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Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

Peltier J, Shaw HA, Couchman EC, Dawson LF, Yu L, Choudhary JS, Kaever V, Wren BW, Fairweather NF - J. Biol. Chem. (2015)

Bottom Line: Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells.This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch.These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.

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Related in: MedlinePlus

Expression and purification of CD2831R-HA from the cell wall of C. difficile ΔzmpI.A, CD2831R-HA construct under control of the inducible tetracycline promoter (Ptet) containing signal peptide (SP), the PPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+). Cleavage sites of SrtB and ArgC are indicated. B, CD2831 is covalently linked to the peptidoglycan of C. difficile. Nonacetylated glucosamine residues (G) and unusual mDAP-mDAP cross-links generated by dl-transpeptidation are predominant in the peptidoglycan of C. difficile (34). Cleavage sites of the CD27L endolysin are indicated by arrowheads. NAM, N-acetylmuramic acid; DAP, diaminopimelic acid. C, cells from ΔzmpI strain expressing CD2831R-HA were suspended in PS buffer and treated with the purified catalytic domain of the CD27L endolysin. Digested cell wall was isolated by centrifugation and subjected to immunoprecipitation using anti-HA. Purified polypeptides were separated on 10% SDS-polyacrylamide gels and Coomassie Blue-stained (left panel) or probed with anti-HA antibodies (right panel).
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Figure 5: Expression and purification of CD2831R-HA from the cell wall of C. difficile ΔzmpI.A, CD2831R-HA construct under control of the inducible tetracycline promoter (Ptet) containing signal peptide (SP), the PPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+). Cleavage sites of SrtB and ArgC are indicated. B, CD2831 is covalently linked to the peptidoglycan of C. difficile. Nonacetylated glucosamine residues (G) and unusual mDAP-mDAP cross-links generated by dl-transpeptidation are predominant in the peptidoglycan of C. difficile (34). Cleavage sites of the CD27L endolysin are indicated by arrowheads. NAM, N-acetylmuramic acid; DAP, diaminopimelic acid. C, cells from ΔzmpI strain expressing CD2831R-HA were suspended in PS buffer and treated with the purified catalytic domain of the CD27L endolysin. Digested cell wall was isolated by centrifugation and subjected to immunoprecipitation using anti-HA. Purified polypeptides were separated on 10% SDS-polyacrylamide gels and Coomassie Blue-stained (left panel) or probed with anti-HA antibodies (right panel).

Mentions: SrtB-dependent cell wall anchoring of CD2831 suggests its covalent anchoring to the peptidoglycan. To determine the cell wall anchor structure of CD2831, a C-terminally modified derivative (CD2831R-HA) was constructed and placed under the control of the inducible Ptet promoter (pJKP095). CD2831R-HA consists of CD2831 with an arginine residue and an HA tag inserted five amino acids upstream of the PPKTG sorting motif (Fig. 5A). pJKP095 was transferred into the ΔzmpI mutant, and upon induction with ATc, CD2831R-HA could be detected in both the supernatant and the cell wall fraction by immunoblotting with anti-CD2831 or anti-HA antibodies, indicating that the subcellular localization of CD2831R-HA is similar to that of CD2831 (data not shown).


Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

Peltier J, Shaw HA, Couchman EC, Dawson LF, Yu L, Choudhary JS, Kaever V, Wren BW, Fairweather NF - J. Biol. Chem. (2015)

Expression and purification of CD2831R-HA from the cell wall of C. difficile ΔzmpI.A, CD2831R-HA construct under control of the inducible tetracycline promoter (Ptet) containing signal peptide (SP), the PPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+). Cleavage sites of SrtB and ArgC are indicated. B, CD2831 is covalently linked to the peptidoglycan of C. difficile. Nonacetylated glucosamine residues (G) and unusual mDAP-mDAP cross-links generated by dl-transpeptidation are predominant in the peptidoglycan of C. difficile (34). Cleavage sites of the CD27L endolysin are indicated by arrowheads. NAM, N-acetylmuramic acid; DAP, diaminopimelic acid. C, cells from ΔzmpI strain expressing CD2831R-HA were suspended in PS buffer and treated with the purified catalytic domain of the CD27L endolysin. Digested cell wall was isolated by centrifugation and subjected to immunoprecipitation using anti-HA. Purified polypeptides were separated on 10% SDS-polyacrylamide gels and Coomassie Blue-stained (left panel) or probed with anti-HA antibodies (right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591827&req=5

Figure 5: Expression and purification of CD2831R-HA from the cell wall of C. difficile ΔzmpI.A, CD2831R-HA construct under control of the inducible tetracycline promoter (Ptet) containing signal peptide (SP), the PPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+). Cleavage sites of SrtB and ArgC are indicated. B, CD2831 is covalently linked to the peptidoglycan of C. difficile. Nonacetylated glucosamine residues (G) and unusual mDAP-mDAP cross-links generated by dl-transpeptidation are predominant in the peptidoglycan of C. difficile (34). Cleavage sites of the CD27L endolysin are indicated by arrowheads. NAM, N-acetylmuramic acid; DAP, diaminopimelic acid. C, cells from ΔzmpI strain expressing CD2831R-HA were suspended in PS buffer and treated with the purified catalytic domain of the CD27L endolysin. Digested cell wall was isolated by centrifugation and subjected to immunoprecipitation using anti-HA. Purified polypeptides were separated on 10% SDS-polyacrylamide gels and Coomassie Blue-stained (left panel) or probed with anti-HA antibodies (right panel).
Mentions: SrtB-dependent cell wall anchoring of CD2831 suggests its covalent anchoring to the peptidoglycan. To determine the cell wall anchor structure of CD2831, a C-terminally modified derivative (CD2831R-HA) was constructed and placed under the control of the inducible Ptet promoter (pJKP095). CD2831R-HA consists of CD2831 with an arginine residue and an HA tag inserted five amino acids upstream of the PPKTG sorting motif (Fig. 5A). pJKP095 was transferred into the ΔzmpI mutant, and upon induction with ATc, CD2831R-HA could be detected in both the supernatant and the cell wall fraction by immunoblotting with anti-CD2831 or anti-HA antibodies, indicating that the subcellular localization of CD2831R-HA is similar to that of CD2831 (data not shown).

Bottom Line: Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells.This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch.These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.

Show MeSH
Related in: MedlinePlus