Molecular phylogeny of Archaea in boreal forest soil, freshwater and temperate estuarine sediment German Jurgens Department of Applied Chemistry and Microbiology Division of Microbiology University of Helsinki Finland ACADEMIC DISSERTATION IN MICROBIOLOGY To be presented with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in Auditorium 2, Viikinkaari 11, Viikki Infocenter, on November 22th 2002, at 12:00 noon Helsinki 2002 Supervisor: PhD Aimo Saano, Docent, University of Helsinki Metsähallitus, Natural Heritage Services, Vantaa, Finland Co-supervisors: PhD Uwe Münster, Docent, University of Helsinki, Lecturer, Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, Finland PhD Kristina Lindström, Docent, University of Helsinki Department of Applied Chemistry and Microbiology, University of Helsinki, Finland Reviewers: PhD Maarit Niemi, Docent, University of Helsinki Finnish Environment Institute, Research Programme for Biodiversity, Helsinki, Finland PhD Hannu Fritze, Docent, University of Helsinki Finnish Forest Research Institute, Vantaa Research Center, Finland Opponent: Professor Vigdis Torsvik, University of Bergen, Department of Microbiology, Bergen, Norway ISSN 1239-9469 ISBN 952-10-0714-1 (paperback) ISBN 952-10-0715-X (PDF) ISBN 952-10-0716-8 (HTML) Electronic publication available at http://ethesis.helsinki.fi Helsinki University Printing House Helsinki, Finland 2002 Front cover: "Archaea's Universe" - collage made by author. Background: combination of same field micrographs made with epifluorescence microscope using filter sets specific for DAPI (blue color) and CY3 (red color, euryarchaeota specific probe EURY499, paper IV), Valkea Kotinen Lake water sample. Center: 16S rRNA secondary structure diagram of the archaeal soil clone FFSB6 - one of the first "non-extreme" crenarchaeota sequences found in forest soil (kindly made by Dr. Robin Gutell in August 1996). To my mother, Candidate of Biological Sciences Ina Leopoldovna Jurgens CONTENTS LIST OF ORIGINAL PAPERS THE AUTHOR'S CONTRIBUTION ABBREVIATIONS 1 INTRODUCTION................................................................................................................. 1 1.1 Microorganisms in the biosphere and the recent evolution of their taxonomy......... 1 1.1.1 Microorganisms as part of the biosphere - widespread and important, but mainly unknown ................................................................................................. 1 1.1.2 History of classification schemes........................................................................ 2 1.1.3 A new era in microorganism studies - introduction of molecular techniques and novel taxonomy .................................................................................................. 3 1.2 The use of molecular methods to study microbes in natural environments.............. 4 1.2.1 Ribosomal RNA: a key to molecular phylogeny ................................................ 4 1.2.2 Universal phylogenetic tree - the molecular tree of life...................................... 6 1.2.3 Molecular microbial ecology approaches to accessing natural diversity............ 8 1.2.4 Inferring phylogenetic relationships from rRNA gene sequences .................... 11 1.2.5 Limitations of molecular microbial ecology..................................................... 12 1.2.6 Combination of traditional culture-dependent methods and modern molecular techniques ......................................................................................................... 14 1.3 Archaea .......................................................................................................................... 15 1.3.1 Features distinguishing Archaea from Bacteria and Eucarya ........................... 15 1.3.2 Archaeal phenotypes and phylogenetic division............................................... 16 1.3.3 Phylum Crenarchaeota ...................................................................................... 17 1.3.4 Phylum Euryarchaeota ...................................................................................... 23 1.3.5 Archaea as "non-extremophiles"....................................................................... 28 2 AIMS OF THE STUDY...................................................................................................... 37 3 MATERIALS AND METHODS........................................................................................ 38 3.1 Sampling and nucleic acid extraction ......................................................................... 38 3.1.1 Soil sampling (Finland)..................................................................................... 38 3.1.2 Water sampling for microbial DNA extraction (Finland)................................. 38 3.1.3 Estuarine sediment sampling (Portugal) ........................................................... 39 3.2 PCR amplification, cloning and clones characterization .......................................... 39 3.3 Conditions for in situ hybridization analysis.............................................................. 41 3.3.1 Sampling for in situ hybridization .................................................................... 41 3.3.2 Hybridization conditions and probes description.............................................. 41 3.4 Phylogenetic analysis .................................................................................................... 42 4 RESULTS............................................................................................................................. 43 4.1 ARB database and Archaea trees reconstruction...................................................... 43 4.2 Phylogeny of Archaea from soil................................................................................... 47 4.3 Phylogeny of Archaea from estuarine sediment ........................................................ 48 v 4.4 Archaea from lake water.............................................................................................. 49 4.4.1 Phylogenetic analysis........................................................................................ 49 4.4.2 In situ hybridization analysis ............................................................................ 50 4.5 Diagnostic signature and feature analysis of the studied sequences ........................ 51 5 DISCUSSION ...................................................................................................................... 52 6 SUMMARY AND CONCLUSIONS.................................................................................. 54 7 TIIVISTELMÄ.................................................................................................................... 56 8 ACKNOWLEDGEMENTS................................................................................................ 57 9 REFERENCES....................................................................................................................59 vi LIST OF THE ORIGINAL PAPERS This thesis is based on the following articles, referred to in the text by their Roman numerals. Additionally, some unpublished results are presented. I. Jurgens, G., Lindström, K. and Saano, A. 1997. Novel group within kingdom Crenarchaeota from boreal forest soil. Applied and Environmental Microbiology, Vol. 63: 803-805. II. Jurgens, G. and Saano, A. 1999. Diversity of soil Archaea in boreal forest before, and after clear-cutting and prescribed burning. FEMS Microbiology Ecology, Vol. 29: 205- 213. III. Abreu, C., Jurgens, G., De Marco, P. , Saano, A. and Bordalo, A.A. 2001. Crenarchaeota and Euryarchaeota in temperate estuarine sediments. Journal of Applied Microbiology, Vol. 90: 713-718. IV. Jurgens, G., Glöckner, F.-O., Amann, R., Saano, A. Montonen, L., Likolammi, M. and Münster, U. 2000. Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization. FEMS Microbiology Ecology, Vol. 34: 45-56. Papers I to IV are reprinted with kind permission of the publishers. THE AUTHOR'S CONTRIBUTION I and II. German Jurgens planned and conducted the experiments, analyzed and interpreted the results as well as wrote the paper, under supervision of project leader Aimo Saano. Kristina Lindström acted as an expert in phylogenetic analysis for paper I. III. The work in this study was done in cooperation with Cristina Abreu (Portugal) while she was on Socrates scholarship in University of Helsinki during spring 1998. Cristina Abreu conducted the experiment and wrote the paper. German Jurgens planned and instructed the research, conducted the phylogenetic analysis and assisted in writing the paper. Aimo Saano, Paolo de Marco and Adriano Bordalo supervised the work. IV. German Jurgens planned and conducted the experiments, analyzed and interpreted the results and wrote the paper. Frank-Oliver Glöckner made the FISH picture and acted as FISH experiments adviser and ARB phylogenetic software expert. Markit Likolammi prepared bacteria biomass and bacteria production measurements and Leone Montonen performed PCR analysis with methyl-coenzyme M reductase primers. Rudolf Amann, Aimo Saano and project leader Uwe Münster supervised the work and assisted in writing. vii ABBREVIATIONS ARB from arbor, Latin: tree ATP adenosine triphosphate ATPase adenosine triphosphatase BAC bacterial artificial chromosome DAPI 4',6-diamidino-2-phenylindole