RNA based research - development, application and analysis within the MIMAS project by Dipl. Biol. Anna Klindworth A thesis submitted in partial fulfilment of requirements for the degree of DOCTOR OF PHILOSOPHY in Biology Approved Thesis Committee: Prof. Dr. Frank Oliver Glöckner (chair) Max Planck Institute for Marine Microbiology, Bremen, Germany Jacobs University, Bremen, Germany Prof. Dr. Matthias Ullrich Jacobs University, Bremen, Germany Prof. Dr. Jack Gilbert University of Chicago, USA Argonne National Laboratory Date of Defense: October, 08th 2012 School of Engineering and Science Statement of Sources DECLARATION I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from published or unpublished scientifc work has been cited in the text and listed in the references. Signature Date I Thesis abstract Every prokaryotic cell contains different sorts of ribonucleic acid (RNA) molecules, which are mainly dedicated to processing and regulating gene expression. The wide palette of functions is reflected by distinct RNA types, and each of them is represented by a complex and comprehensive research field. In particular, different culture-independent applications have attracted attention recently because the majority of microbes still resists cultivation until today. For example, the standard approach for microbial diversity studies is based on the comparative analysis of the evolutionarily conserved 16S ribosomal RNA gene (16S rDNA), and messenger RNA (mRNA) based metatranscriptomics allows culture independent gene expression analysis without prior knowledge of the present microbes or transcripts. 16S rDNA biodiversity studies, metatranscriptomics and other ‘omic’ applications play a central role within the MIMAS project, which aims at characterizing a bacterioplankton community at the long-term ecological research site Helgoland Roads. However, culture- independent applications have their limitations, and a careful design of experimental procedures is crucial to assure that these limitations do not overtly bias the results. Therefore, this thesis outlines the development and application of an improved pipeline for the analysis of metatranscriptomic data and the evaluation of PCR primers used to amplify 16S rRNA. In particular, the outcome of the latter serves as a guideline for enhanced research to find the most suitable primer pair for 16S rDNA biodiversity analysis in any habitat using any currently available sequencing technology. The methods developed were used in a multi ‘omic’ study to characterize the phylogenetic and functional potential of the microbial community. The results identified the key players of an observed bacterioplankton bloom at Helgoland Roads and provided the first insights into taxonomically distinct nutrient strategies. They indicated that Flavobacteria, Gammaproteobacteria and Alphaproteobacteria are specialized for successive degradation of different algal primary products. This provided a series of ecological niches, allowing certain community members to grow. The results helped to uncover the secret of how members of the bacterioplankton can evade extinction despite the limited resources in the habitat. The work accomplished allows future follow-up studies and furnishes scientific society with guidelines to perform accurate diversity studies. Moreover the outcome serves as basis for future ecosystem monitoring. II III Table of contents Statement of Sources ................................................................................................................... I Thesis abstract ........................................................................................................................... II Table of contents ....................................................................................................................... II List of abbreviations ................................................................................................................. VI List of figures ........................................................................................................................... IX List of tables .......................................................................................................................... XIII 1. Chapter .............................................................................................................................. 1 Introduction 1.1. The multiple facets of the RNA ...................................................................................... 1 1.2. 16S rDNA based research ............................................................................................... 4 1.2.1. 16S rDNA as a marker gene ........................................................................................ 4 1.2.2. Sequencing of the 16S rDNA gene .............................................................................. 6 1.2.3. Limitations of PCR based 16S rDNA analysis ............................................................ 8 1.3. mRNA based gene expression analysis in prokaryotes ................................................... 9 1.3.1. Metatranscriptomics in prokaryotes ............................................................................ 9 1.3.2. Limitations of metatranscriptomics ........................................................................... 11 II 1.4. DNA Sequencing technologies ..................................................................................... 13 1.4.1. Next generation sequencing technologies ................................................................. 13 1.5. The MIMAS project ...................................................................................................... 18 1.5.1. Aims of the MIMAS project (the big picture) ........................................................... 18 1.5.2. Project partner and contributions .................................................................................. 18 1.6. Helgoland Roads: one sampling site of choice within the MIMAS project.................. 20 1.7. Rhodopirellula baltica SH1T: one model organism within the MIMAS project .......... 21 1.8. Research aims and thesis structure ................................................................................ 22 1.9. Evaluation of 16S rDNA primer and primer pairs ........................................................ 22 1.10. NGS based 16S rDNA analysis – proof of concept ...................................................... 22 1.11. Functional analysis the of bacterial community at Helgoland Roads in the North Sea 23 1.12. Impact from pure culture studies ................................................................................... 23 1.13. Publication overview ..................................................................................................... 25 2. Chapter ......................................................................................................................... 28 Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next generation sequencing based diversity studies 2.1. Abstract ......................................................................................................................... 28 2.2. Introduction ................................................................................................................... 29 2.3. Material and Methods.................................................................................................... 30 2.4. Results and Diskussion.................................................................................................. 35 2.5. Conclusion ..................................................................................................................... 47 III 3. Chapter ............................................................................................................................ 50 Substrate-controlled succession of marine bacterioplankton populations induced by phytoplankton bloom 3.1. Abstract ......................................................................................................................... 50 3.2. Manuscript ..................................................................................................................... 51 4. Chapter ............................................................................................................................ 60 Comparative metatranscriptome analysis of a diatome-induced bacterioplankton North Sea bloom 4.1. Abstract ......................................................................................................................... 60 4.2. Introduction ................................................................................................................... 60 4.3. Materials and Methods .................................................................................................. 62 4.4. Results and Discussion .................................................................................................. 67 4.5. Conclusion ..................................................................................................................... 76 5. Chapter ......................................................................................................................... 80 Expression of sulfatases in Rhodopirellula baltica and the diversity of sulfatases