MALDI for An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVDP482) with 7-hydroxytropolone is pivotal for its antibacterial activity

Strains and culture conditionsThe strains used in the study are listed in Table 1. Unless otherwise stated, P. donghuensis P482, HYST, and the P482 mutants were routinely grown in Miller’s Lysogeny Broth (LB, Novagen, Germany) or on LB solidified with 1.5 % agar (LB-agar, Novagen, Germany). Pseudomonas spp. and Dickeya solani IFB102 (referred to as Ds) were cultured at 28 °C, and Escherichia coli ST18 at 37 °C. For the P482 mutants: KN1009 and KN3755 defective in pyoverdine synthesis (locus tags: BV82_1009 and BV_3755 respectively), KN4706 and KN4709 defective in 7-hydroxytropolone production (locus tags: BV82_4706 and BV82_4709 respectiverly (Figure S1)), KN4243 defective in emrA gene (later referred to as EmrA), KN3318 defective in gacA gene (later referred to as GacA), KN4870 defective in fur gene (later referred to as Fur) the medium was supplemented with 30 μg ml−1 of kanamycin.For the growth of the auxotrophic strain E. coli ST18, the medium was supplemented with 50 μg ml−1 of 5-aminolevulinic acid (5-ALA, Sigma-Aldrich, USA). When necessary, the medium was, streptomycin (50 μg ml−1).Purification and characterisation of P482 pyoverdine (PvdP482)Pyoverdine was purified from a 200 ml 48-h culture of the P482 strain grown in CAA at 28 ⁰C. The cells were removed by centrifugation, and the supernatant was filter-sterilised on the PES membrane (Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units). The supernatant was lyophilised and resuspended in 20 ml of water. The suspension was filtered through filtration units with MWCO 5000 Da (Concentrators, Spin 5K MWCO, 4 ml, Agilent Technologies).Pyoverdine was purified by using semi-preparative HPLC system (flow rate 5 mL min-1, 223 nm) with C8 column (Phenomenex Luna 100 Å 10 µm 250x10 mm), and gradient 0-10 % B in 10 min; 10-30 % B in 60 min, where A: 0.1 % TFA/Mili-Q water, and B: 0.1 % TFA/acetonitrile. After each purification step, all fractions were checked by MALDI-TOF mass spectrometer using DHB as a sample matrix