Fusidic acid sodium salt

All experiments were carried out in either polymix buffer A (50 mM Tris-OAc (pH 7.5), 100 mM KCl, 5 mM NH₄OAc, 0.5 mM Ca(OAc)₂, 5 mM Mg(OAc)₂, 6 mM 2-mercaptoethanol, 0.1 mM EDTA, 5 mM putrescine and 1 mM spermidine)^{45 (link)} or polymix buffer B (30 mM HEPES pH 7.5, 5 mM MgCl₂, 50 mM NH₄Cl, 5 mM 2-mercaptoethanol, 2 mM spermidine and 5 mM putrescine)^{46 (link)}. A cocktail of triplet-state quenchers (1 mM Trolox, 1 mM nitrobenzyl alcohol and 1 mM cyclooctatetraene) and an enzymatic oxygen scavenging system (protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD)) were used for smFRET experiments. Spectinomycin sulfate was purchased from MP Biomedicals. Fusidic acid sodium salt, GTP and GTPγS were from Sigma-Aldrich. GTP was further purified using a Mono Q 5/50 GL anion exchange column (GE Healthcare Life Sciences). Pyruvate kinase, myokinase and phosphoenolpyruvate (PEP) were purchased from Sigma-Aldrich. All other standard reagents were purchased from Sigma-Aldrich or VWR.

Streptomyces coelicolor strains and DNA primers used in this study are listed in Table S1 in the supplemental material. Spores of each strain, prepared according to standard procedures (4 ), were inoculated in YEME liquid medium containing 5 mM MgCl₂ and 10% sucrose and grown at 30°C with shaking at 180 rpm. For antibiotic stress conditions, a freshly made solution of tetracycline hydrochloride (Sigma) or stock solutions of chloramphenicol (Sigma) or erythromycin A dihydrate (Sigma) at the indicated concentrations were treated to early exponential cells (optical density at 600 nm [OD₆₀₀] of 0.1 to 0.5). For determining MIC, erythromycin (Sigma), tetracycline hydrochloride (Sigma), lincomycin hydrochloride (Fluka), chloramphenicol (Sigma), fusidic acid sodium salt (Sigma), hygromycin B concentrated solution (Duchefa), linezolid (Sigma), streptomycin sulfate salt (Sigma), thiostrepton from Streptomyces azureus (Sigma), puromycin dihydrochloride from Streptomyces alboniger (Sigma), and spectinomycin dihydrochloride pentahydrate (Fluka) were used. E. coli strains BW25113/pIJ790 and ET12567/pUZ8002 were grown and used as previously described (57 (link)), and E. coli DH5α was grown in LB medium for standard plasmid manipulations.