Ludwig-Maximilians-Universität München Fakultät für Biologie CHARACTERIZATION OF THE SYMBIOTIC BACTERIAL PARTNERS IN PHOTOTROPHIC CONSORTIA Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades vorgelegt von Kristina Renate Pfannes Ludwig‐Maximilians‐Universität München Fakultät für Biologie Characterization of the symbiotic bacterial partners in phototrophic consortia Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades vorgelegt von Kristina Renate Pfannes aus Dettelbach München im Juli 2007 „WENN DU EIN SCHIFF BAUEN WILLST, DANN TROMMLE NICHT MÄNNER ZUSAMMEN, UM HOLZ ZU BESCHAFFEN, AUFGABEN ZU VERGEBEN UND DIE ARBEIT EINZUTEILEN, SONDERN LEHRE SIE DIE SEHNSUCHT NACH DEM WEITEN, ENDLOSEN MEER” ANTOINE DE SAINT-EXUPERY FRZ. FLIEGER & SCHRIFTSTELLER (1900-1944) 1. Gutachter: Prof. Dr. Jörg Overmann, LMU München 2. Gutachterin: Prof. Dr. Kirsten Jung, LMU München Tag der Abgabe: 31.07.2007 Tag des Promotionskolloquiums: 22.11.2007 PUBLICATIONS ORIGINATING FROM THIS THESIS CHAPTER 3 Kanzler BEM, Pfannes KR, Vogl K, Overmann J (2005) Molecular characterization of the nonphotosynthetic partner bacterium in the consortium “Chlorochromatium aggregatum”. Appl Environ Microbiol 71:7434‐7441 CHAPTER 4 Pfannes KR, Vogl K, Overmann J (2007) The heterotrophic symbionts of phototrophic consortia belong to a highly diverse subgroup of Betaproteobacteria characterized by a tandem rrn operon structure. Environ Microbiol 9:2782‐2794 CHAPTER 5 Vogl K, Glaeser J, Pfannes KR, Wanner G, Overmann J (2006) Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium “Chlorochromatium aggregatum”. Arch Microbiol 185:363‐372 CHAPTER 6 Pfannes KR, Müller JF, Zikeli K, Bayer AI, Vogl K, Wenter R, Hütz KA, Grossart H‐P, Sandmann G, Takaichi S, Overmann J (2007) Oxygen tolerance and physiological properties of symbiotic and free‐living green sulfur bacteria. (manuscript) CONTRIBUTIONS CHAPTER 3 The chemotaxis experiments were performed by Birgit Kanzler. She also did the in situ hybridization and the phylogenetic analyses under supervision of Kajetan Vogl. Kristina Pfannes established the enrichment and isolation of genomic DNA of the central bacterium via CsCl equilibrium density gradient centrifugation, determined the mol% G+C content and performed the real‐time PCR. CHAPTER 4 Kristina Pfannes performed the amplification and cloning of the rRNA operon. Moreover, she did the screening and analysis of the natural bacterial communities and conducted the in situ hybridization as well as the phylogenetical and statistical analyses. Kajetan Vogl developed the new cultivation approach of consortia cultures. CHAPTER 5 The epibiont was isolated and phylogenetically analysed by Kajetan Vogl in cooperation with Jens Glaeser. Kajetan Vogl also did the substrate test and the growth experiments (temperature optimum, light intensity optimum, pH optimum) and the pigment analyses. Prof. Dr. Gerhard Wanner took the electron microscopy pictures. The contributions of Kristina Pfannes were the determination of the cell surface hydrophobicity and the salinity optimum, the pigment extraction, the KOH‐string test and the Gram‐staining. CHAPTER 6 Kristina Pfannes, Kerstin Zikeli and Johannes Müller conducted the oxygen tolerance test. The enzyme assays were established and performed by Kristina Pfannes and Johannes Müller. Roland Wenter did the RT‐PCR. Kristina Pfannes performed the genome analyses and the phylogenetic studies. Also, she prepared the samples for the analysis of the carotenoids and the determination of the amino acid and carbohydrate excretion. Prof. Dr. Gerhard Sandmann and Prof. Dr. Shinichi Takaichi analysed the carotenoids. Kerstin Zikeli and Katharina Hütz were responsible for the chlorosome analysis. The carbohydrate excretion and the amino acid excretion was measured by Dr. Hans‐Peter Grossart and Prof. Dr. Meinhard Simon, respectively. Anne Bayer did the amino acid utilization experiment. I hereby confirm the above statements Kristina Pfannes Prof. Dr. Jörg Overmann TABLE OF CONTENTS I. ABBREVIATIONS.....................................................................................................................1 1 Summary.....................................................................................................................................1 2 Introduction ...............................................................................................................................3 2.1 Diversity of Symbioses ...........................................................................................................3 2.2 Significance of Prokaryotic Symbioses.................................................................................4 2.3 Photosynthesis and Phototrophy in Bacteria.......................................................................8 2.4 Green Sulfur Bacteria..........................................................................................................10 2.5 Phototrophic Consortia........................................................................................................13 2.6 "Chlorochromatium aggregatum" as a Model Organism..................................................18 2.7 Aim of this Work..................................................................................................................20 2.8 References .............................................................................................................................23 3 Molecular characterization of the non‐photosynthetic partner bacterium in the consortium “Chlorochromatium aggregatum”...................................................................35 3.1 Abstract.................................................................................................................................35 3.2 Introduction ..........................................................................................................................36 3.3 Material and Methods..........................................................................................................37 3.3.1 Source of organisms ........................................................................................................................ 37 3.3.2 Media and growth conditions .......................................................................................................... 37 3.3.3 Enrichment by chemotaxis .............................................................................................................. 38 3.3.4 PCR ................................................................................................................................................. 39 3.3.5 Quantitative PCR............................................................................................................................. 40 3.3.6 Denaturing gradient gel electrophoresis (DGGE)............................................................................ 40 3.3.7 Cloning ............................................................................................................................................ 40 3.3.8 Sequencing ...................................................................................................................................... 41 3.3.9 Phylogenetic analyses...................................................................................................................... 41 3.3.10 Fluorescence in situ hybridization (FISH) .................................................................................. 42 3.3.11 CsCl – bisbenzimidazole density gradient centrifugation ........................................................... 42 3.3.12 Accession numbers...................................................................................................................... 43 TABLE OF CONTENTS 3.4 Results and Discussion ........................................................................................................ 45 3.4.1 Improved chemotactic enrichment of intact phototrophic consortia................................................45 3.4.2 Analysis of the 16S rRNA gene sequence of the central bacterium of "C. aggregatum" ................46 3.4.3 Phylogenetic classification of the central rod ..................................................................................49 3.4.4 GC-content and enrichment of genomic DNA of the central bacterium..........................................50 3.4.5 Relevance for future studies.............................................................................................................53 3.5 Acknowledgements .............................................................................................................. 54 3.6 References ............................................................................................................................ 54 4 The heterotrophic symbionts of phototrophic consortia belong to a highly diverse subgroup of Betaproteobacteria characterized by a tandem rrn operon structure....... 61 4.1 Abstract ................................................................................................................................ 61 4.2 Introduction ......................................................................................................................... 62 4.3 Materials and Methods ....................................................................................................... 64 4.3.1 Source of organisms