Functional investigation of a transcriptional regulator ptrA from Pseudomonas chlororaphis PA23
Chan, Jason Hok Shun
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Pseudomonas chlororaphis PA23 is a promising biological control candidate against Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot in canola. A library of transposon mutants was previously created to understand the molecular mechanisms underlying the antifungal capabilities of PA23. A novel LysR-type transcriptional regulator, called PtrA, was identified as a key global regulator involved in secondary metabolite production. The function of PtrA at the molecular level was investigated in this thesis. Solubility problems encountered during the purification of PtrA redirected efforts to studying a truncated version of the protein instead. A two-step purification of the truncated protein, involving streptomycin sulfate precipitation and immobilized metal-ion affinity chromatography, yielded a highly pure protein. Preliminary crystal growth was achieved for the effector binding domain portion of PtrA. Transcriptional fusions suggested that essential regulatory binding sites of ptrA may lie somewhere between 52 and 198 bp upstream of the translational start site. The research presented in this thesis will help guide future functional studies on PtrA.