Bioinformacijska analiza lokusov vapBC cianobakterije Microcystis aeruginosa PCC 7806 in eksperimentalna karakterizacija potencialnih toksinov VapC29, VapC33 in VapC45

magistrsko delo

Jernej Imperl (Author), Marko Dolinar (Mentor), Miha Pavšič (Thesis defence commission member), San Hadži (Thesis defence commission member), Jurij Lah (Thesis defence commission member)

Abstract

Sistemi toksin-antitoksin (TA) so sestavljeni iz dveh komponent. Toksin je praviloma stabilen protein, ki v celicah, kjer se izraža, deluje citostatično ali citotoksično, antitoksin pa je nestabilen protein ali RNA, ki preprečuje delovanje toksina in omogoča normalno rast celic. Sistemi vapBC predstavljajo skupino sistemov TA tipa II, kjer sta obe komponenti po strukturi proteina. Značilnost toksinov VapC je domena PIN (domena na N-koncu proteina PilT; ang. »PilT N-terminal«), ki jim daje nukleazno aktivnost, s katero v celici zavirajo translacijo. Antitoksini VapB so lahko po strukturi raznoliki, ampak delujejo tako, da s toksinom tvorijo netoksičen kompleks. Cianobakterija Microcystis aeruginosa lahko povzroča cvetenje voda in predstavlja bakterijo z največ predvidenimi lokusi TA v bazi podatkov TADB2.0. Kljub temu pa so zaenkrat sistemi TA cianobakterije M. aeruginoisa še zelo slabo okarakterizirani. V sklopu magistrskega dela smo izvedli bioinforomacijsko analizo, v kateri smo s programoma TAfinder in TASer poiskali predvidene lokuse TA v genomu M. aeruginosa PCC 7806. Skupno smo našli 370 potencialnih lokusov TA, od katerih je bilo 57 sistemov vapBC. Iz nabora sistemov vapBC smo izbrali šest parov TA in zapise za toksin ter za pare toksin-antitoksin uspešno prenesli v klonirne oziroma ekspresijske vektorje. Tri toksine, VapC29, VapC33 in VapC45, smo analizirali tudi eksperimentalno, tako, da smo spremljali njihov vpliv na rast bakterije Escherichia coli, ki so jih izražale. Medtem ko VapC33 verjetno nima toksične aktivnosti, pa naši rezultati kažejo, da bi VapC29 in VapC45 lahko bila aktivna toksina in vivo.

Keywords

cianobakterije;Microcystis aeruginosa PCC 7806;proteini;sistemi toksin-antitoksin;sistemi vapBC;bioinformatika;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2022
Typology:	2.09 - Master's Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[J. Imperl]
UDC:	577.112(043.2)
COBISS:	120684291
Views:	44
Downloads:	20
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Bioinformatics analysis of loci vapBC of the cyanobacteria Microcystis aeruginosa PCC 7806 and experimental characterization of putative toxins VapC29, VapC33 and VapC45
Secondary abstract:	Toxin-antitoxin (TA) systems are composed of two components. The toxin is typically a stable protein, which exhibits cytostatic or cytotoxic activity in the cells that express it. The antitoxin is either an unstable protein or RNA, which inhibits the activity of the toxin and therefore enables normal cell growth. The vapBC systems are classified as a type II TA system, meaning that both components are structurally proteins. VapC toxins contain the nuclease PIN-domain (PilT N-terminal) and act by inhibiting cellular translation. Antitoxins VapB can be structurally variable, however they all act via binding to the toxin and forming a non-toxic complex. Microcystis aeruginosa is a bloom-forming cyanobacterium containing the highest number of TA loci of all bacteria, according to the TADB2.0 database. Despite this, TA systems of M. aeruginosa remain poorly understood. In this work, we have performed a bioinformatics analysis, using TAfinder and TASer to find new putative TA loci in the genome of M. aeruginosa PCC 7806. We have identified 370 potential TA loci, of which 57 are vapBC systems. From our list we selected six TA pairs and successfully constructed cloning and expression vectors containing the genes for either the toxin or the toxin-antitoxin pair. Three toxins, VapC29, VapC33 and VapC45, were also experimentally characterized by assessing their effect on the growth of Escherichia coli cells during expression. Based on our results, VapC33 likely possesses no toxic activity, while VapC29 and VapC45 could potentially act as active toxins in vivo.
Secondary keywords:	toxin-antitoxin systems;vapBC systems;Univerzitetna in visokošolska dela;
Type (COBISS):	Master's thesis/paper
Study programme:	1000377
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages:	68 str.
ID:	16261730