Cellulose tlc

³²P-c-di-GMP and ³²P-c-di-AMP used in the experiments were synthesized from 33 nM ³²P-NTP (NTP = GTP or ATP), catalyzed by 40 μM WspR D70E (Pseudomonas aeruginosa. The D70E substitution mimics the phosphorylated, presumably active, conformation of WspR. WspR is, therefore, constitutively active) or DisA (Bacillus subtilis) at 37°C overnight. The buffer for WspR D70E reaction contains 10 mM Tris-HCl, pH 8.0, 100 mM NaCl and 5 mM MgCl₂ and buffer for DisA reaction contains 40 mM Tris-HCl, pH 7.5, 100 mM NaCl and 10 mM MgCl₂. After incubation, reaction mixture was heated up to 95°C and kept at 95°C for 5 min and cooled down to room temperature in 10 min, followed by filtration through 10 KD filter. The conversion was analyzed by a cellulose TLC (EMD Millipore, MA, USA) with running solvent 1:1.5 (v/v) saturated (NH₄)₂SO₄ : 1.5 M KH₂PO₄.

WspR D70E (P. aeruginosa), DisA (B. subtilis) and YybT (B. subtilis) were purified as described previously [49 ]. 1.5 μM YybT reacted with 10 μM c-di-GMP (³²P labeled + unlabeled) at 37°C for 30 min. 1.5 μM YybT reacted with 10 μM c-di-AMP (³²P labeled + unlabeled) at 37°C for 20 min. YybT reaction buffer contains 100 mM Tris-HCl, pH 8.3, 20 mM KCl, 1 mM DTT and 0.5 mM MnCl₂. Intercalators and cations were added where indicated. Reactions were quenched by heating up at 95°C for 5 min. 0.3 μl reaction mixture was applied on a cellulose TLC (EMD Millipore). TLC was developed in a buffer containing 1.5:1 (v/v) 1.5 M KH₂PO₄ and saturated (NH₄)₂SO₄.

As a step prior to the experiments, we verified that the chaperonins under analysis were able to hydrolyze ATP (Figure S1). The recombinant naïve Hsp60 was obtained from ATGen (Seongnam, South Korea) in stock solution at 16.0 µM (1 mg/ml) (buffer 20 mM Tris pH 8.0 and 10% glycerol (w/w)). Lyophilized GroEL was obtained from SIGMA (St. Louis, MO, USA). ATPase assay was performed as previously described [43] (link). Briefly, recombinant naïve Hsp60 or GroEL was added to ATPase buffer (6.6 mM HEPES (pH 7.6), 0.66 mM EDTA, 0.66 mM 2-mercaptoethanol, 0.033% NP-40, 1.1 mM MgCl₂, 33 µM ATP, 5 µCi (γ-33P) ATP-3000 mmol⁻¹ (Ge Healthcare)), and 100 ng of plasmidic DNA was used as substrate. The buffer was used as control and it was setup in parallel. The reactions were incubated for 30 min at 24°C. Unreacted ATP and free y-phosphate were separated by thin layer chromatography, using TLC cellulose (Merck Millipore, Milan, Italy). ATP hydrolysis quantification was done with a Bio-Rad (Berkeley, CA, USA) Personal Molecular Imager FX System.