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Interspecies Interaction and Diversity of Green Sulfur Bacteria

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Interspecies Interaction and Diversity of Green Sulfur Bacteria Interspecies interaction and diversity of green sulfur bacteria Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Johannes Friedrich Müller am 10 Juli 2012 1. Gutachter: Prof. Dr. Jörg Overmann, DSMZ Braunschweig 2. Gutachter: Prof. Dr. Anton Hartmann, LMU München Tag des Promotionskolloquiums: 11.12.2012 „Wer sich Steine zurechtlegen kann, über die er stolpert, hat Erfolg in den Naturwissenschaften.“ Erwin Chargaff, österreichisch.-amerikanischer Biochemiker u. Schriftsteller (1905 - 2002) Publications originating from this thesis Publication 1: Müller, J.F., Wenter, R., Manske, A.K., Sikorski, J., Teeling, H., Garcia-Gil, L.J., Overmann, J. (2012) Biodiversity and phylogeny of the family Chlorobiaceae based on analyses of different genomic regions. (manuscript) Publication 2: Müller, J.F., Overmann, J. (2011) Close interspecies interactions between prokaryotes from sulfureous environments. Front Microbiol 2:146. (review) Publication 3: Müller, J.F., Eisenreich, W., Bunk, B., Liu, Z., Henke, P., McGlynn, S., Wenter, R., Orphan, J.V., Bryant, D.A., Overmann, J. (2012) The complex exchange of metabolites in the phototrophic consortium "Chlorochromatium aggregatum". (manuscript) Contributions of Johannes Müller to this thesis Johannes Müller cultivated the consortia enrichment and the Chlorobium chlorochromatii CaD3T cultures used. He established and performed the magnetic capture experiments. Johannes Müller prepared the samples for isotopologue analysis by incubating the cultures and separating the consortia by cesium density gradient centrifugation. The subsequent analyses were conducted by Dr. Wolfgang Eisenreich. Johannes Müller prepared the central bacterium DNA used for genome analysis carried out by Prof. Dr. Donald A. Bryant and Dr. Zhenfeng Liu by separating the central bacterium from the epibiont by cesium density gradient centrifugation and extracting the DNA. He prepared the cDNA utilized for transcriptome analysis. Bioinformatic analysis of the results was performed together with Boyke Bunk who also processed the transcriptome raw data. Johannes Müller performed the fluorescence recovery after photobleaching (FRAP) analysis with the aid of Andreas Binder. Petra Henke performed the NanoSIMS analysis, Roland Wenter the amino acid excretion profile of the epibiont. Johannes Müller and Jörg Overmann wrote the review. Johannes Müller calculated and constructed the 16S rRNA gene tree of green sulfur bacteria. The phylogenetic trees for the ITS region, the bchg, sigA and fmoA gene sequences of green sulfur bacteria were constructed using the pre-alignments of Ann Manske and Roland Wenter as base material. Johannes Müller also calculated and constructed the concatenated phylogenetic tree. He prepared the sequence alignments for the distance matrix comparison and the pair-wise sequence dissimilarity analysis and calculation of ka and ks values. The respective calculations and figures were supplied by Johannes Sikorski. Johannes Müller performed the coverage and diversity estimates and constructed the world map containing the sampling sites of the sequences used in the 16S rDNA tree. I herby confirm the above statements ________________________ ________________________ Johannes Müller Prof. Dr. Jörg Overmann Contents Chapter 1: Summary………….………….………….………….………….………….……..1 Chapter 2: Introduction………….………….………….………….………….……………..5 2.1 Green sulfur bacteria…………………………………………………………….....5 2.2 Phylogeny of green sulfur bacteria………………………...……………………….6 2.3 Niche formation by biotic interaction…………………….…….………………….9 2.4 Characterization of phototrophic consortia …………………….……………...…11 2.5 "Chlorochromatium aggregatum" as a model system for symbiotic niche formation…………………….…………………….……………………………….…14 2.6 Aims of this study…………………….…………………….…………………….17 Chapter 3: Experimental procedures…………………….……………………...…………24 3.1 Experimental procedures for phylogenetic analysis of green sulfur bacteria…….24 Cultures of green sulfur bacteria …………………….………………………24 DNA-extraction from cultures and water samples ………………..……….…24 Standard conditions for PCR…………………….………………………...….24 Amplification of 16S rRNA gene sequences………….………….……...…….25 PCR and cloning of the 16S-23S rRNA intergenic spacer (ITS) region………27 Primer design and PCR for group 1 70-type sigma factor gene sequences (sigA) ………….………….………….………….……………...…27 BchG gene sequences………….………….………….………….………...….28 FmoA gene sequences………….………….………….………….……...…….28 Sequencing of PCR products………….………….………….………….….…28 Phylogenetic trees………….………….………….………….……………….28 Concatenated phylogenetic tree………….………….………….………….…29 Distance matrix comparisons (Mantel test) ………….………….…..……….29 Coverage and diversity estimates………….………….………….……...……30 Pair-wise sequence dissimilarity analysis and calculation of ka and ks values………….………….………….………….………….…………………30 Map of sampling sites………….………….………….………….……………30 Nucleotide accession numbers………….………….………….………..…….31 3.2 Experimental procedures to investigate the molecular basis of the interspecies interaction in “C. aggregatum” …………………….………...……31 Bacterial cultures and growth conditions…………………….………………31 Probes…………………….…………………….…………………………….31 Nanoscale secondary ion mass spectrometry (NanoSIMS) …………………32 Magnetic bead separation of 14C - labeled RNA…………………………….33 Cell counting…………………….…………………….……………….…….35 Fluorescence-Recovery-After-Photobleaching (FRAP)-analysis……………36 Sample preparation for Isotopolomics…………………….…………………36 Hydrolysis of cellular protein and silylation of the resulted amino acids......37 Gas chromatography - mass spectrometry (GC/MS) ………………………..37 Genome analysis…………………….…………………….…………….……39 In silico subtractive hybridization …………………….…………………...…39 Transcriptome analysis…………………….…………………….……...……40 HPLC analysis of amino acid excretion…………………….…………...……41 Chapter 4: Results………….………….………….………….………….………….….……47 4.1 Results of the phylogenetic analysis………….………….………….………...….47 16S rRNA gene sequence tree of GSB ………….………….……………...….47 Comparison of the concatenated tree with the 16S rDNA gene tree………….48 Distance matrix comparisons (Mantel test) ………….………….………..….51 Coverage and diversity estimates………….………….………….………..….51 Pair-wise sequence dissimilarity analysis and calculation of ka and ks values………….………….………….………….………….………...…....52 World map of sampling sites………….………….………….………….….…54 4.2 Results for the investigation of the molecular basis of the interspecies interaction in “C. aggregatum” ………….………….………….………………...56 Transfer of 14C with magnetic bead capture………….………….……...……56 NanoSIMS analysis ………….………….………….………….………..…….60 Isotopolome analysis………….………….………….………….…………….60 Excretion of amino acids by epibiont cells………….…………..….…………64 Fluorescence-Recovery-After-Photobleaching (FRAP) analysis…………..…65 Genome analysis………….………….………….………….…………………66 In silico subtractive hybridization ………….………….………….……….…76 Transcriptome analysis 77 Chapter 5: Discussion……………………………………………………………………….88 5.1 Phylogeny of the green sulfur bacteria………….…………………….……….….88 5.1.1 Diversification of green sulfur bacteria………….……………….….………….90 5.1.2 Analysis of the marker genes and the ITS region…………...………….………93 5.1.3 Future sequencing of green sulfur bacterial strains………….…………….……95 5.2 Molecular basis of the symbiosis in the phototrophic consortium “C. aggregatum” ………….………….………….………….………….………..……96 5.2.1 Transfer of metabolites from the epibiont to the central bacterium…………….96 5.2.2 Mechanisms of metabolite exchange………….………….………………..….101 5.2.3 Dependence of the central bacterium on the epibiont………….……..……….103 5.2.4 Evidence of metabolite transfer from the central bacterium to the epibiont…..105 5.2.5 Implications for the origin of phototrophic consortia………….…………..….105 5.2.6 Features of the epibiont that relate to symbiosis………….………………..….107 5.3 Conclusions………….………….………….………….………….………..……111 Ι. Danksagung ………….………….………….………….………….………….…….……123 II. Curriculum vitae ………….………….………….………….………….…………...….125 III. Supplementary material………….………….………….………….……….…...……127 Summary Chapter 1 Chapter 1 Summary The following work is shedding light on the phylogenetic classification on the family of the Chlorobiacea, the members of which are showing signs of preadaptation to symbiosis. Symbioses consisting of purely prokaryotic associations between phylogenetically distinct bacterial species have been widely documented. Only few are available as a laboratory culture to elucidate the molecular basis of their interaction. One of these few model organisms is the phototrophic consortium “Chlorochromatium aggregatum”. It consists of 12-20 green sulfur bacteria epibionts surrounding a central, Betaproteobacterium in a highly ordered fashion. The phototrophic partner bacterium, belonging to the green sulfur bacteria, is available in pure culture and its physiology has been studied in detail. In this work, novel insights into the physiology of the central bacterium that was previously uncharacterized are provided. The family of the Chlorobiaceae represents a phylogenetically coherent and isolated group within the domain Bacteria. Green sulfur bacteria are obligate photolithoautotrophs that require highly reducing conditions for growth and can utilize only a very limited number of carbon substrates. These bacteria thus inhabit a very narrow ecologic niche. For the phylogenetic studies on green sulfur bacteria, 323 16S rRNA gene sequences, including cultured species as well as environmental sequences were analysed. By rarefaction analysis and statistical projection, it
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