Limits...
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.

Show MeSH

Related in: MedlinePlus

Intracellular c-diGMP regulates the production and the subcellular localization of CD2831.A, concentration of c-diGMP in the cytoplasm of the wild-type strain (WT) carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA) (gray bar) was quantified by LC-MS/MS. Cyclic dinucleotide concentrations are presented as nanograms of c-diGMP/mg of bacterial dry weight, and the average and standard deviation of three values are plotted. B, supernatant (Sn), cell wall (Cw), membrane (Mb), and cytosolic (Cy) compartments of wild-type (WT), sortase mutant (ΔsrtB), and complemented sortase (ΔsrtBcomp) strains carrying pECC12 (constitutively expresses DccA) were analyzed by Western blotting with anti-CD2831 antibodies. Supernatant and whole cell lysate (WL) fractions of wild-type strain carrying an empty plasmid (vector) were also analyzed as negative controls. Proteins were separated on 6% SDS-polyacrylamide gels. C, supernatant (Sn) and whole cell lysate (WL) fractions of wild-type strain (WT) carrying an empty plasmid (vector) or pECC17 (expresses DccA in the presence of ATc) and induced with the indicated concentrations of ATc were immunoblotted with anti-CD2831 antibodies. Proteins were separated on 6% SDS-polyacrylamide gels. D, supernatant (Sn) and whole cell lysate (WL) fractions of zmpI mutant strain (ΔzmpI), carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc, and of wild-type strain (WT), carrying pECC12 (constitutively expresses DccA), were immunoblotted with anti-CD2831 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591827&req=5

Figure 8: Intracellular c-diGMP regulates the production and the subcellular localization of CD2831.A, concentration of c-diGMP in the cytoplasm of the wild-type strain (WT) carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA) (gray bar) was quantified by LC-MS/MS. Cyclic dinucleotide concentrations are presented as nanograms of c-diGMP/mg of bacterial dry weight, and the average and standard deviation of three values are plotted. B, supernatant (Sn), cell wall (Cw), membrane (Mb), and cytosolic (Cy) compartments of wild-type (WT), sortase mutant (ΔsrtB), and complemented sortase (ΔsrtBcomp) strains carrying pECC12 (constitutively expresses DccA) were analyzed by Western blotting with anti-CD2831 antibodies. Supernatant and whole cell lysate (WL) fractions of wild-type strain carrying an empty plasmid (vector) were also analyzed as negative controls. Proteins were separated on 6% SDS-polyacrylamide gels. C, supernatant (Sn) and whole cell lysate (WL) fractions of wild-type strain (WT) carrying an empty plasmid (vector) or pECC17 (expresses DccA in the presence of ATc) and induced with the indicated concentrations of ATc were immunoblotted with anti-CD2831 antibodies. Proteins were separated on 6% SDS-polyacrylamide gels. D, supernatant (Sn) and whole cell lysate (WL) fractions of zmpI mutant strain (ΔzmpI), carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc, and of wild-type strain (WT), carrying pECC12 (constitutively expresses DccA), were immunoblotted with anti-CD2831 antibodies.

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).


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)

Intracellular c-diGMP regulates the production and the subcellular localization of CD2831.A, concentration of c-diGMP in the cytoplasm of the wild-type strain (WT) carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA) (gray bar) was quantified by LC-MS/MS. Cyclic dinucleotide concentrations are presented as nanograms of c-diGMP/mg of bacterial dry weight, and the average and standard deviation of three values are plotted. B, supernatant (Sn), cell wall (Cw), membrane (Mb), and cytosolic (Cy) compartments of wild-type (WT), sortase mutant (ΔsrtB), and complemented sortase (ΔsrtBcomp) strains carrying pECC12 (constitutively expresses DccA) were analyzed by Western blotting with anti-CD2831 antibodies. Supernatant and whole cell lysate (WL) fractions of wild-type strain carrying an empty plasmid (vector) were also analyzed as negative controls. Proteins were separated on 6% SDS-polyacrylamide gels. C, supernatant (Sn) and whole cell lysate (WL) fractions of wild-type strain (WT) carrying an empty plasmid (vector) or pECC17 (expresses DccA in the presence of ATc) and induced with the indicated concentrations of ATc were immunoblotted with anti-CD2831 antibodies. Proteins were separated on 6% SDS-polyacrylamide gels. D, supernatant (Sn) and whole cell lysate (WL) fractions of zmpI mutant strain (ΔzmpI), carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc, and of wild-type strain (WT), carrying pECC12 (constitutively expresses DccA), were immunoblotted with anti-CD2831 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Intracellular c-diGMP regulates the production and the subcellular localization of CD2831.A, concentration of c-diGMP in the cytoplasm of the wild-type strain (WT) carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA) (gray bar) was quantified by LC-MS/MS. Cyclic dinucleotide concentrations are presented as nanograms of c-diGMP/mg of bacterial dry weight, and the average and standard deviation of three values are plotted. B, supernatant (Sn), cell wall (Cw), membrane (Mb), and cytosolic (Cy) compartments of wild-type (WT), sortase mutant (ΔsrtB), and complemented sortase (ΔsrtBcomp) strains carrying pECC12 (constitutively expresses DccA) were analyzed by Western blotting with anti-CD2831 antibodies. Supernatant and whole cell lysate (WL) fractions of wild-type strain carrying an empty plasmid (vector) were also analyzed as negative controls. Proteins were separated on 6% SDS-polyacrylamide gels. C, supernatant (Sn) and whole cell lysate (WL) fractions of wild-type strain (WT) carrying an empty plasmid (vector) or pECC17 (expresses DccA in the presence of ATc) and induced with the indicated concentrations of ATc were immunoblotted with anti-CD2831 antibodies. Proteins were separated on 6% SDS-polyacrylamide gels. D, supernatant (Sn) and whole cell lysate (WL) fractions of zmpI mutant strain (ΔzmpI), carrying pJKP041 (expresses CD2831 in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc, and of wild-type strain (WT), carrying pECC12 (constitutively expresses DccA), were immunoblotted with anti-CD2831 antibodies.
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).

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