Xad16 resin

Wild-type Microcin J25 (MccJ25) was used in this study as formation of the lasso ring is not possible synthetically. The peptide was expressed using MccJ25 biosynthetic gene cluster carrying plasmid pTUC202 (a gift from Rutgers University USA) in competent E. coli MC4100 cells [25 (link)]. It was grown in 2L M9 minimal media for 18 h at 37 °C. The culture supernatant was obtained by centrifugation at 4000 g for 15 min, and then subjected to two successive purification steps. First, it was applied on the flex column filled with XAD16 resin (Sigma-Aldrich, St. Louis, USA). Two successive elution steps were performed with (30:70, v/v) and (80:20, v/v) methanol/water mixtures. MccJ25 is eluted in the (80:20, v/v) methanol/water mixture. MccJ25 was purified using reversed-phase (RP) HPLC (Varian Prostar 210, SpectraLab Scientific Inc. Markham, Canada). The purity of the peptides was confirmed by MALDI-TOF mass spectrometry.

Myxococcus xanthus strains were cultivated in 50-mL CTT medium supplemented with 2% XAD-16 resin (Sigma-Aldrich). After 96 h of shaking at 200 rpm and 30 °C, the cells and XAD resin were harvested by centrifugation (20 min, 5000 rpm, 20 °C). The pellet was extracted with 3 mL methanol overnight, and the extracts were dried in vacuum and resuspended in 3 mL distilled methanol. Three replicates were performed for each experiment.