Interactions between Diatoms and Bacteria from phototrophic Biofilms of the littoral Zone of Lake Constance
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Zusammenfassung
In this study we developed methods to purify diatoms from associated bacteria. Therefore most diatoms could be purified by short term harsh antibiotics treatment followed by single cell care. Diatoms that are difficult to purify could be made axenic via an intermediate defined co-culture with Escherichia coli. Phylogenetic analysis via 16S rRNA gene profiling of diatom associated bacteria indicated that Alphaproteobacteria and Bacteroidetes have adapted to the micro environment diatom biofilm. Defined co-cultures of diatoms and bacteria support this hypothesis by indicating that different bacteria utilize different fractions of secreted diatom carbohydrates. Bacteria were found to influence diatom growth strongly, either direct or by constitutively released soluble substances. We found concentrations of dissolved free amino acids (DFAA), analyzed by HPLC via ortho-phthaldialdehyde derivatization, to correlate with diatom growth when influenced by bacteria, and thus postulate that DFAA may be one class of such substances, regulating growth rate and growth density of diatoms. Further bacteria have a strong influence on the secretion of extracellular polymeric substances (EPS) by the diatoms. Investigating diatom carbohydrates by HPAE-PAD and spectrophotometric assays revealed that diatom carbohydrate secretion is influenced predominately quantitatively by bacteria. By quantifying diatom protein secretion and separating diatom proteins via SDS-PAGE we found diatom protein secretion to be influenced qualitatively and quantitatively by bacteria. The model organisms Phaeodactylum tricornutum and Escherichia coli were used to identify via Maldi-tof mass spectrometry peptide mass fingerprinting extracellular bacteria and diatom proteins that are induced during biofilm formation as an interaction of these organisms. These proteins could be related to different functions that are thought to play an important role during this interaction.
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Im Rahmen dieser Arbeit wurden systematisch Methoden entwickelt, um Diatomeen von assoziierten Bakterien zu reinigen. Die meisten Kieselalgen konnten dabei durch kurzzeitige Behandlung mit hochkonzentrierten Antibiotika-Kombinationen von Bakterien befreit werden, wobei in schwierigen Fällen eine intermediäre Escherichia coli Co-Kultur hilfreich war. Phylogenetische Studien indizieren, dass Alphaproteobakteria und Bacteroidetes sich an den Mikrolebensraum Kieselalgenbiofilm anpassen konnten. Definierte Co-Kulturen aus Diatomeen und Bakterien unterstützen diese Hypothese, da verschiedene dieser Bakterien unterschiedliche Fraktionen der von den Kieselalgen sekretierten Kohlenhydrate verstoffwechselten. Ferner konnte gezeigt werden, dass Bakterien das Wachstum der Diatomeen stark beeinflussen, entweder direkt oder über konstitutiv freigesetzte lösliche Faktoren. Mittels HPLC-Quantifizierung über Ortho-Phthaldialdehyd Derivatisierung freier löslicher Aminosäuren (DFAA) fanden wir Korrelationen zwischen DFAA Konzentrationen und Diatomeenwachstum unter bakteriellem Einfluss. DFAA könnten daher eine Substanzklasse darstellen, die einen regulativen Einfluss auf Kieselalgen ausübt und Wachstumsgeschwindigkeit und Wachstumsdichte der Diatomeen steuert. Weiterhin üben Bakterien einen starken Einfluss auf die Sekretion extrazellulärer polymerer Substanzen durch die Kieselalgen aus: mittels spektrophotometrischer Quantifizierung und HPAE-PAD fanden wir Diatomeenkohlenhydratsekretion vor allem quantitativ beeinflusst, Proteinsekretion aber quantitativ wie qualitativ. Die Modellorganismen Phaeodactylum tricornutum und Escherichia coli wurden verwendet um mittels Maldi-tof Massenspektrometrie-Peptidmassenabgleich Proteine zu identifizieren, welche bei der Biofilmbildung als Funktion der Wechselwirkung zwischen den Organismen induziert werden. Diese Proteine konnten umfassenden Funktionen zugeordnet werden und indizieren dadurch wichtige Vorgänge während der Biofilmbildung durch Kieselalgen und Bakterien.
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BRUCKNER, Christian G., 2008. Interactions between Diatoms and Bacteria from phototrophic Biofilms of the littoral Zone of Lake Constance [Dissertation]. Konstanz: University of KonstanzBibTex
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