions tested. It was noted from the Western blot that MecA-Ox showed an extra band that slightly bigger than the MecA. The feature of this extra band was unclear. One possibility was that over-expressed MecA might also bind to a small, unknown protein or peptide in S. mutans. Alternatively, this band might be a non-specific reaction. We then began to determine the effect of an overexpressed MecA on the cellular levels of SigX in response to CSP. The results revealed that the overexpression of MecA negatively affected the cellular levels of SigX in this strain, since addition of CSP induced very low levels of SigX, as detected by Western blotting using the anti-SigX antibody (Figure 2B). Consistent with this result, the constitutive expression of MecA resulted in hundred-fold reduction in transformation efficiency (Figure 1B). The results suggest that the overexpressed MecA enhances MecA-mediated proteolysis of SigX, accelerating 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