Vpliv proteina gp1 na aktivnost promotorjev P1 in P2 bakteriofaga GIL01

Popović, Maja (author), Butala, Matej (mentor)

Bakteriofag GIL01 je temperatni tektivirus katerega gostitelj je bakterija Bacillus thuringiensis serovar israelensis. Fag GIL01 ob vstopu v celico lahko vzpostavi lizogenijo znotraj katere lahko ostane vse dokler ne pride do večjih poškodb DNA. Te sprožijo bakterijski odziv SOS. Za regulacijo lizogenije in izražanja genov bakteriofag GIL01 namreč izrablja bakterijski trankripcijski faktor LexA in fagni protein gp7. Ob sprožitvi odziva SOS pride do cepitve proteina LexA in s tem njegove inaktivacije. Inaktivacija LexA sproži litični cikel faga GIL01. Z beta-galaktozidaznimi testi in površinsko plazmonsko resonanco smo dokazali, da poleg LexA tudi protein gp1 faga GIL01 uravnava izražanje genov za replikacijo in regulacijo, ki so pod kontrolo promotorjev P1 in P2 faga GIL01. Protein gp1 tako sodeluje pri vzpostavitvi in ohranjanju lizogenije faga GIL01. Gre za prvi dokaz, da ima fag GIL01 tudi lasten represor za regulacijo promotorjev P1 in P2, ki je deloma neodvisen od gostiteljevega odziva SOS. S površinsko plazmonsko resonanco smo dokazali, da se predvidoma 5 do 6 monomerov proteina gp1 stabilno in z visoko afiniteto veže na promotor P2. Monomeri proteina gp1 najverjetneje tvorijo filament, ki sterično ovira vezavo RNA polimeraze in tako zniža aktivnost promotorja P2. Z beta-galaktozidaznimi testi smo ponovno potrdili, da je za uravnavanje izražanja genov za vzpostavitev lizogenije pri fagu GIL01 nujno potrebna prisotnost korepresorja gp7. Iz rezultatov je razvidno tudi, da promotorja P1 in P2 vplivata na aktivnost drug drugega.

• Language: Slovenian
• Keywords: protein gp1, temperatni bakteriofag GIL01, Bacillus thuringiensis, promotor P1 in P2, lizogeni in litični življenjski cikel, odziv SOS
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [M. Popović]
• Year: 2022
• PID: 20.500.12556/RUL-138094
• UDC: 577
• COBISS.SI-ID: 114958339
• Publication date in RUL: 10.07.2022

POPOVIĆ, Maja, 2022, Vpliv proteina gp1 na aktivnost promotorjev P1 in P2 bakteriofaga GIL01 [online]. Master’s thesis. Ljubljana : M. Popović. [Accessed 25 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=138094

Secondary language

• Language: English
• Title: The role of gp1 protein on the activity of bacteriophage GIL01 promoters P1 and P2

Abstract

Bacteriophage GIL01 is a temperate tectivirus that infects Bacillus thuringiensis serovar israelensis. When bacteriophage GIL01 infects a cell, it establishes the lysogenic cycle and remains dormant, until the SOS response is inducted due to DNA damage. To establish lysogeny, phage GIL01 uses the bacterial transcription regulator LexA, which in complex with phage protein gp7 represses the phage lytic cycle. When the SOS response is activated, LexA undergoes autocatalytic self cleavage, which induces the lytic cycle of bacteriophage GIL01. Here we show, by using beta-galactosidase assay and surface plasmon resonance spectrometry, that LexA is not the only transcription factor regulating promotor P1 and P2 activity. We show that phage protein gp1 represses gene expression of promoter P2 and is involved in maintaining lysogeny of bacteriophage GIL01. This is the first account of bacteriophage GIL01 having its own repressor partially independent of the host SOS response. With the use of surface plasmon resonance we showed that at promoter P2, protein gp1 forms a high affinity complex. According to our in vitro data we predict that approximately 5 or 6 monomers of gp1 bind to promoter P2 via a filament that lowers promotor activity by sterically restricting access of RNA polymerase for the P2 promoter elements. With the use of beta-galactosidase assay we reconfirmed that gp7 is critical for GIL01 lysogeny. We also show that promoters P1 and P2 influence each other’s activity.

• Keywords: protein gp1, temperate bacteriophage GIL01, Bacillus thuringiensis, promoter P1 and P2, lysogenic and lytic life cycle, SOS response