The regulation of the selected type II toxin-antitoxin systems in Escherichia coli cells

Živič, Zala (avtor), Hadži, San (mentor)

Toxin-antitoxin systems are small genetic elements with a wide variety of proposed biological functions, uses in biochemistry and potential applications in medicine. Of the eight types currently known, type II is the most studied and abundant toxin-antitoxin system type, consisting of a protein toxin which is inactivated by direct binding of its protein antitoxin. This thesis was centred on the regulation mechanism of these type II toxin-antitoxin systems. Their regulation is notable due to its often-complex nature. Most commonly, antitoxin binds and represses its own promoter region. If toxin is present, its binding to antitoxin affects antitoxin-DNA binding capabilities. We focused on two effects that toxin binding can achieve: 1. toxin association weakens DNA-binding and causes de-repression (anti-cooperative regulation) and 2. toxin association can enhance DNA-binding up to a certain molar ratio followed by de-repression (conditionally cooperative regulation). While many type II regulation mechanisms have been studied in great detail in vitro, there are few extensive investigations of their in vivo behaviour. We aimed to establish an expression system which would allow for toxin/antitoxin ratio-dependent control of reporter protein production. For this purpose, we tested IPTG and L-arabinose as potential antitoxin and toxin inducers, respectively. IPTG and L-arabinose are prone to producing an “all-or-nothing” response when added to cells. In order to achieve tuneable gene expression, we used LacY-deficient strain Tuner[DE3] and constant expression of araE transporter gene. These modifications proved to be effective and allowed us to independently express sfGFP in a tuneable manner. Based on these results, we constructed a pRAT-sfGFP vector base for the study of type II toxin-antitoxin systems in E. coli. The work of our research group and collaborators then allowed us to elucidate the anti-cooperative regulation mechanism of VcHigBA2. In vitro data detailing the disorder-based anti-cooperativity was corroborated by data obtained from constitutive expression of system in E. coli along with pRAT-sfGFP-based experiments. The main driving force of anti-cooperativity is antitoxin N-terminus, being both a stabilizing agent in DNA-binding and the main toxin-antitoxin interface. When bound to toxin, this N-terminus no longer stabilizes DNA-binding and thus leads to de-repression. We then investigated systems PvHigBA, PpGraTA, EcMqsRA, EcRelBE, EcMazEF, EcCcdBA and EpPhdDoc using pRAT-sfGFP. We were able to compare promoter strength and expression profiles of these systems. We found that plasmid-borne system promoter strength is stronger. We also noticed that regulation mapping of previous work qualitatively aligned with our results in the case of PvHigBA and PpGraTA, whereas EcMqsRA, EcRelBE, EcMazEF, EcCcdBA and EpPhdDoc require further investigation. While promising, there are still issues that need to be addressed for more accurate regulation mapping, namely the potential residual toxicity of enzymatically inactive toxins, the correct inducer levels for conditional cooperativity, antitoxin leaking and the detection of toxin and antitoxin protein molecules from expression experiments. Still, our results provide an interesting new framework for the study and comparison of type II toxin-antitoxin systems.

• Jezik: Angleški jezik
• Ključne besede: toxin-antitoxin, tuneable gene expression, VcHigBA2, regulation mapping, IPTG, L-arabinose
• Vrsta gradiva: Doktorsko delo/naloga
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-174625
• COBISS.SI-ID: 254116099
• Datum objave v RUL: 07.10.2025

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Regulacija izbranih sistemov toksin-antitoksin tipa II v celicah Escherichia coli

Izvleček

Sistemi toksin-antitoksin so majhni genetski elementi s širokim naborom predlaganih bioloških funkcij ter uporabe v biokemiji in medicini. Izmed do sedaj znanih osem tipov sistemov toksin-antitoksin, je najbolj preučevan tip II. Sestavljata ga gena za protein toksin in protein antitoksin. Antitoksin zavira delovanje toksina preko neposrednih interakcij. V doktorskem delu sem se osredotočila preučevanje zapletenega regulacijskega mehanizma sistemov tipa II. Pogosto je v regulacijo vpleten antitoksin, ki se veže na lasten promotor in zavira izražanje genov za antitoksin in toksin. Vezava toksina na antitoksin vpliva na njegovo sposobnost vezave DNA. Pri delu sem obravnavala predvsem ne-kooperativne sisteme, kjer toksin ošibi interakcijo z DNA, ter pogojno kooperativne sisteme, sisteme, pri katerih toksin ošibi vezanje antitoksina na DNA (ne-kooperativna regulacija), ter sisteme, pri katerih je vezava kompleksa toksin-antitoksin na promotor vezana na njuno molsko razmerje (pogojno kooperativna regulacija). Raziskave mehanizmov regulacije delovanja so pogosto izvedene in vitro, brez podrobne karakterizacije obnašanja regulacije sistema v celičnem okolju. Želela sem vzpostaviti sistem za spremljanje odziva promotorja na molsko razmerje toksin/antitoksin v E. coli. Kot potencialna induktorja za nadzorovano izražanje genov antitoksina in toksina sem ovrednotila IPTG ter L-arabinozo. Oba sta ob dodatku v celično populacijo znana po odzivu »vse-ali-nič«, kar je bilo za načrtovane raziskave nezaželeno. Takega odziva sem se znebila z uporabo seva Tuner[DE3] ter s konstanto produkcijo transporterja AraE, kar mi je omogočilo neodvisno nadzorovano izražanje poročevalca sfGFP. Na osnovi pridobljenih rezultatov sem zasnovala ogrodni vektor pRAT-sfGFP, ki omogoča preučevanje regulacijskega mehanizma sistemov tipa II v E. coli. Raziskave, v sodelovanju z drugimi komplementarnimi skupinami so nam omogočile karakterizacijo ne-kooperativnega mehanizma regulacije sistema VcHigBA2. Značilnosti izražanja s konstitutivnimi promotorji in ogrodjem pRAT-sfGFP se ujemajo z mehanizmoim predpostavljenim na osnovi eksperimentov izvedenih in vitro. Regulacija sistema VcHigBA2 je nadzorovana predvsem s strani neurejene domene antitoksina. Tak nadzor ni mogoč, ko je neurejena domena antitoksina vezana na toksin. Naslednji korak je bilo raziskovanje v sistemih PvHigBA, PpGraTA, EcMqsRA, EcRelBE, EcMazEF, EcCcdBA in EpPhdDoc na ogrodju pRAT-sfGFP. Primerjala sem moč promotorjev in odziv sistemov na prisotnost induktorjev. Sistemi, ki izvirajo s plazmidov, imajo močnejše promotorje od kromosomalnih sistemov. Odziv izražanja poročevalca je za posamezen sistem unikaten in se v veliki meri ujema s podatki pridobljenimi v prejšnjih poskusih za sistema PvHigBA in PpGraTA. Sistemi EcMqsRA, EcRelBE, EcMazEF, EcCcdBA in EpPhdDoc proizvedejo bolj kompleksen, ki zahteva bolj podrobno obravnavo. Pri ugotavljanju mehanizma regulacije delovanja sistemov toksin-antitoksin v E. coli so se pojavili tudi novi izzivi. Predvsem je potrebna podrobna obravnava toksičnosti encimsko neaktivnih toksinov, koncentracij proteinov, ki določajo pogojno kooperativnost, »puščanja« antitoksina in zaznavanja ravni proizvedenih proteinov pri poskusih izražanja. Kljub temu menim, da rezultati mojega doktorskega dela predstavljajo dobro osnovo za raziskave delovanja sistemov toksin-antitoksin v celičnem okolju.

• Ključne besede: toksin-antitoksin, nadzorovano izražanje gena, VcHigBA2, profil izražanja, IPTG, L-arabinoza