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.

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.

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The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation. In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response. / Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar. DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.   I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras. Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript