Eksperimentalna karakterizacija šestih kandidatnih sistemov toksin-antitoksin tipa I cianobakterije Microcystis aeruginosa PCC 7806 s poudarkom na MsoT1/MsoA1

Ruparčič, Matija (avtor), Dolinar, Marko (mentor)

Sistemi toksin-antitoksin (TA) so genetski elementi, ki zapisujejo relativno stabilen toksin in nestabilen antitoksin. Pri tipu I so toksini kratki membranski ali citosolni proteini, ki povzročijo smrt celic oziroma zavrejo njihovo rast, antitoksini pa so kratke nekodirajoče RNA, ki se vežejo na toksinsko mRNA in s tem preprečijo translacijo toksina. Cianobakterija Microcystis aeruginosa, ki spada med najbolj zloglasne povzročitelje cvetenja jezer, je zelo zanimiva z vidika sistemov TA, saj ima sodeč po bazi podatkov TADB 2.0 največ lokusov TA tipa II od vseh bakterij. Kljub temu je trenutno znanje o sistemih TA v M. aeruginosa pomanjkljivo, saj je bil do sedaj eksperimentalno okarakteriziran le en sistem tipa II, medtem ko sistemov tipa I pri tej vrsti še niso odkrili. V magistrski nalogi smo želeli poiskati kandidatne sisteme TA tipa I v M. aeruginosa in jih eksperimentalno okarakterizirati. Izvedli smo bioinformacijsko analizo, pri čemer smo z uporabo parametrov, ki so značilni za tip I, v genomu M. aeruginosa PCC 7806 našli petnajst kandidatnih lokusov, ki bi lahko predstavljali sisteme TA tipa I. Za eksperimentalno karakterizacijo smo izbrali šest kandidatnih toksinov, in sicer BH695_0311, BH695_0320, BH695_3336, BH695_4017, BH695_4989 in BH695_5020. Zapise za kandidatne toksine smo vstavili v ekspresijske vektorje, z njimi transformirali celice Escherichia coli BL21(DE3) pLysS ter z merjenjem OD600 opazovali, kako izražanje kandidatnih toksinov vpliva na rast celic. Ugotovili smo, da ima izražanje BH695_0320 in BH695_4017 negativen vpliv na rast celic, izražanje ostalih kandidatnih toksinov pa nima vpliva. V nadaljevanju smo se osredotočili na par BH695_4017/MaT1A_4017, ki smo ga preimenovali v MsoT1/MsoA1. S testom viabilnosti z eritrozinom B smo dodatno potrdili toksičnost MsoT1, z imunodetekcijo pa smo potrdili izražanje MsoT1 med testom toksičnosti. Ugotovili smo tudi, da ima izražanje toksina MsoT1 v prisotnosti kandidatnega antitoksina MsoA1 zakasnjen toksični učinek. Za konec smo pripravili sinteznobiološke vektorje, v katerih smo pod promotorja kandidatnega antitoksina MsoA1 vstavili zapis za ß-laktamazo oziroma mRFP. S spremljanjem rasti celic E. coli BL21(DE3) v gojiščih z različnimi koncentracijami antibiotika ampicilina oziroma z merjenjem fluorescence mRFP smo ugotovili, da sta se reporterska proteina izražala pod nadzorom promotorjev PmsoA1, s čimer smo posredno dokazali izražanje antitoksina MsoA1. Rezultati magistrske naloge nakazujejo, da MsoT1/MsoA1 najverjetneje predstavlja novo družino sistemov TA tipa I ter prvi odkrit sistem tipa I pri cianobakterijah.

• Jezik: Slovenski jezik
• Ključne besede: sistemi toksin-antitoksin, Microcystis aeruginosa PCC 7806, sistemi toksin-antitoksin tipa I
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-149277
• COBISS.SI-ID: 167889923
• Datum objave v RUL: 06.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Experimental characterization of six candidate type I toxin-antitoxin systems of the cyanobacterium Microcystis aeruginosa PCC 7806 with emphasis on MsoT1/MsoA1

Izvleček

Toxin-antitoxin (TA) systems are genetic elements that encode a relatively stable toxin and a labile antitoxin. In the case of type I systems, the toxin is a short membrane or cytosolic protein which exhibits cytostatic or cytotoxic activity while the antitoxin is a short non-coding RNA which binds to the toxin mRNA and thus prevents its translation. The notorious bloom-forming cyanobacteria Microcystis aeruginosa is especially interesting in terms of TA systems as it contains the highest number of type II TA loci of any bacteria, according to the TADB 2.0 database. Despite this, TA systems od M. aeruginosa remain poorly understood as only one type II system has been experimentally studied up to now while type I TA systems were not found in this species so far. In this thesis, we aimed to find putative type I TA systems in M. aeruginosa and to characterize them experimentally. We performed a bioinformatic analysis in which we searched the M. aeruginosa PCC 7806 genome for candidate loci using a set of search parameters based on characteristics of known type I loci. We found fifteen candidate type I TA loci and experimentally characterized six of them, namely BH695_0311, BH695_0320, BH695_3336, BH695_4017, BH695_4989, and BH695_5020. We prepared expression vectors carrying sequences for candidate type I toxins and used them to transform Escherichia coli BL21(DE3) pLysS cells. The effect of candidate toxin expression on cell growth was then tested by measuring OD600. Our results show that the expression of BH695_0320 and BH695_4017 has a negative effect on cell growth while the expression of the remaining candidate toxins has no effect. We continued our research focusing on the BH695_4017/MaT1A_4017 pair, which we renamed to MsoT1/MsoA1. By performing an immunodetection assay and a cell viability assay using erythrosin B, we were able to confirm the expression as well as the toxicity of MsoT1. Additionally, we found the expression of MsoT1 to have a delayed toxic effect when the cognate candidate antitoxin MsoA1 is also present on the vector. We concluded the research by constructing synthetic-biology vectors carrying the seqeunce for either ß-lactamase or mRFP under the MsoA1 candidate antitoxin promoters. By monitoring E. coli BL21(DE3) cell growth in media containing different concentrations of the antibiotic ampicillin and by measuring mRFP fluorescence, we were able to detect the expression of the two reporter proteins under the control of the PmsoA1 promoters, thus indirectly proving the expression of the MsoA1 antitoxin. The results of this thesis indicate MsoT1/MsoA1 to be a novel type I toxin-antitoxin family as well as the first type I system to be discovered in cyanobacteria.

• Ključne besede: toxin-antitoxin systems, Microcystis aeruginosa PCC 7806, type I toxin-antitoxin systems