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: Immunofluorescence microscopy analysis was performed to detect CD2831. In the wild-type strain carrying pECC12 (constitutive expression of dccA) or pECC17 and grown in the presence of 100 ng/ml ATc (inducible expression of dccA), fluorescence was not detected (data not shown). Phase contrast microscopy revealed that cells carrying pECC12 grew as chains compared with the wild-type strain carrying an empty vector (Fig. 9A). These chains contained incomplete cell septa as visualized by staining with MitoTracker Green, suggesting that increased intracellular levels of c-diGMP result in aberrant cell division. Chains of cells were also observed in the strain carrying pECC17 upon induction with 100 ng/ml ATc, but to a much lesser extent (Fig. 9B). Indeed, in the strain carrying pECC17, chain length was found to correlate to the inducer concentration. Thus in C. difficile 630, formation of long chains of cells is likely the direct consequence of the elevation of c-diGMP levels. The absence of CD2831 immunolabeling at the surface of cells expressing high levels of c-diGMP might be related to the formation of chains or to insufficient levels of surface-localized CD2831. To explore this second hypothesis, the levels of CD2831 in strains 630 (pECC12) and 630 Δzmp1 (pJKP041) were compared. The levels of CD2831 were extremely low in 630 (pECC12) compared with 630 Δzmp1 (pJKP041), after induction with ATc, and were therefore probably too low to be detected by immunofluorescence (Fig. 8D).
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.