These results suggested that a putative transcription factor of the phtD operon is present in P. syringae pv. phaseolicola NPS3121 during growth at both temperatures. The putative transcription factor of the phtD operon is encoded Selleck GSK126 outside of the Pht cluster In general, genes that participate in the synthesis of phytotoxins are grouped together in a particular chromosomal region, within which are encoded both structural genes and regulatory proteins involved in the process . However, in the case of P. syringae
pv. phaseolicola it is unknown whether all genes necessary for the synthesis and regulation of phaseolotoxin are found within the Pht cluster. We performed a bioinformatic analysis for each of the predicted ORFs of the Pht cluster, in a search for DNA binding motifs using the Pfam database (http://pfam.sanger.ac.uk/) . According BYL719 solubility dmso to this analysis, no DNA binding motif was found in the Pht gene cluster (data not shown). In order to assess
whether the putative transcription factor of the phtD operon as revealed through the mobility shift analysis was encoded outside or within the Pht region, gel-shift assays were performed using crudes extracts from P. syringae pv. phaseolicola strain CLY233, a non-toxigenic strain lacking the Pht cluster and P. Selleckchem Luminespib syringae pv. tomato DC3000 (non phaseolotoxin-producer) grown at 18°C and 28°C in M9 minimal medium. Incubation
of the radiolabeled P phtD fragment with crude protein extracts of the above mentioned strains demonstrated the presence TCL of a retarded mobility complex similar to that obtained with protein extracts of P. syringae pv. phaseolicola NPS3121 (Figure 2). Mobility shift competition assays with specific and non-specific probes indicated that the observed DNA-protein binding was specific for the P phtD region (data not shown). These results indicated that the putative transcription factor binding upstream of phtD was encoded by a gene located outside of Pht region that is shared with other pathovars and thus is not specific for phaseolotoxin synthesis, and also that its presence is independent of temperature. Therefore, these results suggest that upon transfer of the Pht cluster horizontally, the regulation of phaseolotoxin synthesis adapted to pre-existing regulatory mechanisms of P. syringae pv. phaseolicola NPS3121. Figure 2 Gel shift assays with crude extracts of different pathovars of P. syringae. Radiolabeled P phtD fragment was incubated with protein extracts of P. syringae pv. phaseolicola strains NPS3121and CLY233, and P. syringae pv. tomato DC3000, grown at 18°C and 28°C in M9 minimal medium. Gel shift assays were carried out under conditions similiar to those used with crude extracts of the wild-type strain. The arrow indicates the DNA-protein complex.