Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.
Bottom Line: The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem.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.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.Show MeSH
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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).
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.