These results validate the bioinformatic prediction that did not reveal a DNA-binding motif for any predicted Pht cluster ORF, suggesting that none of these proteins encode a DNA-binding protein, although evidence in some cases suggests a regulatory role for the product of some genes found within the Pht cluster [10]. Our findings resemble previous reports in P. syringae pv. syringae, in which the syr-syp gene clusters

involved in syringomycin (syr) and syringopeptin (syp) synthesis, are regulated by the SalA and GacS/GacA proteins, which are localized outside of this region and also present in other pathovars. However, the regulation of these phytotoxins also depends on regulatory proteins present in the syr-syp region [24]. An approximation for the identity of the phtD binding protein was obtained by supershift and shift-western assays, which indicated that DNA-binding proteins of the DNABII family (HU or IHF) are involved in the formation of the protein-DNA complex MI-503 observed

in the phtD promoter region. These results are consistent with the bioinformatic analysis, which revealed the presence of a potential binding site for the IHF protein, at position -64 to -44, Selleckchem Staurosporine relative to the start of phtD transcription. Finally, the identity of the phtD binding protein was determined by mobility shift assays using E. coli strains mutated in each of the genes encoding subunits of HU and IHF proteins. The absence of retardation signal in ihfA – and ihfB – mutants clearly indicates a role for these proteins in the formation of the DNA-protein complex, thus demonstrating that IHF protein binds to the phtD promoter region. Further evidence for the binding of IHF to this region was provided by cell extracts from a complemented E. coli ihfA – strain, in which the retarded signal was restored by the

Urocanase presence of the P. syringae pv. phaseolicola ihfA gene acting in trans. Finally, mobility shift assays using purified IHF protein confirmed that the protein binding the phtD promoter region was IHF. IHF is a small basic DNA-binding protein conserved in Gram-negative bacteria that belongs to the class of so-called nucleoid associated proteins (NAP’s) [39, 40]. The IHF protein consists of two heterologous subunits, IHFα and IHFβ which are encoded in different transcription units by the homologous ihfA (himA) and ihfB (himD) genes, respectively. Both subunits also share significant homology with the subunits of the HU protein, a nonspecific DNA binding protein that also belongs to the same protein family. Unlike the HU protein, the IHF protein recognizes a specific consensus sequence: WCARNWNNTTR (where W represents A or T and R represents A or G), which introduces a bend of 180° into the DNA, centered at the 5′end of the 5′-WWWCAR-3′ element in the binding site [35, 39]. In addition, 5′-proximal bases with high dA-dT content, are also thought to be required for binding of this protein at some sites [34, 41].