Previously, it was reported that the promoter region of the algD gene in P. aeruginosa contains a functional binding site for the IHF protein [32]. This site has also been found in the promoter region of the orthologous gene in P. syringae pv. Vistusertib phaseolicola 1448A. For that reason, we decided to use the promoter region of the algD gene of 1448A, which contains a putative IHF binding site, as a competitor in gel shift assays. The results showed that the retarded mobility signal progressively decreased, compared to the DNA-protein complex,

indicating that increasing concentrations of competitor DNA titrated the protein. However, when the promoter region of the same gene without the putative IHF binding site was used as a competitor, the retarded signal intensity was find more not altered (see Additional file 1). Additionally, a second Selonsertib price experiment was conducted where the P. syringae pv. phaseolicola NPS3121 IHF alpha subunit gene was cloned in the pCR4-TOPO vector, creating the plasmid pPihfA, which was then introduced

into the E. coli ihfA – mutant. Crude extracts of the complemented E. coli strain were used in mobility shift assays to analyze the binding activity of the P phtD fragment. Mobility shift assays showed the presence of a retarded signal similar to that obtained with our P. syringae pv. phaseolicola strain, indicating that the presence of the ihfA gene in trans is capable of restoring the formation of the DNA-protein complex (Figure 4A). Finally, strong evidence concerning the identity of the P phtD binding protein was obtained through gel shift assays using IHF protein purified from E. coli, which showed the presence of a retarded signal whose position was identical to that formed with the protein present in extracts of P. syringae pv. phaseolicola NPS3121 (Figure 4B). These results unambiguously demonstrate that the IHF protein interacts with the phtD promoter region and is probably involved in regulation of this operon. The IHF protein exerts a negative effect on the

expression of the phtD operon in E. coli To assess the participation of the IHF protein OSBPL9 in regulating phtD operon expression, a transcriptional fusion of the phtD promoter was made to the gfp reporter gene creating the pJLAG plasmid with the intention of evaluating the expression from this construct in an IHF- background of our P. syringae pv. phaseolicola strain. However, despite the fact that several strategies were attempted to obtain mutations in the subunits of the P. syringae pv. phaseolicola NPS3121 IHF protein, these mutants could not be obtained. Nevertheless, because the amino acid sequences of the P. syringae pv. phaseolicola IhfA and IhfB proteins are 86% and 73% identical to the E. coli IhfA and IhfB proteins respectively (data not shown), and since previous reports demonstrated that the E.