MALDI for An iron fist in a velvet glove: The cooperation of a novel pyoverdine from <i>Pseudomonas donghuensis</i> P482 (PVD_P482) 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, <i>P. donghuensis </i>P482, HYS^T, 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). <i>Pseudomonas</i> spp. and <i>Dickeya solani </i>IFB102 (referred to as <i>Ds</i>) were cultured at 28 °C, and <i>Escherichia</i> <i>coli</i> ST18 <i>at </i>37 °C. For the P482 mutants: KN1009 and KN3755 defective in pyoverdine synthesis (locus tags: <i>BV82_1009 </i>and <i>BV_3755</i> respectively), KN4706 and KN4709 defective in 7-hydroxytropolone production (locus tags: <i>BV82_4706 </i>and <i>BV82_4709 </i>respectiverly (Figure S1)), KN4243 defective in <i>emrA </i>gene (later referred to as EmrA), KN3318 defective in <i>gacA</i> gene (later referred to as GacA), KN4870 defective in <i>fur </i>gene (later referred to as Fur) the medium was supplemented with 30 μg ml⁻¹ of kanamycin.For the growth of the auxotrophic strain <i>E. coli</i> 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 (Pvd_P482)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