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Exploring Microbial Dark Matter in East Antarctic Soils

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Exploring Microbial Dark Matter in East Antarctic Soils EXPLORING MICROBIAL DARK MATTER IN EAST ANTARCTIC SOILS Mukan Ji A thesis in fulfilment of requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Science Faculty of Science UNSW Australia ORIGINALITY sYfrEMENT 'I hereby declare that this submission is my own work and to the best of my knowledge it contains no·materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presenta · and linguistic expression is acknowledged.' Signed Date COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media. now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of ::: Date AUTHENTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format. Date ..... C' 1 Table of contents 2 3 Table of contents ............................................................................................................ i 4 Abstract ........................................................................................................................ v 5 Acknowledgement ...................................................................................................... vii 6 List of Tables ............................................................................................................. viii 7 List of Figures .............................................................................................................. ix 8 List of Appendices ...................................................................................................... xii 9 List of abbreviations .................................................................................................. xiii 10 Chapter 1 General introduction ..................................................................................... 1 11 1.1 Terrestrial Antarctica harbours abundant and diverse microbial communities ... 1 12 1.2 Cold adaption strategies of Antarctic bacteria .................................................. 6 13 1.3 Functional capacities of Antarctic soil bacteria ................................................ 8 14 1.4 The role of biotic and abiotic factors in shaping Antarctic soil microbial 15 communities ........................................................................................................ 11 16 1.5 Next generation sequencing technologies and expanding our understanding of 17 microbial diversity and function .......................................................................... 12 18 1.6 The Windmill Islands, an under-explored ice free region ............................... 18 19 1.7 Aims .............................................................................................................. 21 20 Chapter 2 Microbial diversity at Mitchell Peninsula, East Antarctica: a potential 21 microbial dark matter “hotspot” .................................................................................. 22 22 2.1 INTRODUCTION ......................................................................................... 22 23 2.2 MATERIAL AND METHODS ...................................................................... 23 24 2.2.1 Soil sampling ...................................................................................... 23 25 2.2.2 Physicochemical analyses ................................................................... 23 i 1 2.2.3 DNA extraction................................................................................... 24 2 2.2.4 Multiplex 454 pyrosequencing and data processing ............................. 24 2.2.5 Quantitative PCR (qPCR) targeting bacterial SSU RNA and fungal SSU 3 2.2.5 Quantitative PCR (qPCR) targeting bacterial SSU RNA and fungal SSU 4 RNA genes .................................................................................................. 27 5 2.2.6 Statistical analysis ............................................................................... 27 6 2.2.7 Network analysis ................................................................................ 28 7 2.3 RESULTS ..................................................................................................... 29 8 2.3.1 Soil properties ..................................................................................... 29 9 2.3.2 Soil microbial abundance and diversity ............................................... 30 10 2.3.3 Microbial community similarity across Mitchell Peninsula ................. 33 2.3.4 Network analysis of bacterial and fungal communities and measured 11 2.3.4 Network analysis of bacterial and fungal communities and measured 12 environmental parameters ............................................................................ 40 13 2.4 DISCUSSION ............................................................................................... 42 C hapter 3 Atmospheric chemotrophy: A unique functional capacity of East Antarctic 14 Chapter 3 Atmospheric chemotrophy: A unique functional capacity of East Antarctic 15 microbial communities ................................................................................................ 45 16 3.1 Introduction ................................................................................................... 45 17 3.2 Material and methods .................................................................................... 46 18 3.2.1 Site description ................................................................................... 46 19 3.2.2 Metagenome sequencing and assembly ............................................... 49 20 3.2.3 Population genome binning and assessment ........................................ 49 21 3.2.4 Metabolic annotation .......................................................................... 50 22 3.2.5 Microbial community profile .............................................................. 50 23 3.2.6 Phylogenetic inference ........................................................................ 50 24 3.2.7 Comparative genomics analysis .......................................................... 51 3.2.8 Gene cluster and phylogenetic analysis of RubisCO, hydrogenase and 25 3.2.8 Gene cluster and phylogenetic analysis of RubisCO, hydrogenase and 26 ammonia monooxygenase subunit C (amoC) genes ..................................... 51 27 3.3 Results .......................................................................................................... 52 ii 1 3.3.1 The Robinson Ridge metagenome ....................................................... 52 2 3.3.2 Recovery of draft genomes from Robinson Ridge soil ......................... 55 3.3.3 Energy and nutrient dynamics inferred from the Robinson Ridge 3 3.3.3 Energy and nutrient dynamics inferred from the Robinson Ridge 4 metagenome ................................................................................................ 59 5 3.4 Discussion ..................................................................................................... 70 6 Chapter 4 Characterisation of candidate phylum WPS-2 ............................................. 73 7 4.1 Introduction ................................................................................................... 73 8 4.2 Materials and methods ................................................................................... 74 9 4.2.1 Genome analysis ................................................................................. 74 10 4.2.2 Probe design and optimisation............................................................. 74 11 4.2.3 Construction of a WPS-2 specific clone .............................................. 75 12 4.2.4 Clone-FISH ........................................................................................ 78 13 4.2.5 WPS-2 specific Clone-FISH optimisation ........................................... 78 14 4.2.6 Epi-fluorescence microscopy .............................................................. 79 15 4.2.7 Extraction of cells from soil using Nycodenz ...................................... 79 16 4.3 Results .........................................................................................................
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