Multitron standard shaker

The optimal Fe(III)citrate concentration (within the boundaries 0–2 g L⁻¹) for IMPI expression was determined using a seven-level factorial design at concentrations of 0, 0.33, 0.67, 1, 1.33, 1.67, and 2 g L⁻¹. The cultures were inoculated at ΔOD₆₀₀ = 0.1 and incubated at 37°C in 500 mL baffled shake flasks containing 50 mL medium, shaking at 250 rpm on a Multitron Standard shaker (Infors). At ΔOD₆₀₀ = 1, IMPI expression was induced by addinig isopropyl-β-d-thiogalactopyranoside (IPTG; Carl Roth) at a final concentration of 1 mM followed by incubation for 4 h as above. The cells were then harvested by centrifugation (5,236 × g for 10 min at 4°C) in a Sigma 6-16 KS centrifuge equipped with a 11,650 rotor and four 13,650 cups (Sigma, Osterode am Harz, Germany). The pellet was disrupted for IMPI activity testing as described above. The results were evaluated using Design Expert v11 (Stat Ease, Minneapolis, MN, USA).

The microorganisms used in this report were P. taiwanensis VLB120ΔCeGFP and P. taiwanensis VLB120ΔCeGFP Δ04710 (Schmutzler et al., 2015, 2016). Pseudomonas taiwanensis VLB120 is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), deposition number: DSM 24711. For pre‐cultures, 5 ml of Lysogeny broth (LB) (Bertani, 1951) was inoculated from −80°C glycerol stocks and incubated overnight at 30°C and 200 r.p.m. (2.5 cm amplitude; Multitron standard shaker Infors HT, Bottmingen, Switzerland). Afterwards, 1 ml of the pre‐culture was centrifuged and washed with sterile 0.9% (wt/vol) NaCl solution to reduce the amount of residual LB medium. The cells were diluted 1:100 (vol/vol) in M9 minimal medium (Sambrook and Russel, 2001) supplemented with 0.5% (wt/vol) glucose and 100 μg ml⁻¹ streptomycin. Cultures were subsequently cultivated overnight at 30°C and 200 r.p.m. (2.5 cm amplitude; Multitron standard shaker Infors HT) in baffled shaking flasks (airphase/liquid 10:1 (vol/vol).