Purification and analysis of achromobactin The protocol for achromobactin purification was adapted from Berti and Thomas . Briefly, 200 ml of standard M9 minimal medium, with succinic acid as the carbon source, was inoculated with 10 ml pvd – P. syringae 1448a from a C59 wnt supplier stationary phase culture grown in the same medium. The resulting culture was grown for 72 h (22°C, 200 rpm) following which cells were removed by centrifugation (5000 g, 30 min). The supernatant was then sterilised
by passing through a 0.22 μm filter and then the volume reduced to 20 ml by rotary evaporation (temperature not exceeding 45°C). Methanol (180 ml) was then added, whereupon salt from the culture medium precipitated out of solution. Precipitate was removed by centrifugation (12,000 rcf, 20 min) followed by filtration using a 0.45 μm filter. The solution was then mixed 1:1 with ethyl acetate and 100 ml of the resulting MK-8776 chemical structure solution applied to a glass chromatography column containing 40 cc silica beads pre-equilibrated with solvent A (9:1:10 v/v methanol:H2O:ethyl acetate). 100 ml Solvent A was then applied find more to the column, followed by 100 ml solvent B (9:1 v/v methanol:H2O). The elutate from the solvent B step was
captured in 10 ml fractions. Siderophore activity of the fractions was then assessed by adding 30 μL CAS reagent to a 150 μL aliquot of each fraction and incubating for 10 min at room temperature. The fraction which resulted in the greatest discolouration of the CAS dye was then reduced in volume to 2 ml by rotary evaporation (temperature not exceeding 40°C) and 1 ml of the solution removed. The remaining 1 ml was evaporated to dryness and resuspended in 1 ml ddH2O. Both of these 1 ml samples were then sent to the Centre for Protein Research at the University of Otago for MALDI-TOF analysis. Construction of gene knockout and over-expression plasmids Gene sequences were retrieved from the Pseudomonas genome database . Primers were designed using Vector NTI (Invitrogen) to amplify 400 bp regions from the 5′ and 3′ regions of the NRPS genes (including the putative yersiniabactin cluster
gene hmwp1) such that when they were fused no frame shift would result ioxilan (all primers used in this study are listed in Additional file 1, Table S1). For deletion of acsA, which is much smaller, 400 bp regions immediately upstream and downstream of the gene, including the first and last 3 codons of the gene on either side, were amplified. The upstream primer of the 3′ fragments contained a region complementary to the downstream primer of the 5′ fragment for use in splice overlap extension (SOE) PCR . The outer-most primers contained restriction enzyme sites to enable directional cloning of the spliced fragments into the suicide vector pDM4 , following which gene knockout was performed as described below.