Prepoznava genskih skupkov za razgradnjo hemiceluloz in pektinov v bakterijskem rodu Prevotella s transkriptomskim pristopom : doktorska disertacija

Murovec, Urška (author), Accetto, Tomaž (mentor)

Bakterije iz rodu Prevotella so ena najbolj dominantnih skupin v mikrobioti vampa. Njihova glavna funkcija je razgradnja hemiceluloz in pektinov rastlinske celične stene ter založnih polisaharidov. Te substrate razgrajujejo s pestrim naborom encimov, ki so kodirani v skupkih, odgovornih za razgradnjo polisaharidov (angl. Polysaccharide Utilisation Loci – PULs). To so lokusi ko-reguliranih genov, katerih produkti zaznavajo, vežejo, prenašajo in encimsko razgrajujejo kompleksne ogljikove hidrate. S konstituivno ekspresijo genov sistema PUL lahko te bakterije spremljajo substrate v okolju in če jih zaznajo, začnejo z ekspresijo genov na višjem nivoju. PUL se aktivira ob zaznavi substrata, katerega so produkti aktiviranega PULa sposobni razgraditi. Bioinformacijske analize vampnih vrst Prevotella so pokazale, da vsebujejo raznolik nabor PULov v svojih genomih, transkriptomski in genetski pristopi pa so napovedi potrdili na omejenem številu substratov. V tej študiji smo transkriptomsko identificirali različne PULe v Prevotella ruminicola KHP1 in Prevotella bryantii TF1-3, da bi potrdili njihovo specifičnost in preiskali raznolikost prisotnih PULov. Poleg tega smo z RT-qPCR preverili substratne preference in ocenili, da specifična hitrost rasti in količina biomase, ki jo sev lahko pridobi na substratu, vplivata na izbiro substratov za rast. Preferenčni substrati so bili uporabljeni v obeh bakterijah istočasno. Opazili smo tudi izrabljanje substratov, za katere ni bilo napovedanih in identificiranih PULov s transkriptomiko. V takšnem primeru sklepamo, da razgradnja substrata poteka z delovanjem več PULov hkrati. Prav tako smo preverjali homologijo in strukturno podobnost odkritih PULov s transkriptomiko še v 75 sevih Prevotella, da smo dobili vpogled v dinamiko spreminjanja genoma v tem rodu.

• Language: Slovenian
• Keywords: polisaharidi, transkriptomika, Prevotella, vamp, prežvekovalci, hemiceluloze, pektin, RNAseq, bioinformacijska analiza, genski skupki
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2024
• PID: 20.500.12556/RUL-164757
• COBISS.SI-ID: 214812675
• Publication date in RUL: 11.11.2024

Secondary language

• Language: English
• Title: Identification of hemicellulose and pectin ultilization loci in Prevotella using transcriptomics : doctoral dissertation

Abstract

Bacteria of the genera Prevotella are among the most dominant groups in the rumen microbiota. They are characterized by the ability to utilize different hemicelluloses and pectin of plant cell-wall as well as plant energy storage polysaccharides. The degradation is possible with the use of cell envelope bound multiprotein apparatuses coded in polysaccharide utilization loci (PULs), which have been shown to be substrate specific. PULs are constitutively expressed at low levels, but when they detect their cognate substrate, the expression of PUL genes increases singnificantly. The knowledge of PUL presence in rumen Prevotella based on bioinformatic analyses is already established and transcriptomic and genetic approaches confirmed predicted PULs for a limited number of substrates. In this study, we transcriptomically identified additional different PULs in Prevotella ruminicola KHP1 and Prevotella bryantii TF1-3. We also identified substrate preferences and found that specific growth rate and extent of growth impacted the choice of substrates preferentially used for degradation. These preferred substrates were used by both strains simultaneously as judged by their PUL upregulation. Lastly, β-glucan and xyloglucan were used by these strains in the absence of bioinformatically and transcriptomically identifiable PUL systems. We also analysed homology and synteny of transcriptomicaly confirmed PULs in 75 more strains of Prevotella to gain the insight of dynamics of PUL evolution in this genus.

• Keywords: polysaccharides, transcriptomics, Prevotella, rumen, ruminants, hemicellulose, pectin, RNAseq, bioinformatic analysis, genetic loci