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Dizertační Práce MASARYKOVA UNIVERZITA Přírodovědecká fakulta Ústav experimentální biologie Dizertační práce Brno 2019 Stanislava Králová MASARYKOVA UNIVERZITA Přírodovědecká fakulta Ústav experimentální biologie Typizace a taxonomie psychrofilních prokaryot z Antarktidy Dizertační práce Stanislava Králová Školitel: doc. RNDr. Ivo Sedláček, CSc. Brno 2019 Bibliografický záznam Autor: RNDr. Stanislava Králová Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Česká sbírka mikroorganismů Název práce: Typizace a taxonomie psychrofilních prokaryot z Antarktidy Studijní program: Experimentální biologie Studijní obor: Mikrobiologie Školitel: doc. RNDr. Ivo Sedláček, CSc. Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Česká sbírka mikroorganismů Akademický rok: 2018/2019 Počet stran: 66 + publikace Klíčová slova: systematika, taxonomie, klasifikace, typizace, Antarktida, druh, mastné kyseliny, diverzita, adaptace Bibliografický záznam Autor: RNDr. Stanislava Králová Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Česká sbírka mikroorganismů Názov práce: Typizácia a taxonómia psychrofilných prokaryot z Antarktídy Štúdijný program: Experimentálna biológia Štúdijný odbor: Mikrobiológia Školiteľ: doc. RNDr. Ivo Sedláček, CSc. Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Česká sbírka mikroorganismů Akademický rok: 2018/2019 Počet strán: 66 + publikácie Kľúčové slová: systematika, taxonómia, klasifikácia, typizácia, Antarktída, druh, mastné kyseliny, diverzita, adaptácia Bibliografický záznam Author: RNDr. Stanislava Králová Faculty of Science, Masaryk University Department of Experimental Biology Czech Collection of Microorganisms Title of Dissertation: Typing and taxonomy of psychrophilic prokaryotes from Antarctica Degree Programme: Experimental Biology Field of study: Microbiology Supervisor: Assoc. prof. RNDr. Ivo Sedláček, CSc Faculty of Science, Masaryk University Department of Experimental Biology Czech Collection of Microorganisms Academic Year: 2018/2019 Numer of Pages: 66 + scientific papers Key Words: systematics, taxonomy, classification, typing, Antarctica, species, fatty acids, diversity, adaptation Abstrakt Předkládaná dizertační práce je koncipováná jako soubor komentovaných publikací, jejichž hlavním objektem jsou zástupci početného rodu Flavobacterium. Flavobakterie jsou významnými kolonizátory chladných oblastí, a to hlavně vod a půd. Z toho důvodu představují jeden z nejvíce zastoupených a zároveň kultivovatelných rodů kolonizujících antarktické habitaty. Dizertační práce představuje taxonomické studium flavobakterii vyizolovaných z abiotických vzorků získaných právě na Antarktidě. Izolované kmeny byly charakterizovány polyfázovým taxonomickým přístupem, s využitím fenotypových (biotypizace), chemotaxonomických (mastné kyseliny, polární lipidy, menachinony, polyaminy) a molekulárních metod (PCR, repetitivní PCR, DNA-DNA hybridizace, sekvenování genů pre 16S rRNA, celogenómové sekvenování). Mimo jiné, chemotaxonomické analýzy, konkrétně analýza mastných kyselin, umožnily charakterizovat velmi diskutované adaptační mechanizmy chladnomilných baktérií. Výsledkem této práce byla rozsáhlá charakterizace žlutě pigmentujících izolátů, která vedla až k popisům nových taxonů, a také podrobná charakterizace adaptačních mechanizmů cytoplazmatické membrány, které umožňují přežití flavobakterii v extrémnich antarktických podmínkách. Abstrakt Predkladaná dizertačná práca je koncipovaná ako súbor komentovaných publikácií, ktorých hlavným objektom sú zástupcovia početného rodu Flavobacterium. Flavobaktérie sú významnými kolonizátormi chladných oblastí, a to najmä vôd a pôd. Z tohto dôvodu predstavujú aj jeden z naviac zastúpených a zároveň dobre kultivovateľných rodov kolonizujúcich antarktické habitaty. Dizertačná práca predstavuje taxonomické štúdium flavobaktérií vyizolovaných z abiotických vzoriek odoberaných práve na Antarktíde. Izolované kmene boli charakterizované polyfázovým taxonomickým prístupom, s využitím fenotypových (biotypizácia), chemotaxonomických (mastné kyseliny, polárne lipidy, menachinóny, polyamíny) a molekulárnych metód (PCR, repetitívna PCR, DNA-DNA hybridizácia, sekvenovanie génov pre 16S rRNA, celogenómová sekvenácia). Okrem toho, chemotaxonomické analýzy, kontrétne analýza mastných kyselín, umožnili charakterizovať veľmi diskutované adaptačné mechanizmy chladnomilných baktérií. Výsledkom tejto práce bola rozsiahla charakterizácia žlto pigmentovaných izolátov, ktorá vyústila až k popisom nových taxónov, a taktiež podrobná charakterizácia adaptačných mechanizmov cytoplazmatickej membrány, ktoré umožňujú prežitie flavobaktérií v extrémnych antarktických podmienkach. Abstract Present Ph.D thesis is compiled as a collection of commented scientific papers focused on members of the genus Flavobacterium. Flavobacterium spp. are important colonizers of cold areas, especially waters and soils. For this reason, they are also found as one of the most abundant and cultivable genus in Antarctica. This Ph.D thesis is compiled as a taxonomic study of Flavobacterium spp. isolated from abiotic samples collected in Antarctica. Isolates were characterized with polyphasic taxonomic approach. Methods including phenotyping (biotyping), chemotaxonomy (fatty acids, polar lipids, menaquinones, polyamines) and methods of molecular biology (PCR, repetitive PCR, sequencing of the 16S rRNA genes, DNA-DNA hybridization, whole genome sequencing) were all used in this study. Moreover, chemotaxonomic approach, analysis of fatty acids, was used in order to determine cold adaptation of this cold-loving bacteria. Findings of this study led to extensive characterization of yellow pigmented isolates followed by description of novel Flavobacterium species. Besides, analysis of fatty acids resulted in characterization of membrane adaptation mechanisms enabling these bacteria to survive in extreme Antarctic conditions. © Stanislava Králová, Masarykova Univerzita, 2019 Poďakovanie Na tomto mieste by som sa chcela úprimne poďakovať vedúcemu svojej dizertačnej práce docentovi Ivo Sedláčkovi za odborné rady, trpezlivosť a vedenie počas nielen doktorandského šúdia ale aj počas spracovávania dizertačnej práce a publikovania prvých článkov, čo si nesmierne vážim. RNDr. Pavlovi Švecovi srdečne ďakujem za podporu, každú radu, mnohé konzultácie a vylepšenie nálady v stresových obdobiach štúdia. Ďalšie poďakovanie venujem celému kolektívu Českej zbierky mikroorganizmov, nielen za poskytnutie podmienok pre experimenty, ale taktiež za príjemné pracovné podmienky a podporu počas celého doktorandského štúdia. V neposlednom rade by som sa chcela poďakovať všetkým spoluautorom za výborne odvedenú prácu a mnohé konzultácie, ktoré viedli k úspešným publikačným výstupom. Moja veľká vďaka patrí prof. Bussemu na Veterinárnej Univerzite vo Viedni, nakoľko bol ochotný poskytnúť zázemie a taktiež svoj cenný čas a rady pre uskutočnenie chemotaxonomických analýz, súvisiacich nielen s touto dizertačnou prácou. Menovite by som sa tiež rada poďakovala RNDr. Danielovi Krskovi na SZÚ v Prahe za snímky z elektrónového mikroskopu, bez ktorých by ani jedna z tu prezentovaných taxonomických publikácií nevznikla. Prehlásenie Prehlasujem, že som svoju dizertačnú prácu vypracovala samostatne s využitím informačných zdrojov, ktoré sú v práci a v jej prílohách citované. Brno 01.01. 2019 ………………………. Stanislava Králová OBSAH: ZOZNAM POUŽITÝCH SKRATIEK ..................................................................................... 12 1. ÚVOD ............................................................................................................................... 13 1.1 Mikrobiálna kolonizácia chladných habitatov .......................................................... 13 1.2 Život na Antarktíde ................................................................................................... 13 1.3 Štúdium antarktických baktérií ................................................................................. 14 1.4 Ekológia psychrofilov na Antarktíde ........................................................................ 15 1.4.1 Akvatické habitaty .............................................................................................. 15 1.4.2 Mikrobiálne povlaky .......................................................................................... 17 1.4.3 Pevninské habitaty .............................................................................................. 17 1.5 Adaptácia a fyziológia psychrofilných baktérií ........................................................ 18 1.5.1 Environmentálne adaptácie................................................................................. 19 1.5.1.1 Výber habitatu ............................................................................................. 19 1.5.1.2 Dormantný stav ........................................................................................... 19 1.5.2 Molekulárne adaptácie ........................................................................................ 20 1.5.2.1 Genóm ......................................................................................................... 20 1.5.2.2 Proteíny a enzýmy ....................................................................................... 20 1.5.2.3 Odpovede na teplotné šoky ......................................................................... 21 1.5.3 Fyziologické adaptácie ......................................................................................
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