Charles University in Prague Faculty of Science Department of Genetics and Microbiology ...

176 Pages·2015·5 MB·English

Charles University in Prague
Faculty of Science
Department of Genetics and Microbiology
Microbiology
Mgr. Tomáš Větrovský
Combination of biochemical and high-throughput-sequencing approaches to study the role
of Actinobacteria and fungi in the decomposition of plant biomass
Studium úlohy Aktinobakterií a hub účastnících se degradace rostlinné biomasy kombinací
biochemických a moderních sekvenačních metod
Ph.D. Thesis
Supervisor: RNDr. Petr Baldrian, Ph.D.
Institute of Microbiology
of the Czech Academy of Sciences
Prague, 2015

Declaration:
I declare that I wrote this work on my own and all sources and literature are properly cited. I
also declare, that this work or its major part was not previously used for obtaining of the
same or any other academic degree.

Prague, 23.11.2015 Tomáš Větrovský

Table of contents

Acknowledgements 7
Abstract 8
Abstrakt 10
List of Abbreviations 13
1. Introduction 14
2. Scientific background 17
2.1. Dead plant biomass composition 17
2.2. Mechanisms of lignocellulose degradation 19
2.3. Actinobacteria as lignocellulose degraders 22
2.4. Fungal and bacterial communities in soils 24
2.5. The use of ribosomal genes for fungal and bacterial community analyses 26
2.6. Next generation sequencing data processing and analysis 29
3. List of Publications 35
4. Methods 45
5. Results and discussion 46
6. Conclusion 63
7. References 65

Acknowledgements

First I would like to acknowledge my supervisor RNDr. Petr Baldrian, Ph.D. for his great help
and friendly attitude over the period of my project and for the opportunity to obtain
valuable skills in various areas of my research. Further, I would like to thank all my
colleagues and friends from the Institute of Microbiology for their advice, assistance and
collaboration. I have learned a lot from them and they were creating a pleasant working
environment. My thanks also go to Doc. RNDr. Jiří Gabriel, DrSc., Mgr. Miroslav Kolařík Ph.D
and Doc. Kari Steffen Ph.D. and his team at the University of Helsinky, where the
experiments with radiolabelled substances were carried out. Finally I would also like to thank
my wife for her patience and support and my family for the permanent encouragement.

This work was supported by the Czech Science Foundation (Projects No. 526/08/0751, 13-
06763S), by the Czech Academy of Sciences (Project No. IAA603020901), and by the Ministry
of Education, Youth and Sports of the Czech Republic (Project No. LD12048).

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Abstract
Dead plant biomass is a key pool of carbon in terrestrial ecosystems. Its decomposition in
soil environments is thus an essential process of the carbon cycle. Fungi are considered to be
the primary decomposers in soil ecosystems because of their physiological adaptations and
enzymatic apparatus composed from highly effective oxidative and hydrolytic enzymes.
Many recent works show that in addition to fungi, bacteria may also play a significant role in
lignocellulose decomposition and among bacteria, the members of the phylum
Actinobacteria are often regarded to significantly contribute to cellulose and lignocellulose
decomposition.
This thesis is focused on the evaluation of the role that fungi and Actinobacteria play
in dead plant biomass degradation. First, it explored mechanisms involved in degradation, in
particular the enzymatic breakdown of major lignocellulose components as cellulose,
hemicelluloses and lignin. Enzymatic apparatus of the saprotrophic fungus Fomes
fomentarius was explored both in vitro as well as in vivo. Several Actinobacteria were
isolated from soil and comparative experiments, investigating production of hydrolytic
enzymes, were carried out to track the transformation of polysaccharides and lignin by these
strains.
To explain the roles of lignocellulose decomposers in complex environments like soil,
the community composition of fungi and bacteria, with special focus on Actinobacteria, was
investigated. The use of next generation sequencing (NGS) methods to cover this task
required implementation and design of appropriate tools for data handling, not available at
the time when the studies were conducted. This resulted in the development of the
software pipeline SEED. With the help of this tool, active degraders in forest soil were
identified by the comparison of RNA and DNA communities. Further, it was demonstrated
that some microbial taxa show high RNA/DNA ratio or were detected only in RNA pool and
thus they are underestimated or missing in studies based on DNA analysis. Results also
confirmed the importance of Actinobacteria showing that they belong to the most active
bacterial groups especially in soil organic horizon.
To provide an in-depth analysis of actinobacterial communities along the gradient of
heavy metal contamination, where they were expected to represent the most metabolically
active group, the method for a selective community composition analysis of the
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Actinobacteria using 454 pyrosequencing was developed that allows community
composition at a high resolution. The study showed that diversity of Actinobacteria was
unaffected by heavy metal content, but the contamination changed the community
composition significantly. Results also confirmed that Actinobacteria thrive better than other
bacteria in contaminated soils and may thus serve as important degraders of lignocellulose.
Finally, to get the most accurate estimates of the relative abundance of fungal and
bacterial taxa obtained by NGS, methods improving these estimates were proposed. This
part of study investigated the impact of the variability of 16S copy numbers among the
bacterial genomes on the diversity and abundances of different taxonomical groups and
steps to refine the abundance estimates were suggested. In the case of fungal communities,
an alternative marker derived from the single-copy gene rpb2 was compared with the widely
used multi-copy ITS with the aim to reduce the problems of community analysis due to the
intragenome variability and multicopy nature of the latter marker. Broad taxonomic
coverage, suitability for taxonomic assignments and sufficient variation for the use in
phylogenetic analyses were confirmed for the newly proposed marker.

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Abstrakt
Odumřelá rostlinná biomasa je klíčovým zdrojem uhlíku v pevninských ekosystémech, a
proto je její rozklad pro koloběh tohoto prvku na zemi esenciální. Mezi nejvýznamnější
rozkladače jsou řazeny především houby, a to zejména díky jejich schopnosti produkovat
vysoce účinné oxidativní a hydrolytické enzymy. Mnoho současných prací dále ukazuje, že
kromě hub mohou být dalšími důležitými rozkladači celulózy a lignocelulózy také bakterie,
zejména zástupci kmene Actinobacteria.
Tato disertační práce je zaměřena na zhodnocení role hub a aktinobakterií při
rozkladu odumřelé rostlinné biomasy. V rámci jejího řešení byly nejdříve zkoumány
mechanismy tohoto rozkladu, týkající se především rozkladu hlavních komponent
lignocelulózy, celulózy, hemicelulóz a ligninu pomocí extracelulárních enzymů. U saprotrofní
houby Fomes fomentarius byl důkladně popsán enzymový aparát a to jak in vitro, tak in vivo.
Schopnost produkce hydrolytických enzymů byla dále testována u aktinobakterií isolovaných
z půdy a u vybraných kmenů byly provedeny experimenty sledující osud polysacharidů a
ligninu v průběhu jejich dekompozice.
Pro objasnění úlohy rozkladačů lignocelulózy v komplexních prostředích, jako je půda,
bylo zkoumáno složení společenstev hub a bakterií se speciálním zaměřením na
aktinobakterie. K dosažení tohoto cíle bylo využito metod sekvenování nové generace (NGS),
které vyžadovalo navržení a implementaci nástrojů pro práci se získanými daty, v dané době
ještě nedostupných. Tato snaha vyústila ve vytvoření programu pro zpracování sekvenačních
dat SEED. S pomocí tohoto programu byli na základě porovnání RNA a DNA komunit odhaleni
aktivní mikrobiální rozkladači v lesní půdě. Z porovnání je patrné, že mnohé významné
taxonomické skupiny mikroorganismů mají vysoký poměr RNA/DNA nebo jsou obsaženy
pouze v RNA, a tak mohou být podceněny nebo úplně chybět ve studiích, kde byla
analyzována pouze DNA. Výsledky také potvrdily význam aktinobakterií jako jedné z
nejaktivnějších bakteriálních skupin, zejména v organickém horizontu půdy.
Pro získání detailního popisu společenstva aktinobakterií žijících v půdách
kontaminovaných těžkými kovy, ve kterých mohou představovat metabolicky nejaktivnější
skupinu mikroorganismů, byla vyvinuta metoda pro jejich selektivní analýzu pomocí 454
pyrosekvenace s vysokým rozlišením. Použití této metody ukázalo, že diversita aktinobakterií
není ovlivněna obsahem těžkých kovů, avšak koncentrace těchto kovů významně ovlivňují
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