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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

Role of SrtB in anchoring CD2831 to the cell wall. Supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-CD2831 polyclonal antibodies. Supernatant and whole cell lysate (WL) fractions of ΔzmpI pJKP041 strain cultivated in the absence of ATc (−ATc) were also analyzed as negative controls.
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Figure 4: Role of SrtB in anchoring CD2831 to the cell wall. Supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-CD2831 polyclonal antibodies. Supernatant and whole cell lysate (WL) fractions of ΔzmpI pJKP041 strain cultivated in the absence of ATc (−ATc) were also analyzed as negative controls.

Mentions: The ZmpI cleavage sites within CD2831 are not randomly distributed along the protein sequence but are confined in the C-terminal extremity, directly upstream of the putative PPKTG sorting motif (30). Thus, it is possible that the CD2831 protein is anchored to the cell wall through a sorting reaction catalyzed by SrtB and subsequently secreted into the extracellular medium after its cleavage by the ZmpI protease. To test this hypothesis, the srtB gene in the ΔzmpI mutant strain was inactivated by deletion. pJKP041 was then introduced into the ΔzmpIΔsrtB double mutant, and the subcellular localization of CD2831 in this strain grown in the presence of inducer was then analyzed by immunoblotting and immunofluorescence microscopy. Cell wall extracts from the ΔzmpIΔsrtB mutant displayed undetectable levels of CD2831, whereas the protein was strongly detected in the supernatant fraction (Fig. 4). In agreement with these data, the anti-CD2831 antibodies did not surface-label the ΔzmpIΔsrtB mutant strain (Fig. 3), demonstrating that SrtB is required for anchoring of CD2831 to the cell wall.


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)

Role of SrtB in anchoring CD2831 to the cell wall. Supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-CD2831 polyclonal antibodies. Supernatant and whole cell lysate (WL) fractions of ΔzmpI pJKP041 strain cultivated in the absence of ATc (−ATc) were also analyzed as negative controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Role of SrtB in anchoring CD2831 to the cell wall. Supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-CD2831 polyclonal antibodies. Supernatant and whole cell lysate (WL) fractions of ΔzmpI pJKP041 strain cultivated in the absence of ATc (−ATc) were also analyzed as negative controls.
Mentions: The ZmpI cleavage sites within CD2831 are not randomly distributed along the protein sequence but are confined in the C-terminal extremity, directly upstream of the putative PPKTG sorting motif (30). Thus, it is possible that the CD2831 protein is anchored to the cell wall through a sorting reaction catalyzed by SrtB and subsequently secreted into the extracellular medium after its cleavage by the ZmpI protease. To test this hypothesis, the srtB gene in the ΔzmpI mutant strain was inactivated by deletion. pJKP041 was then introduced into the ΔzmpIΔsrtB double mutant, and the subcellular localization of CD2831 in this strain grown in the presence of inducer was then analyzed by immunoblotting and immunofluorescence microscopy. Cell wall extracts from the ΔzmpIΔsrtB mutant displayed undetectable levels of CD2831, whereas the protein was strongly detected in the supernatant fraction (Fig. 4). In agreement with these data, the anti-CD2831 antibodies did not surface-label the ΔzmpIΔsrtB mutant strain (Fig. 3), demonstrating that SrtB is required for anchoring of CD2831 to the cell wall.

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