Production of an uncharacterized siderophore by Pseudomonas fluorescens CFBP13502 reduced the growth of the plant pathogen Xanthomonas campestris pv. campestris

Transcriptome analysis of Pseudomonas fluorescens CFBP13502 was performed during co-culture with two strains (Stenotrophomonas rhizophila CFBP13503 and Xcc8004) that produced a cell-free supernatant (CFS) with bacterial growth inhibition potential. These transcriptomes profiles were compared to co-culture of P. fluorescens CFBP13502 with two strains (Pantoea agglomerans CFBP13505 and Pseudomonas viridiflava CFBP13507) that did not trigger CFS with inhibitory activity.All cultures were performed in Tryptic Soy Broth 1/10 strength (17 g.l-1 tryptone, 3 g.l-1 soybean peptone, 2.5 g.l-1 glucose, 5 g.l-1 NaCl and 5 g.l-1 K2HPO4) at 28 degree C. Four independent replicates were performed per co-culture at a starting OD600 = 0.02.Total RNA of each strain was isolated as followed. A 2-ml suspension of co-culture after 6 h growth was centrifuged 30 s at 14,000 X g. The pellet was resuspended in 600 micro l of 65 degree C Trizol. Cell debris was removed by centrifugation (2,500 X g, 5 min at 15 degree C) and the supernatant was transferred to a fresh tube. Chloroform (120 micro l) was added to 600 micro l of the supernatant and the mixture was vortexed vigorously 30 s. After 2 min at room temperature, the phases were separated by centrifugation at 12,000 X g for 15 min at 4 degree C and the upper phase (300 micro l) was removed to a fresh tube. Isopropyl alcohol (250 micro l) was added and the mixture was incubated at room temperature for 10 min. The precipitated RNA was collected by centrifugation at 12,000 X g for 20 min at 4 degree C. The supernatant was removed and the pellet was washed with 500 micro l of 75% ethanol. A new pellet of RNA was collected by centrifugation at 7,500 X g for 5 min at 4 degree C and was air dried to near completion. The RNA was dissolved in 89 micro l of sterile double-distilled H2O, and the preparation was treated with 1 micro l of DNase during 1 h at 37 degree C. The preparation was heated 10 min at 75 degree C to inactivate the DNAse and RNA was ethanol precipitated and dissolved in 50 micro l of double-distilled H2O.RNA-Seq libraries were prepared with the QIAseq FastSelect -5S/16S/23S and QIAseq stranded total RNA Lib kits following manufacturer's instructions. RNA fragmentation was performed at 89 degree C for 6 min. The quality of the resulting libraries was assessed with a Bioanalyzer and concentration of libraries and subsequent equimolar pool was quantified with quantitative PCR using Illumina primers. The equimolar pool was sequenced with Illumina NextSeq550 using High Output 150 cycles cartridge.