Identification and characterisation of the gene cluster for the anti-MRSA antibiotic bottromycin: expanding the biosynthetic diversity of ribosomal peptides

William J. K. Crone, Finian J. Leeper, Andrew W. Truman
2012 Chemical Science  
Materials All chemicals were analytical grade and obtained from Sigma Aldrich unless otherwise specified. Bacterial Strains Streptomyces scabies DSM 41658 and Streptomyces bottropensis DSM 40262 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) and were kept either on agar plates at 4 °C or in 30-40% glycerol stocks at -80 °C. NovaBlue competent cells (Novagen) were used for genetic manipulation and the methylation deficient strain of E. coli, ET12657 1 ,
more » ... oli, ET12657 1 , was used for transfer of genetic material to S. scabies by conjugation. Cells transformed with plasmids were stored in a 30-40% glycerol stock at -80 °C. Antibiotics Antibiotics were used at final concentrations as follows: kanamycin (Melford) was used at 50 µg mL -1 , apramycin (Duchefa) at 50 µg mL -1 , carbenicillin (Melford) at 30 µg mL -1 , chloramphenicol at 25 µg mL -1 and nalidixic acid at 25 µg mL -1 . Production of bottromycin GYM medium (0.4% glucose (Fisher), 0.4% yeast extract, 1.0% malt extract, in Milli-Q (MQ) water 2 ) in a 250 mL flask was inoculated with a small piece (approx. 1 cm 2 ) of bacteria grown on SFM-agar (Soy Flour Medium: 20 g L -1 soy flour, 20 g L -1 mannitol, 20 g L -1 agar, 10 mM MgCl 2 in tap water 3 ). This was incubated at 250 rpm, 30 °C for 2 days. This starter culture was then used to inoculate production medium 2 (PM: 1% glucose, 1.5% starch, 0.5% yeast extract, 1.0% soy flour, 0.5% NaCl (Breckland), 0.3% CaCO 3 (Breckland) in MQ water), at 5% of the PM volume. 1.05 mM of CoCl 2 was often included in the PM as it was found to increase production of bottromycin. LCMS analysis of cell extract Spectra were obtained using a Hewlett-Packard HPLC 1100 series instrument coupled to a Finnigan MAT LCQ ion trap mass spectrometer fitted with a positive mode ESI source. PM cell culture (100 µL) was diluted with methanol (100 µL) at various time points and centrifuged to pellet cell debris. 40 µL of the supernatant was added to 5 µL of 0.5 mM desvancosaminyl vancomycin (DVV), which was used as a reference compound. Samples were injected onto a Phenomenex Luna C18(2) column (250 mm x 2.0 mm, 5 µm), eluting with a linear gradient of 5 to 95% acetonitrile (Fisher, Electronic Supplementary Material (ESI) for Chemical Science This journal is
doi:10.1039/c2sc21190d fatcat:mo4753mpdjaf3midw7bd42gwxa