1,721,021 research outputs found
Production of synthetic protein microcompartment for the isolation of nucleoprotein complexes from bacteria Escherichia coli
Get PDFKompartmenizacija v bakterijah je pomembna, saj omogoči ločitev specifičnih procesov, ki potekajo v mikro-razdelkih in so ločeni od preostalih procesov v celici. Proteinski mikro-razdelki predstavljajo pomembno orodje v sintetični biologiji. Namen naše raziskave je bil sintetizirati proteinski mikro-rezdelek Pdu iz bakterije Citrobatcter freudii v bakteriji Escherichia coli. V ta namen smo pripravili derivat plazmida pACBSR-DL1, ki je vključeval gene operona pdu pod kontrolo promotorja pBAD, ki se aktivira ob dodatku L-arabinoze. Slednji plazmid, sicer pod kontrolo istega promotorja nosi zapis za še dva proteina, prvi nam omogoči sprostitev izbranega nukleoproteinskega kompleksa iz genoma, da bi ga lahko vključili v mikro-razdelek, drugi protein pa stabilizira DNA v kompleksu. V plazmid pACBSR-DL1 smo v več korakih klonirali nukleotidno zaporedje za proteine PduA-B-J-K-N-U, ki gradijo mikro-razdelek Pdu. Za izolacijo rekombinantnega mikro-razdelka smo uporabili gradientno centrifugiranje in s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata analizirali izolirane proteine.As compartmentalization of cells allows for separation of specific processes, which take place in micro-compartments and are separated from other events in the cell, it is essential for functioning of each cell. Protein micro-compartments are present in bacteria and are an important tool for synthetic biology. In this work, we sought so synthesise protein micro-compartment Pdu from Citrobatcter freudii in Escherichia coli cells. We cloned genes for the Pdu subunits PduA-B-J-K-N-U into plasmid pACBSR-DL1 under the control of pBAD promotor which is triggered by L-arabinose. Such a plasmid construct harboured also genes that enable liberation of a selected nuleoprotein complex to be packaged into a microcompartment. We applied gradient centrifugtion to isolate the L-arabinose induced microcompartment Pdu from E. coli and analysed the isolated proteins by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS PAGE)
The influence of temperature on interaction between Dickeya fangzhongdai and Podoviridae bacteriophage BF25/12
No full textSevi bakterije Dickeya fangzhongdai povzročajo bolezen mehkih gnilob, ki je v Sloveniji odgovorna za veliko ekonomsko škodo v tržnih obratih vzgoje orhidej. Ena od možnosti za obvladovanje bolezni je, da bi bakteriofag BF25/12, ki specifično okužuje nekatere seve D. fangzhongdai, uporabili kot sredstvo za obvladovanje bolezni orhidej. Pred implementacijo uporabe bakteriofaga v praksi je potrebno dobro poznavanje lastnosti interakcije med bakteriofagom in gostiteljsko bakterijo pri različnih okoljskih pogojih, kot so pH, UV sevanje in temperatura. Namen magistrske naloge je bil raziskati vpliv temperature na interakcijo med bakterijo D. fangzhongdai in bakteriofagom BF25/12. Vpliv temperature na interakcijo smo proučevali z analizo pojavljanja plakov, analizo adsorpcije in s spremljanjem rasti bakterij v tekoči kulturi v prisotnosti bakteriofaga. Ugotovili smo, da so sevi D. fangzhongdai B16, MK7 in JS5T bolj občutljivi za okužbo z bakteriofagom pri nižjih (20 in 28 °C) kot pri višjih (37 in 42 °C) temperaturah. Skladno s tem smo ugotovili, da se pri nižji temperaturi (28 °C) na površino D. fangzhongdai B16 adsorbira več bakteriofagov kot pri višji (37 °C). S spremljanjem rasti bakterij smo opazili, da se med različnimi sevi D. fangzhongdai pojavljajo razlike v poteku okužbe pri različnih temperaturah. Pri 28 °C sta seva B16 in MK7 občutljiva, sev JS5T zmerno občutljiv, seva S1 in NCPPB 3274 pa neobčutljiva za okužbo z bakteriofagom. Pri 37 °C pa je bilo vseh pet sevov neobčutljivih za okužbo. Ocenjujemo, da ima bakteriofag pri 28 °C aplikativen potencial za uporabo pri obvladovanju bolezni orhidej, vendar moramo pri tem upoštevati možnost razvoja odpornosti bakterij.Strains of bacteria Dickeya fangzhongdai cause soft-rot disease, which is responsible for high economic loss in commercial production of orchids in Slovenia. One of the disease control options is the bacteriophage BF25/12, which specifically infects some of the strains of D. fangzhongdai and could therefore be used to control soft-rot in orchids. Before the bacteriophage can be used in practice, the interactions with the host bacterium under different environmental conditions such as pH, UV radiation and temperature need to be studied. The aim of this master thesis was to investigate the effect of temperature on the interaction between D. fangzhongdai and bacteriophage BF25/12. The study was conducted by plaque assay, adsorption test and by monitoring bacterial growth in liquid media in the presence of bacteriophage. Strains B16, MK7 and JS5T were found to be more susceptible to bacteriophage infection at lower temperatures (20 and 28 °C) than at higher temperatures (37 and 42 °C). In addition, it was found that more bacteriophages were adsorbed on the surface of D. fangzhongdai B16 at 28 °C than at 37 °C. By monitoring the bacterial growth, the differences in infection kinetics between different D. fangzhongdai strains at different temperatures were observed. At 28 °C, strains B16 and MK7 were sensitive, strain JS5T was moderately sensitive, and strains S1 and NCPPB 3274 were insensitive to bacteriophage infection. Howevert, at 37 °C, all five strains were insensitive to infection. It is concluded that bacteriophage BF25/12 has potential application for orchid disease control at 28 °C, but the possibility of bacterial resistance should be considered
In vivo encapsulation of specific DNA fragments in bacterial microcompartment
No full textBakterijski mikrorazdelki so primitivni proteinski organeli, ki jih sintetizira okrog 20 % bakterijskih vrst, katerih zaporedje genoma je določeno. Njihova glavna naloga je izboljševanje učinkovitosti posameznih metabolnih reakcij ali preprečitev akumulacije toksičnih metabolitov v citoplazmi. V osnovi jih delimo na tiste, ki sodelujejo v anabolizmu in na tiste, ki sodelujejo v katabolizmu celice. Med slednjimi je najbolj znan mikrorazdelek Pdu, ki je bil zaradi modularnosti uporabljen že za več biotehnoloških aplikacij. V magistrskem delu smo prikazali novo aplikacijo, in sicer enkapsulacijo specifičnega fragmenta DNA, kar bi lahko služilo kot osnova metode za odkrivanje interakcij protein-DNA ali kot ogrodje za metabolni inženiring. Za lažjo izolacijo smo podenoti mikrorazdelka PduA dodali peptidno značko Twin-Strep-tag, ki omogoča afinitetno izolacijo intaktnih mikrorazdelkov. Pokazali smo, da se fuzijski protein PduD(1-18)-LacI-EGFP učinkovito enkapsulira v mikrorazdelke in vivo ter po afinitetni izolaciji mikrorazdelkov ostane funkcionalen. Pripravili smo sistem dveh plazmidov v Escherichia coli, ki je aktiviran z dodatkom arabinoze v gojišče. Z enega plazmida se začnejo izražati geni za kvasno restriktazo I-SceI, protein Gam, podenote mikrorazdelka Pdu in gen za fuzijski protein PduD(1-18)-LacI-EGFP. Na drugem plazmidu sta eno ali dve restrikcijski mesti za kvasno restriktazo I-SceI, ki obdajata vezavna mesta za LacI. Z visokozmogljivim sekvenciranjem smo dokazali, da je DNA fragment z vezavnimi mesti za protein LacI v afinitetno izoliranih mikrorazdelkih Pdu obogaten glede na druge regije genoma, kar kaže na to, da je bil ta fragment uspešno enkapsuliran. Dokažemo tudi, da lahko v mikrorazdelek Pdu enkapsuliramo DNA z vezavnimi mesti za vsaj dva transkripcijska faktorja.Bacterial microcompartments are primitive proteinaceous organelles synthesized by approximately 20 % of bacterial species whose genomes have been sequenced. Their main function is to improve the efficiency of metabolic reactions or to accumulate toxic metabolic byproducts. In general, depending on their function, they are divided into those that perform anabolic or catabolic reactions. Among the latter, the Pdu microcompartment is the most studied, as it is used in many biotechnological applications due to its modularity. In this master\u27s thesis, we demonstrate a new application, more specifically, the encapsulation of a specific DNA fragment in the microcompartment that could be used to study protein-DNA interactions or as a scaffold for metabolic engineering. To facilitate isolation, we tagged the major subunit of the microcompartment PduA with the peptide tag Twin-Strep-tag, which allows affinity isolation of intact microcompartments. We show that the fusion protein PduD(1-18)-LacI-EGFP is efficiently encapsulated in microcompartments in vivo and remains functional after affinity isolation of the microcompartments. We prepared a system of two plasmids in Escherichia coli. The first plasmid carries selected components under an arabinose-inducible promoter, genes for: Yeast nuclease I-SceI, phage protein Gam, subunits of the Pdu microcompartment and the fusion protein PduD(1-18)-LacI-EGFP. On the second plasmid, there are one or two restriction sites for the yeast endonuclease I-SceI flanking the binding sites for LacI. Using high-throughput sequencing, we demonstrate that in the affinity-isolated microcompartments, the fragment carrying LacI binding sites is enriched relative to other regions of the genome, suggesting that this fragment was truly encapsulated. In addition, we show that we can encapsulate a DNA fragment carrying at least two different transcription factors in the microcompartments
The analysis of bacteriophage GIL01 gp7 protein characteristics
Get PDFProteina RecA in LexA sta ključna proteina tako imenovanega odziva SOS, ki omogoča ohranitev integritete in strukture genoma bakterij, ki rastejo v stresnih, genotoksičnih razmerah. V tem odzivu pa se lahko sproži tudi prehod nekaterih bakteriofagov iz lizogenega v litičen cikel. Bakteriofag GIL01, ki okužuje bakterijo Bacillus thuringiensis, za vzpostavitev lizogenega cikla izkorišča gostiteljev protein LexA. Protein LexA se veže na tarčna mesta v promotorski regiji P1 bakteriofaga GIL01, to vezavo pa dodatno stabilizira protein gp7 tega bakteriofaga. Kompleks LexA-gp7 prepreči bakteriofagu prehod v litični cikel. V magistrski nalogi smo preverili, ali lahko protein gp7 prosto prehaja iz gojišča v bakterijo. Z afinitetno kromatografijo smo očistili protein gp7 in analogni protein DdrR iz bakterije Acinetobacter baumannii. Nadalje smo očistili protein gp7, katerega smo sklopili s signalnim zaporedjem YKKSNNPFSD, ki naj bi omogočilo vnos proteinov v bakterijo Bacillus subtilis. Za odstranitev afinitetne značke na amino-terminalnem koncu proteinov smo uporabili encim enterokinaza. Zasnovali smo test prehoda proteinov gp7 v Bacillus thuringiensis serovar israelensis, ki temelji na analizi promotorske aktivnosti P1 z β-galaktozidaznimi testi. Dokazali smo, da je protein gp7 stabilen v izrabljenem gojišču bakterije B. thuringiensis. Rezultati nakazujejo, da gp7 ali gp7 s signalno sekvenco ne prehajata prosto v bakterijo.The proteins RecA and LexA are important factors in the SOS response that enables bacterial genome integritiy under stressful genotoxic conditions. SOS response also induces bacteriophages transition from the lysogenic to the lytic cycle. The bacteriophage GIL01 infects the bacterium Bacillus thuringiensis and requires host`s LexA repressor to establish and maintain the lysogenic cycle. LexA binds to specific sites in the P1 promoter region of the GIL01 bacteriophage. The LexA protein can bind gp7 which additionally enhances the binding of LexA to DNA and prevents the bacteriophage from switching to the lytic cycle. We tested whether gp7 could freely enter into the bacterium. We purified gp7 and its analog from the bacterium Acinetobacter baumannii, the protein DdrR with affinity chromatography and also gp7 with the signal sequence YKKSNNPFSD, which presumably allows the fusion protein to enter into the bacterium Bacillus subtilis. To remove the histidine affinity tag at the amino-terminal part of the protein, we used enterokinase. We designed the gp7 protein transformation in Bacillus thuringiensis serovar israelensis based on analyzes of the activity of the P1 promoter with β-galactosidase assays. We proved that gp7 is stable in the spent culture medium. The results show that gp7 or its analog carrying the signal sequence cannot freely migrate into the bacterium
The function and molecular mechanisms of small proteins that control the bacterial SOS response to genomic stress
No full textPorast bakterijskih patogenov, odpornih proti več antibiotikom, je velik zdravstveni problem. Ker novih antibiotikov ni lahko najti, se veliko raziskav usmerja v iskanje možnosti za povečanje učinkovitosti že znanih antibiotikov. Številni antibiotiki v subinhibitornih koncentracijah izzovejo poškodbe DNA v bakterijah. Te v odgovor na poškodovano DNA sprožijo odziv SOS, pri katerem najprej sintetizirajo encime, ki natančno popravijo DNA. Ob hujših poškodbah DNA, če te niso bile odpravljene, pa se kasneje v odzivu SOS zgodi sinteza mutagenih DNA-polimeraz, ki omogočajo popravljanje DNA, podvrženo napakam. Tako uvedene mutacije v bakterijskem genomu pa lahko vodijo do razvoja odpornosti proti antibiotikom. Dolgo je veljala predpostavka, da sta transkripcijski faktor LexA in protein RecA edina regulatorja odziva SOS. Nedavno je bilo dokazano, da bakteriofag GIL01, ki okužuje bakterijo Bacillus thuringiensis serovar israelenis, nosi zapis za mali protein gp7, ki se neposredno veže z represorjem LexA in zveča njegovo afiniteto do tarčnih nukleotidnih zaporedij. Prepoznava funkcije proteina gp7 je spodbudila teorijo o obstoju malih proteinov, ki delujejo kot koregulatorji LexA in s tem zagotavljajo dodatno raven regulacije odziva SOS. V doktorskem delu smo dokazali, da je protein gp7 globalni regulator prepisa genov v bakteriji B. thuringiensis, saj zavira prepis 1,2 % bakterijskih genov. Naši rezultati so pokazali, da je gp7 ključni dejavnik za inhibicijo prehoda faga pBtic235, ki z GIL01 sobiva v bakteriji, v litični cikel. Protein gp7 tako zagotovi fagu GIL01, da po poškodbi DNA proizvede več potomcev kot fag pBtic235. Razrešili smo mehanizem delovanja proteina gp7, ki z vezavo na LexA reorientira DNA-vezavni domeni v dimeru LexA v konformacijo, potrebno za vezavo na DNA, kar zviša afiniteto represorja LexA do DNA. Naši rezultati dokazujejo obstoj po funkciji podobnih proteinov, kot je protein gp7, tudi v drugih bakterijah: (i) homologov gp7 nekaterih drugih tektivirusov, ki okužujejo bakterije iz skupine Bacillus cereus sensu lato, in (ii) proteina DdrR bakterije Acinetobater baumannii. Naši rezultati torej nakazujejo obstoj malih proteinov s podobnim delovanjem, kot ga ima gp7, tudi v drugih bakterijah, ki kot koregulatorji LexA in LexA podobnih proteinov predstavljajo dodatno raven uravnavanja prepisa genov v odzivu SOS.The increase in bacterial pathogens resistant to multiple antibiotics is a major health problem. Since new antibiotics are not easy to find, much of the research is focused on finding ways to increase the effectiveness of already known antibiotics. At subinhibitory concentrations, many antibiotics induce DNA damage in bacteria. In response to the damaged DNA, bacteria initiate a SOS response in which they first produce enzymes that precisely repair the DNA. In the case of severe DNA damage, and later in the SOS response, if the DNA damage has not been repaired, synthesis of error-prone DNA polymerases occurs that support replicative bypass of damaged bases that arrest high-fidelity repair. Mutations introduced into the bacterial genome in this manner can lead to the development of antibiotic resistance. For a long time, it was assumed that the transcription factor LexA and the protein RecA were the only regulators of the SOS response. Recently, it was shown that the bacteriophage GIL01 infecting Bacillus thuringiensis serovar israelenis carries a gene for the small protein gp7 that binds directly to the LexA repressor and increases its affinity for target nucleotide sequences. Recognition of the gp7 protein suggested the existence of small proteins that act as coregulators of LexA, providing an additional level of regulation of the SOS response. In this dissertation, we demonstrated that the gp7 protein is a global regulator of gene transcription in the bacterium B. thuringiensis, as it inhibits the transcription of 1.2 % of bacterial genes. Our results show that gp7 is the key factor that inhibits the transition of phage pBtic235, which coexists with GIL01 in the bacterium, into the lytic cycle. Thus, the gp7 protein ensures that the phage GIL01 produces more progeny than the phage pBtic235 after DNA damage. We have elucidated the mechanism of action of the gp7 protein, which, by binding to LexA, reorients the DNA binding domain of the LexA dimer into the conformation required for binding to DNA, which increases the affinity of the LexA repressor for DNA. Our results show that functionally similar proteins, such as the gp7 protein, exist in other bacteria: (i) gp7 homologs of other tectiviruses infecting bacteria from the group Bacillus cereus sensu lato and (ii) the DdrR protein of the bacterium Acinetobater baumannii. Thus, our results suggest the existence of small proteins with a similar function to gp7 in other bacteria as well, which, as co-regulators of LexA and LexA-like proteins, represent an additional level of regulation of gene transcription in the SOS response
Characteristics of Spirosoma linguale aegerolysin
Get PDFEgerolizini so družina majhnih (13-20 kDa) β-strukturiranih proteinov, ki jih najdemo pri predstavnikih evkariontov in prokariontov. Večina egerolizinov interagira z membranskimi lipidi, pri določenih pa so odkrili lastnost porotvornosti v interakciji s proteinskim partnerjem. Bakterije, ki imajo v operonu poleg gena za egerolizin še gen za protein, ki intreragira z egerolizinom, izkazujejo insekticidne lastnosti. Ena izmed bakterij, pri kateri so zasledili operon z genom za egerolizin in z genom za protein, ki domnevno tvori insekticidni kompleks, je bakterija Spirosoma linguale. Namen mojega magistrskega dela je bil pridobiti rekombinantni egerolizin SlinAg in protein SlinB, ki imata gena na skupnem operonu bakterije S. linguale. Izolirana proteina nam omogočita karakterizacijo interakcije teh dveh proteinov, med seboj in v interakciji z lipidnimi membranami. Iz genomske DNA bakterije S. linguale smo pomnožili gen za egerolizin SlinAg in gen za proteinski partner SlinB. Proteina smo izrazili v heterolognem ekspresijskem sistemu v bakteriji Escherichia coli. Z nikljevo afinitetno kromatografijo smo izolirali egerolizin v topni obliki. Z uporabo cirkularnega dikroizma smo določili sestavo sekundarne strukture rekombinantnega egerolizina in dokazali predominantno β-strukturiranost proteina. Proteina SlinB nismo pridobili v topni obliki, zato nismo preverili v kolikor proteina tvorita kompleks na membrani.Aegerolysin protein family comprises small (13-20 kDa) and mostly β-structured proteins that are present in organisms from different kingdoms (bacteria, eukaryotes). Some aegerolysins interact with membranes and when complexed with a protein partner exhibit pore-forming abilities. In bacteria, egerolysin and its protein partner are encoded on an operon and this bacterial egerolysin complexes exert insecticidal characteristics. The genome of a soil bacterium Spirosoma linguale carries such an operon encompassing an aegerolysin and a putative protein partner genes. The aim of this master theses was to isolate recombinant S. linguale aegerolysin and its protein partner and to characterize the interaction between the two proteins and the interaction of the proteins with the lipid membrane. We successfully constructed recombinant plasmids containing both genes, for the aegerolysin SlinAg and for its putative protein partner SlinB, however we managed to purify only the aegerolysin protein. We characterized the SlinAg protein by circular dichroism spectroscopy which showed that SlinAg is composed of mainly β-strands, which is typical characteristic of aegerolysins. Since we could not isolate SlinB we were not able to assay if the two proteins form a complex on the membrane
The influence of small protein DdRr on characteristics of the LexA-like proteins at Acinetobacter baumanii
No full textEncimi, sintetizirani v odzivu SOS, omogočijo bakterijam popravilo poškodb DNA in s tem adaptacijo na stresne okoljske razmere. Sevi bakterije Acinetobacter baumannii, ki so odporni proti številnim antibiotikom, sintetizirajo mali protein DdrR, ki je specifičen le za rod Acinetobacter, in skupaj s proteinom UmuDAb v tej bakteriji uravnava prepis genov odziva SOS. V delu smo želeli preveriti ali se protein DdrR neposredno veže s proteinom UmuDAb. Poleg tega smo želeli preveriti ali protein DdrR interagira tudi z represorjema, ki nadzirata lizogeni cikel dveh temperantnih profagov v tej bakteriji. V študiji smo z uporabo površinske plazmonske resonance ugotovili, da se DdrR z visoko afiniteto veže na vse analizirane represorje in, najverjetneje na tak način koregulira prepis bakterijskih in bakteriofagnih genov.Enzymes, synthesized in bacterial SOS response, enable bacterial organisms to repair DNA damage and therefore easily adapt to stresful enviroments and chemical agenses. Acinetobacter baumannii strains are resistant to a large range and variety of antibiotics. During SOS response Acinetobacter baumannii synthesize small protein molecule, named DdrR, which is specific only for genus Acinetobacter and can be found no where else. When synthesized, DdrR co-regulates transcription of SOS damage inducible genes together with main bacterial protein regulator, UmuDAb. In the study we examined if DdrR binds directly to UmuDAb protein and to two other phage repressors, which regulate lizogenic cycle of two temperate prophages, situated in bacterial chromosomal DNA. Surface plasmon resonance analysis indicated that DdrR protein binded succesfully to all analyzed protein repressors, with high affinity, and, in this way, it most likely co-regulates transcription of bacterial and bacteriophage genes
Deletion of aegerolysin gene in strain of bacteria Pseudomonas aeruginosa PA01
Get PDFThe dissertation discusses the proteins from the aegerolysin family, specifically the RahU protein of the opportunistic pathogen bacteria, Pseudomonas aeruginosa. The biology of P. aeruginosa, which exploits its host’s weak immune system to cause numerous infections, is described. The main focus of the dissertation was the preparation of the deletion mutants. The rahU gene was deleted from the P. aeruginosa genome by replacing it with the kanymicin cassette. The gene replacement was carried out by using the Lambda Red recombinase system. The purpose of this molecular tool was to gain the rahU deletion mutants, which enable us to obtain new insights into the biology of the RahU protein
The role of gp1 protein on the activity of bacteriophage GIL01 promoters P1 and P2
No full textBakteriofag 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.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
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