Protein gp6 bakteriofaga GIL01 sproži litični cikel v bakteriji Bacillus thuringiensis

Pavlin, Anja (author), Butala, Matej (mentor)

Temperatni bakteriofag GIL01 inficira bakterijo Bacillus thuringiensis. V lizogenem ciklu se bakteriofag podvojuje v bakteriji kot samostojni linearni replikon. V nasprotju z večino ostalih temperatnih fagov, ki uporabijo fagni protein, GIL01 za ohranjanje lizogenega življenskega cikla izkorišča bakterijski trankripcijski faktor LexA. Poškodba DNA sproži litičen cikel faga GIL01. V magistrskem delu smo dokazali, da so litični geni, ki kodirajo proteine kapside in lizo gostiteljske bakterije, prepisani kasneje kot geni, ki uravnavajo replikacijo fagnega genoma in lizogenijo. Natančneje smo preučili uravnavanje litičnega promotorja P3, da bi prepoznali genetsko stikalo za prehod iz lizogenega v litičen cikel faga. Pokažemo, da fagni protein gp7 asistira represorju LexA gostitelja, da v kompleksu preprečita aktivacijo promotorja P3. Dokažemo, da je ključ za prehod faga v litični cikel mali fagni protein gp6. Z metodo na osnovi površinske plazmonske resonance dokažemo, da gp6 interagira s promotorskim področjem P3 tako, da prekriva element -35 promotorja in deluje kot aktivator transkripcije tipa II. Z bioinformatskimi analizami smo pokazali, da je gp6 homolog represorja LexA. Na podlagi modela kompleksa gp6-DNA smo pripravili mutanto gp6 K38A, za katerega smo predvideli, da bo izgubil zmožnost interakcije z zaporedjem promotorja P3. Slednje eksperimentalno potrdimo in dokažemo, da mutanta ne aktivira promotorja P3. Tako kot prvi opišemo molekularni mehanizem časovno uravnanega prepisa litičnih genov faga GIL01 in prvi primer promotorja, katerega aktivnost uravnavata dva proteina iz iste družine, vendar z nasprotujočima si funkcijama.

• Language: Slovenian
• Keywords: temperatni fag GIL01, Bacillus thuringiensis, odziv SOS, LexA represor, preklop iz lizogenega v litični cikel
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2018
• PID: 20.500.12556/RUL-102833
• COBISS.SI-ID: 4890191

Note

Univerzitetna Prešernova nagrada / University Prešern Award 2018

• Publication date in RUL: 08.09.2018

Secondary language

• Language: Unknown
• Title: The lytic life cycle of the bacteriophage GIL01 infecting Bacillus thuringiensis is activated by the gp6 protein

Abstract

Bacteriophage GIL01 is a temperate phage that infects bacterium Bacillus thuringiensis. During the lysogenic cycle, it resides in the host as a dormant independent linear replicon. In contrast to most other temperate bacteriophages that maintain lysogeny by a dedicated phage repressor, GIL01 lysogeny is maintained by a host transcriptional factor LexA. The GIL01 lytic cycle is induced when host genome is severly damaged. In this thesis we show that in comparison to the lysogenic promoter activity the expression from the lytic promoter P3 is considerably delayed. We further examined the regulation of the P3 promoter. We show that LexA in conjunction with phage protein gp7 binds P3 promoter region to repress lytic functions. However, the key to establish the lysogenic/lytic switch is the small bacteriophage-born protein gp6. We used surface plasmon resonance spectroscopy to demonstrate that gp6 binds P3 promoter region overlapping the -35 promoter element and thus acts as a type II transcription activator. The bioinformatics analysis suggests that gp6 is homologous to LexA. We generated the gp6-DNA complex model and according to the model, purified the gp6 K38A mutant that was indeed unable to interact with its binding site at the P3 promoter and failed to activate transcription from P3. Thus, we provide the first study showing that the interplay between two LexA family members, with opposing functions, ensures the timely expression of a phage late genes.

• Keywords: temperate phage GIL01, Bacillus thuringiensis, SOS response, LexA repressor, lysogenic/lytic switch