Analysis of the promoter regions identified in the Pht cluster showed that the divergent promoters for argK and phtA contain canonic sequences of σ70-type promoters, while the promoter regions for phtD, phtL and phtM did not show similarity to consensus sequences for bacterial sigma factors. However, a common mechanism of transcriptional regulation for phtD and phtM has been suggested due to the presence of conserved regions in the promoters of these operons. Furthermore, analysis of transcriptional fusions of the Pht cluster promoter regions suggest that temperature regulation

occurs at the transcriptional level since maximal transcriptional activity occurs at 18°C and is significantly lower at 28°C [10]. In bacteria, transcriptional regulation is JQ1 commonly mediated by regulatory proteins that control gene expression in response to internal metabolic MK-8669 price changes or external signals such as temperature, pH, and carbon source [21, 22]. Previous

reports proposed that argK regulation is under negative control mediated by a repressor protein present at 28°C, although the identity of this regulatory protein has not been elucidated [23]. Similarly, a regulatory function for the PhtL protein has been suggested based on the lack of phtM operon expression in a phtL – background, although this still requires experimental confirmation [10]. Despite our knowledge of the effect of low temperature on phaseolotoxin synthesis, the regulatory mechanisms that control toxin production remain poorly understood. So far it is not known whether all the genes involved in the regulation of phaseolotoxin synthesis are located within the Pht cluster, or whether there are any other genes outside the Pht cluster involved in this process. In the latter case, it would

be interesting to know whether any regulatory gene found outside the Pht cluster is specifically required for phaseolotoxin synthesis, or whether the synthesis of the toxin has adapted its expression to the regulatory mechanisms of the bacteria during horizontal gene transfer. For these reasons, this study was undertaken with the objective of identifying Janus kinase (JAK) regulatory proteins that could participate in the regulation of genes for phaseolotoxin synthesis, with a focus on the regulation of the phtD operon. Results The promoter region of the phtD operon contains a binding site for a putative regulatory protein The phtD operon includes eight genes from phtD to phtK, whose expression can be driven either from the promoter upstream of phtD, or from read-through from the phtA promoter located upstream (Figure 1A). The transcription initiation site for the phtD operon was determined to be 127 bp upstream of the probable initiation codon, and analysis of this promoter region did not show any similarity with binding sites reported for bacterial sigma factors [10].