C di gmp

Interaction between c-di-AMP and targeted GBS proteins was tested by DRaCALA [47 (link)] on whole E. coli protein extract. Expression of the candidate GBS protein was done in Bli5 containing pET-28a or pIVEX expression vector (S6 Table). Expression of the tagged-GBS protein was induced with IPTG (1 mM) for 6 hours at 30°C. Bacterial pellet from 1 ml culture is suspended in 100 μl binding buffer (40 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.5 mg/ml lysozyme, 20 μg/ml DNase), lysed by 3 freeze-thaw cycles, and directly used for DRaCALA and Western blot analysis using anti-His-tag antibodies. For DraCALA, 1 nM ³²P-labeled c-di-AMP, synthetized as described in reference [22 (link)], was added to the whole protein extract, incubated at room temperature for 5 min, and 2.5 μl was spotted onto nitrocellulose membrane. Membranes are revealed with radiographic films (Amersham Hyperfilm ECL, GE Healthcare) and signal intensity quantified with ImageJ (NIH). The c-di-AMP bound fraction was calculated as described [47 (link)]. For competition assay 200 μM of cold nucleotides (c-di-AMP, c-di-GMP, cAMP, cGMP, AMP, and ATP; BioLog Life Science Institute, Germany) were added to the protein extract altogether with radiolabelled c-di-AMP.

A 293 base pair gbpA promoter fragment was amplified from V. cholerae C6706 genomic DNA using primers gbpAP2F + Vc1R2. A 133 base pair non-specific control DNA fragment internal to the gbpA ORF was amplified with gbpAqF2 + gbpAqR2. EMSAs were done as previously described [56 (link)]. Binding reactions were done in 10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 0.1 mM DTT, 5 % glycerol (v/v) and 0.01 mg/ml BSA (final concentrations). The gbpA promoter fragment, CRP, and cAMP (Sigma-Aldrich) were used at final concentrations of 2.5 ng/μl, 11 ng/μl, and 33 μM, respectively. C-di-GMP (Biolog) was used at 33 μM, or 333 μM when in competition with cAMP. The non-specific DNA fragment (75 ng) was added to every mixture. After 1 h incubation at room temperature (~25 °C), the samples were electrophoresed on a 6 % TAE polyacrylamide gel. The gel was stained with GelRed (Biotium, Inc.) in TAE for 10 min, and then visualized under UV light.

Motility assays were performed as previously described (12 (link)). When appropriate, the plates were supplemented with 10 μM c-di-GMP (BioLog), pGpG (BioLog), guanosine (Sigma), cGMP (Sigma), GTP (Sigma), and cyclic AMP (cAMP; Sigma). Pictures were taken using ImageScanner III (GE Healthcare Life Sciences).

Cells were grown to an optical density at 750 nm (OD₇₅₀) of 1 and transferred to new 250 ml flasks with BG-11/HEPES medium containing 0.2 M sorbitol for the osmotic stress, or 0.2 M NaCl for the ionic stress, except for halophile Synechocystis for which the NaCl concentration was 0.6 M. As a control, BG-11/HEPES medium without sorbitol or NaCl added was utilized. Cells were maintained under osmotic or salt stress for 24 h. After 24 h, c-di-AMP and c-di-GMP were quantified as described (Massie et al., 2012 (link); Agostoni et al., 2013 (link); Barker et al., 2013 (link)). In brief, c-di-AMP and c-di-GMP were quantified by UPLC-MS/MS. Prior to analysis, an aliquot of each sample was dried under vacuum to remove extraction buffer and the pellet was resuspended in an equal volume of water. A 10-μl volume of the resuspended sample was analyzed together with an eight-point standard curve of purified c-di-AMP or c-di-GMP (Biolog). c-di-AMP and c-di-GMP concentrations determined for samples were normalized to total soluble protein content from an equal volume of cells from which second messengers were extracted as previously described (Agostoni et al., 2013 (link); Zhu et al., 2016 (link)). Growth over time of WT, OE DAC and OE PDE Synechocystis stains in the presence of sorbitol (0.5 M) or NaCl (0.6 M) was measured using OD₇₅₀ as described above.