Criterion polyacrylamide gel

Sulfate-starved and control S. elongatus PCC 7942 liquid cultures (50 mL) were harvested via centrifugation (4000 x g, 20 min, 4°C), flash-frozen in liquid nitrogen and stored at −80°C for future processing. Pellets were lysed via sonication and clarified by centrifugation (15,000 x g, 20 min, 4°C). Clarified lysates were analyzed using 4–20% Criterion polyacrylamide gels (Bio-Rad) and visualized by silver staining using Pierce Silver Stain Kit (ThermoFisher Scientific). Gel bands were excised and sent to UC Davis Proteomics Core Facility. In-gel proteolytic digestion of the samples was performed followed by LC/MS analysis with a Q Exactive Hybrid Quadrupole-Orbitrap. Spectra were searched against the S. elongatus PCC 7942 proteome and analyzed using Scaffold 4.

Cell pellets (5 g dry cell mass) were thawed at room temperature and resuspended in 50 mL of lysis buffer (20 mM Tris-HCl pH 8, 150 mM NH₄Cl, 20 mM MgCl₂) supplemented with 50 mg lysozyme, 20 U DNaseI, 100 μg RNase A. Samples were lysed by three passages through an Avestin EmulsiFlex-C3 homogenizer and clarified via centrifugation (15,000 × g, 30 min, 4°C). The clarified lysate was then spun at 110,000 × g for 3 hr at 4°C. The supernatant was discarded and the resulting pellet was resuspended with wash buffer (20 mM Tris pH 8, 150 mM NH₄Cl, 20 mM MgCl₂) supplemented with 1X Cell Lytic B (Sigma-Aldrich). The sample was then spun at 4000 × g at 4°C for 10 min followed by removing the supernatant and resuspension of the pellet in 4 mL of 50 mM Tris-HCl pH 8, 300 mM NaCl. The sample was then incubated at room temperature for 10 min to allow for solubilization and then centrifuged at 4000 × g at 4°C for 10 min to remove insoluble material. The resulting supernatant was then concentrated using Vivaspin 6 100000 MWCO concentrator columns (Sartorius). The sample was then purified via size-exclusion chromatography using a Superose 6 Increase column (GE Life Sciences), and fractions were analyzed by SDS-PAGE using 4–20% Criterion polyacrylamide gels (Bio-Rad) and visualized with GelCode Blue stain (Thermo Fisher).