A natural depsipeptide antibiotic binds the E-site of the bacterial ribosome

Bacterial strains and plasmids Bacterial strains and plasmids used in this study are listed in Supplementary Table 7 . Screening of WAC library for antimicrobial activity The pre-fractionation library from the WAC 6 was screened against hyperpermeable efflux-deficient E. coli BW25113 Δ tolC Δ bamB in 384-well microtitre plates (Corning 3701). Each well contained 49 μl of inoculated Mueller–Hinton broth (MHB) medium (cation-adjusted MHB (BD Difco)) and 1 μl of crude methanolic extract, fractions, or conditioned medium. A Biomek FXP Integrated Liquid Handler was used to dispense the fractions, extracts, and inoculated media into the plates. Plates were incubated at 37 °C for 20 h in a static incubator. Cell growth was measured by OD at 600 nm using EnVision, SpectraMax, or Biotek Neo microtitre plate readers. Purification of MKMs S. rimosus WAC 7405 was routinely cultured in tryptic soy broth (TSB) medium (Difco) in 225 ml flasks for 16 h, before inoculating at 1% (v/v) into ASM medium 58 in 2.8 l flasks for 4 days. Cultures were maintained at 30 °C, shaking at 220 rpm. During initial discovery, the active compound, as defined by routine testing against E. coli BW25113 Δ tolC Δ bamB , was isolated from conditioned medium mixed with 5% (v/v) Diaion HP-20 resin, mixed for 2.5 h. HP-20 resin was filtered using a milk filter and extracted with 300 ml of methanol for 2 h. The extract was collected and dried by rotary evaporation. …