Erythromycin a dihydrate

Erythromycin A dihydrate (analytical standard), CLA (≥95% standard), buspirone hydrochloride, dimethylsulfoxide (DMSO, 1.100 g/mL), and the reduced form of β-nicotinamide dinucleotide phosphate (NADPH, ≥93% standard) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Desmethyl-erythromycin A and N-desmethyl clarithromycin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Potassium phosphate buffer (0.1 M, pH 7.4) was purchased from Biosesang (Seongnam, Korea). HPLC-grade methanol and acetonitrile (ACN) were supplied by J.T. Baker Chemical (Radnor, PA, USA). Chicken liver microsomes (PL-MIC-201) were purchased from PRIMACYT GmbH (Schwerin, FRG, Germany) and stored at −80 °C before use. All chemicals were of the highest purity available.

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.

Bacterial strains used in this study are listed in Table S1A in the supplemental material. S. coelicolor strains were grown and maintained by a standard protocol (46 ). Proper culture media (S. coelicolor in YEME with 5 mM MgCl₂, 10% sucrose; E. coli, B. subtilis, and C. glutamicum in LB broth; V. vulnificus in LB with 2.5% NaCl; M. smegmatis in 7H9 medium without ferric ammonium citrate and supplemented with 0.05% Tween 80, 0.2% glucose, 0.5% bovine serum albumin, 0.085% NaCl) were used. For growth of S. coelicolor under anaerobic conditions, S. coelicolor spores were germinated and grown to mid-exponential phase (OD at 600 nm [OD₆₀₀] of ∼2.0) in YEME medium and then diluted 4-fold into YEME medium that had been placed in an anaerobic chamber (Coy) after being autoclaved overnight, followed by cultivation at 30°C under hypoxic condition.
For antibiotic treatment, freshly made solutions of antibiotics (kanamycin disulfate salt, tetracycline hydrochloride, chloramphenicol, erythromycin A dihydrate, fusidic acid sodium salt, polymyxin B sulfate, novobiocin sodium salt, rifampin; all from Sigma) at the indicated concentrations were applied to the early exponential-phase cells (OD₆₀₀ of ∼0.3).