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: Transcription of CD2831 is controlled by a type II c-diGMP riboswitch (Cdi2_3) and is increased in the presence of high levels of c-diGMP (20). Interestingly, expression of the adjacent gene CD2830 encoding ZmpI protease is negatively regulated by c-diGMP through a type I riboswitch (Cdi1_12) (20). Because zmpI and CD2831 are inversely regulated by c-diGMP, we investigated whether CD2831 is anchored to the cell surface in the presence of high levels of c-diGMP. The dccA gene encoding a c-diGMP synthetase was cloned in a plasmid under the control of the constitutive Pcwp2 promoter, leading to pECC12. Measurement of the intracellular c-diGMP concentration by LC-MS/MS confirmed the presence of high levels of c-diGMP in the wild-type strain containing pECC12 (142.57 ± 9.57 ng/mg bacterial dry weight), whereas c-diGMP was undetectable in the strain carrying an empty vector (Fig. 8A). Western blot analysis of subcellular fractions revealed that expression of CD2831 was induced in the presence of a high level of c-diGMP, as the protein could be detected without using any expression in trans (Fig. 8B). Moreover, CD2831 was almost exclusively detected in the cell wall fraction as the two predominant polypeptides of ∼140 and ∼155 kDa, suggesting that production of the ZmpI protease was abolished in these conditions. CD2831 could not be detected in the supernatant or the whole cell protein extract from the wild-type strain carrying an empty plasmid. When pECC12 was introduced into 630 ΔsrtB, CD2831 could be detected but was mostly found in the culture supernatant, consistent with the requirement of the sortase to anchor CD2831 to the cell wall. To complement the ΔsrtB mutant, the srtB gene was restored to its native locus using allelic replacement. To distinguish the complemented strain from the wild-type strain, the original TAA stop codon of srtB was replaced with a TAG stop codon in the complemented srtB strain. Plasmid pECC12 was transferred into the resulting strain, srtBcomp, and wild-type CD2831 localization was restored in this strain (Fig. 8B).
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.