syringae pv. phaseolicola NPS3121, which suggests that regulation

of gene expression within the Pht cluster has integrated into the global regulatory mechanisms. However, it is still necessary to dissect in detail the regulatory mechanism of the IHF protein and identify other regulators that will enable us to elucidate the regulatory pathway for phaseolotoxin production in P. syringae pv. phaseolicola NPS3121. Methods Bacterial strains, media and growth conditions The bacterial strains and plasmids Pevonedistat used in this study are listed in Additional file 2, Table S1. P. syringae strains: pv. phaseolicola NPS3121, pv. phaseolicola CLY233 and pv. tomato DC3000 were grown on M9 minimal medium at 18°C or 28°C. Pre-inoculums (25 ml) of P. syringae strains were grown overnight at 28°C in M9 medium with glucose (0.8%) as the carbon source. The cells were inoculated into 50 ml M9 minimal medium at OD600 nm 0.1 and the cultures were incubated at 18°C and 28°C until they reached the transition phase (OD600 nm 1.0). Escherichia coli wild type and mutant derivative strains, were routinely grown on Luria-Bertani (LB) medium at 37°C. When required, the following antibiotics were added: carbenicillin 100 μg μl-1, kanamycin 50 μg μl-1,

rifampin 50 μg μl-1. Molecular biology techniques Routine techniques were performed using standard protocols [48]. Genomic DNA of P. syringae pv. phaseolicola NPS3121 was isolated as described find more previously [49]. Plasmid DNA was isolated from E. coli using the QIAGEN®: plasmid midi kit following the manufacturer’s instructions. PCR products were amplified with High Fidelity DNA Polymerase and Platinum supermix (Invitrogen, California USA) and purified with the Methocarbamol QIAquick® gel extraction kit (QIAGEN). Restriction enzymes were used according to manufacturer’s instructions. Primers were designed using Vector NTI Software (Invitrogen, California USA)

with reference to the previously reported Pht cluster sequence (Gen Bank DQ141263) [10]. The oligonucleotide primers used in this study are listed in Additional file 2, Table S2. Gel mobility shift Liproxstatin-1 concentration assays The probes used in gel shift assays were obtained by PCR amplification using the oligonucleotide pairs shown in Additional file 2. The double-stranded probes were end-labeled with ( 32P)-ATP using T4 polynucleotide kinase enzyme (Invitrogen, California USA). Gel shift assays were performed as previously described, with some modifications [50]. Briefly, protein extracts were prepared from P. syringae pv. phaseolicola NPS3121 grown in M9 minimal medium at 18°C and 28°C until reaching the transition phase (OD600 nm of 1.0). Cultures were centrifuged and the pellet was rinsed once with 1/20 volume of cold extraction buffer (25 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DTT, 10% glycerol and 0.