Mini-Tn5TransposonScreenPyoverdineLackofR569.md

Mini-Tn5 transposon mutant screen for lack of pyoverdine fluorescence of R569

R569 mini-Tn5 mutants were cultured in 96 well plates at 25 °C and 180 rpm for five days in 50% TSB medium. For the initial mutant screen, fluorescence was acquired at 𝜆excitation=395 nm and 𝜆emission=470 nm in a microplate reader (Infinite M200 PRO, Tecan). Out of 2,000 mutants analysed, the fluorescence-based screening identified twelve R569 mutants that showed severely reduced pyoverdine-specific fluorescence but retained median-like growth behaviour (Thresholds: -6x MAD fluorescence, >-1x MAD Abs600~). For validation, cultures were pre-grown for five days in 50% TSB before sub-culturing into fresh siderophore medium (see Table S5) and growth for five additional days. Finally, bacterial culture density (Abs600) was determined, and pyoverdine-specific fluorescence of bacterial culture supernatants was captured at 𝜆excitation=410 nm and 𝜆emission=500 nm. For comparison between genotypes, fluorescence capacity was calculated by dividing the pyoverdine-specific fluorescence by culture density.