GoKawiilBacterial 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 WAC6 was screened against hyperpermeable efflux-deficient E. coli BW25113 ΔtolCΔbamB 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 medium58 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 ΔtolCΔbamB, 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. After reconstitution in 10 ml water, compounds were separated by Sephadex LH-20 column (300 ml bed volume) and eluted with 50% methanol to yield 7× 50 ml fractions. Active fractions were analysed by the liquid chromatography–tandem mass spectrometry (LC–MS/MS) method; for this analysis, mass range was set to 150–2,500 m/z at a scan rate of 1 spectrum s−1. Three collision energies of 10, 30, 60 eV were selected with a medium isolation width of 4 atomic mass units. The liquid chromatography was performed using a gradient of H 2 O (0.1% formic acid v/v) and acetonitrile on an Eclipse SDB-C8 column (2.1 mm ID × 100 mm, 3.5 µm). The flow rate was 0.4 ml min−1, and the gradient started with 10% B for 2 min, followed by a linear gradient to 100% B over 15 min. After this, the fractions were assessed by GNPS8 to identify known compounds. Masses consistent with oxytetracycline were identified in fractions 3, 4 and 5. Fractions 1 and 2 were loaded as a liquid load on reverse-phase Combi Flash column (RediSep Rf C18 High performance Gold-50g, Teledyne) and eluted with a linear gradient of H 2 O (0.07% trifluoroacetic acid, solvent A) and acetonitrile (0.07% trifluoroacetic acid, solvent B). Active fractions were purified further by preparative reversed-phase high-pressure liquid chromatography (RP-HPLC- Agilent technologies) using C8 column (Eclipse XDB C8 Semi Prep 9.4 × 250 mm, 5 μm, Agilent Technologies) with a gradient of 5% to 20% of solvent B in 20 min. MKM-A and MKM-B were eluted at retention times of 17.5 and 18.5 min, respectively.
Later purifications were optimized as follows. Seed cultures in TSB were cultured for 2 days and inoculated into ASM at 10% (v/v). After HP-20 extraction, the sample was processed using SP-Sepharose cation-exchange chromatography. The column was pre-equilibrated with a 10 mM ammonium acetate buffer (buffer A; pH 5.0–5.2). The sample pH was adjusted to the same range. The column was washed with buffer A and 1 M NaCl in buffer A at pH 5.0. MKMs were eluted in 1 M NaCl in buffer A at pH 8.5–9.5. Fractions were neutralized using 0.6 N HCl during the elution. Following combiflash separation, as described above, analogues of MKM were resolved on a Chromatik Sunniest C28 RP-Aqua Semi Prep column for HPLC (10 × 250 mm, 5 µm) Purity of the compounds (>95%) was confirmed with a C28 analytical column (Sunniest RP-Aqua C28 4.6 × 100 mm, 5 µm). MKM-A (most abundant product), MKM-B, and MKM-E were purified as single peaks. Yield variations are shown in Supplementary Fig. 25.
Structural characterization of MKMs
High-resolution electrospray ionization mass spectra were acquired using an Agilent 1290 UPLC separation module and a qTOF 6550 mass detector in positive ion mode. For general liquid chromatography separation an Agilent Eclipse XDB C8 column (2.1 × 150 mm; 3.5 µm) and the following method were used: from 0 to 1 min 75% A (0.1 v/v formic acid in water), from 1 to 7 min a linear gradient to 100% B (0.1 v/v formic acid in acetonitrile) at a flow rate of 0.4 ml min−1. The NMR spectra were recorded on an AVIII 700 MHz NMR spectrometer, equipped with a cryoprobe. The compounds used in this study were dissolved in deuterated water as a solvent (Cambridge Isotope Laboratories) to a concentration of approximately 5.0 mg ml−1. Chemical shifts are reported in ppm relative to tetramethylsilane using the residual solvent signal at ppm. Chemical shifts values are expressed in ppm (δ), coupling constants (J, Hz) and peak patterns are reported as broad singlet (bs), singlet (s), doublet (d), triplet (t), quartet (q), pentet (p) and multiplet (m). MKM-A (1 mg) was treated with 6 N HCl (1 ml) in a sealed tube at 110 °C for 24 h. The reaction mixture was then extracted with ethyl acetate and the aqueous solution was dried under nitrogen. To the aqueous residue (10 µl) was added 1 M NaHCO 3 (10 µl), followed by Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-d-alanine amide) (50 µl, 1% solution in acetone). The reaction was then carried out for 1 h at 40 °C and stopped by the addition of 1 N HCl (10 µl) and methanol (420 µl)59,60.
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