coli. We examined

the expression of the phtD::gfp transcriptional fusion (pJLAG) in wild type E. coli Ferroptosis inhibitor K12 and ihfA – mutant backgrounds. The expression of phtD::gfp was increased in the ihfA – background, in comparison to the expression observed in the wild type E. coli K12 strain. On other hand, when the expression of the phtD::gfp transcriptional fusion was examined in the ihfA – mutant complemented with the ihfA gene of P. syringae pv. phaseolicola NPS3121, we observed a clear reduction in fluorescence levels, suggesting a decrease in gene expression (Figure 5). However, to investigate the possibility that the decrease in phtD promoter expression was related to the decrease in growth rate observed in this strain, possibly due to over-expression of the ihfA gene, we evaluated the expression of the phtD::gfp fusion in the ihfA – mutant transformed with the PCR 4-TOPO vector (without ihfA gene). The results of these experiments showed that the decrease in the growth rate was possibly due

to the presence of an additional plasmid and not Selleck Temsirolimus to the presence of the ihfA gene, which excludes a possible toxic effect. Likewise, the results showed that the decrease in the expression observed from the phtD::gfp fusion in the complemented ihfA – mutant was, due solely to the presence of the ihfA gene in trans, and not to the observed decrease in growth (Figure 5). These results indicate that the IHF protein negatively regulates expression of the phtD operon in E. coli. Figure 5 Promoter activity of the phtD operon in Escherichia coli background. (A) Growth curve of E. coli strains carrying the phtD::gfp

transcriptional fusion grown in LB broth. (B) Fluorescence activity of phtD::gfp in the E. coli background. Mutations in the putative IHF binding site affect the DNA-protein interaction Since the IHF site found in the phtD operon promoter region has 83% similarity with the Nutlin-3a chemical structure reported consensus sequence, we evaluated the role of STK38 this sequence on the DNA-protein interaction. To this end, 104 bp synthetic oligonucleotides corresponding to the minimum binding region for IHF were designed with mutations at bases previously reported to be necessary for IHF protein binding. The selected mutations were based upon those previously shown to severely affect IHF binding [34]. Two mutant probes were analyzed. Mutant probe 1 (L100271-L100272) has changes in the dA-dT rich upstream region as well as changes of C to A and G to T of the consensus sequence. Gel mobility shift assays with mutant probe 1 clearly show a dramatic decrease in the amount of retarded signal (89%) as compared to the amount of signal obtained with the wild type probe (Figure 6A). These results indicate that the changes introduced in this probe decrease the P phtD -IHF interaction.