Cultivation and Characterization of Synechocystis

Synechocystis sp. 6803 substrain PCC-M²² (link) was used in all experiments. The axenic strain was maintained on agar plates supplemented with BG11 mineral medium²³ at 30°C under constant illumination. Transformants were initially selected on media containing 10 µg ml⁻¹ kanamycin (Km; Sigma), while the segregation of clones and cultivation of mutants was performed at 50 µg ml⁻¹Km. For physiological characterization, axenic cultures of the different strains (Supplementary Table S1) were grown photoautotrophically in BG11 medium, either under slight shaking in Erlenmeyer flasks at 50 µmol photons s⁻¹m⁻², or under bubbling with CO₂-enriched air (5% [v/v]) in batch cultures at 29°C under continuous illumination of 180 µmol photons s⁻¹m⁻² (warm light, Osram L58 W32/3). Contamination by heterotrophic bacteria was evaluated by microscopy or spreading of 0.2 mL culture on LB plates. The E. coli strains TG1, TOP10, and DH5α were used for routine DNA manipulations. E. coli was cultured in LB medium at 37°C. Growth was followed by measurements of the optical density at 750 nm (OD₇₅₀) for Synechocystis and at 500 nm (OD₅₀₀) for E. coli.