Extended Data Fig. 1: Biochemical analysis of SMS system.
From: An early origin of iron–sulfur cluster biosynthesis machineries before Earth oxygenation
A. Production of SMS hybrid fusion proteins used for Bacterial 2-hybrid. E. coli strains transformed with the indicated plasmids were grown in LB at 37 °C and expression was induced with 0.5 mM IPTG for two hours. Total cell extracts were separated on a 10% SDS–PAGE and proteins were detected by Western blot with an anti-T18 monoclonal antibody (sc-13582, Santa Cruz) or an anti-T25 polyclonal antibody (gift from D. Ladant). B. Quantification of the two-hybrid assay. BTH101 strain transformed by the indicated two-hybrid plasmids together with a placZ-GFP compatible plasmid were incubated overnight at 30 °C in LB supplemented with 0.5 mM IPTG. The activities correspond to the ratio between GFP fluorescence and the OD600 of 4 replicates and are given in arbitrary units (A.U.). The mean is represented and the error bars stand for standard deviations. C. UV-visible spectroscopy analysis of reconstituted Methanococcus jannaschii SmsCB (2.3 mg/ml) in 100 mM Tris-HCL, 150 mM NaCl pH 8. Iron and sulfur content: 3.7 ± 0.1 Fe and 3.5 ± 0.15 S/ SufC2B2.
