Cyanobacterial Stress Responses and Signaling

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.