magistrsko delo
Anže Vozelj (Author), Hrvoje Petković (Reviewer), Tjaša Danevčič (Mentor)

Abstract

Širjenje odpornosti proti obstoječim protimikrobnim učinkovinam predstavlja vedno večji svetovni problem, zato je nujno iskanje alternativnih, doslej še neraziskanih virov protimikrobnih učinkovin. Zanimiv sistem za preučevanje predstavlja rizosfera, s hranili bogato ozko področje okrog korenine, kjer najdemo posebno skupino bakterij, t.i. bakterije PGPR (ang. Plant Growth Promoting Rhizobacteria), ki pozitivno vplivajo na rast in donos rastlin. Mednje se uvršča tudi bakterija Bacillus subtilis, ki je predmet raziskav našega magistrskega dela, kjer smo pripravili mutante bakterije B. subtilis z izbrisom genov, ki so vključeni v sintezo surfaktina in bacilena. Ker je sekundarni metabolizem energetsko zelo zahteven, smo želeli preučiti, kako okvara biosintezne poti za sintezo bacilena, vpliva na sintezo surfaktina in obratno. V ta namen smo promotor gena za sintezo surfaktina (srfAA) in bacilena (pksC) spojili z genom za rumeni fluorescenčni protein (yfp). Meritve izražanja omenjenih konstruktov na mikročitalcu potrjujejo postavljeno hipotezo, da okvara biosintezne poti za bacilen vodi k večjemu izražanju promotorja gena za surfaktin in obratno. S surfaktinskim kapljičnim testom smo pokazali, da bacilenska mutanta producira količinsko več surfaktina, kar potruje hipotezo o pozitivni korelaciji med nivojem prepisovanja in prevajanja. Na primeru bacilena smo s konfokalno lasersko mikroskopijo pokazali, da je izražanje promotorja gena pksC v populaciji heterogeno, saj je za njegovo povečano produkcijo specializiran le del bakterijske populacije.

Keywords

Bacillus subtilis;protimikrobne učinkovine;sekundarni metaboliti;bacilen;surfaktin;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Vozelj]
UDC: 579.25:579.852.11:615.33
COBISS: 119928323 Link will open in a new window
Views: 24
Downloads: 21
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Other data

Secondary language: English
Secondary title: Modulation of antimicrobial compounds produced by Bacillus subtilis
Secondary abstract: Tackling against spreading of antimicrobial resistance, which is an intimidating global problem, must focus on undiscovered systems, that may have a potential to discover new antimicrobial compounds. Rhizosphere has enormous potential and can be described as nutrient-rich environment that offers optimal growth conditions for soil-dwelling bacteria such as PGPR (Plant Growth Promoting Rhizobacteria), a specialised group of bacteria that promote plant growth and yield. Bacillus subtilis, an improtant member of the PGPR group, is used in our master's thesis, where we constructed B. subtilis mutants with deletions in specific genes involved in the bacillaene or surfactin biosynthesis. According to the findings, secondary metabolism is energy consuming, and master's thesis focused mainly on the assumption that the deletion of one gene in surfactin or bacillaene biosynthesis affects the production of the other. Therefore, srfAA or pksC gene promoter was fused with a gene encoding for yellow fluorescent protein (yfp), which enabled us to measure the yellow fluorescence intensity on spectrofluorimeter or observe cells under confocal laser scanning microscope. We hypothesised that deletion of pks or srfAA gene reduces energetic burden, resulting in higher levels of pksC promoter expression after deleting surfactin (srfAA) pathway as well as in higher levels of srfAA promoter expression, when deleting bacillaene pathway (pks). Based on yellow fluorescence intensity measurements our hypothesis was confirmed. In addition, surfactin dropplet test was performed to assess the amount of surfactin produced by B. subtilis wild type strain or bacillaene mutant. We found that the bacillaene mutant produces quantitatively more surfactin compared to the wild type and therefore confirmed our third hypothesis, where we inffered on positive correlation between surfactin transcriptional and translational level. The expression of the pksC promoter in the population is heterogenous, confirming our second hypothesis. We have demonstrated by confocal laser scanning microscopy that only a specialised B. subtilis sub-population is responsible for higher levels of pksC promoter expression.
Secondary keywords: Bacillus subtilis;antimicrobial compounds;secondary metabolites;bacillaene;surfactin;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo
Pages: 1 spletni vir (1 datoteka PDF (XIV, 77 str., [7] f. pril.))
ID: 16336353