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. Author manuscript; available in PMC: 2017 Jul 13.
Published in final edited form as: Cell Host Microbe. 2016 Jul 13;20(1):49–59. doi: 10.1016/j.chom.2016.06.003

Figure 2. CdnP degrades extracellular c-di-AMP.

Figure 2

(A) GBS synthesizes only c-di-AMP. The intracellular concentration of cyclic di-nucleotides was quantified by LC-MS/MS in total WT GBS extracts.

(B) GBS degrades extracellular c-di-AMP. At t = 0, 100 μM c-di-AMP was added to early stationary phase cultures of GBS previously washed and resuspend in Tris-HCl (pH 7.5) containing 5 mM MnCl2. Kinetics of extracellular c-di-AMP degradation (mean and S.D. from at least three independent bacterial cultures) were followed by RR-HPLC for the WT (black dots), the ΔnudP (blue triangles), and the ΔcdnP* (red squares) ectonucleotidase mutants.

(C) Extracellular c-di-AMP is degraded into adenosine by the sequential activity of the NudP and CdnP ectonucleotidases. Same experiments as in (B) monitoring the formation of the reaction products at the end of the experiment (t = 100 min) for the WT (black), the ΔnudP (blue), and the ΔcdnP* (red) ectonucleotidase mutants.

(D) Schematic representations of NudP and CdnP. The archetypal metallophosphatase (Metallophos, pfam domain 00149) and substrate-binding (5-Nucleotid_C, pfam domain 02872) domains of ectonucleotidases are colored (blue for NudP, red for CdnP). Numbers indicate the amino acid positions of the domains in the proteins. Transmembrane domains are indicated as grey boxes and are necessary for secretion (SP: signal peptide) and cell wall anchoring through the LPxTG motif (black rhombus). The black asterisk indicates the position of the conserved histidine residue at position 199 of CdnP essential for metallophosphodiesterase activity. The corresponding mutation to alanine in the catalytically inactive (cat−) mutant CdnP* is shown.

(E) CdnP activity limits extracellular c-di-AMP accumulation by GBS. Extracellular c-di-AMP in the GBS culture supernatants was quantified by an enzyme-linked assay followed by RR-HPLC. The two control GBS strains (black: WT and white: WTbk) are compared in parallel with the ΔcdnP* mutant (red). Mean and S.D. in arbitrary units (AU) are calculated from at least three independent experiments. See also Figure S2.