Genetic tools for the investigation of Roseobacter clade bacteria

<p>Abstract</p> <p>Background</p> <p>The <it>Roseobacter </it>clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools.</p> <p>Results</p> <p>Conjugation and electroporation methods for the efficient and stable genetic transformation of selected <it>Roseobacter </it>clade bacteria including <it>Dinoroseobacter shibae</it>, <it>Oceanibulbus indolifex</it>, <it>Phaeobacter gallaeciensis</it>, <it>Phaeobacter inhibens</it>, <it>Roseobacter denitrificans </it>and <it>Roseobacter litoralis </it>were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene <it>dnr </it>from <it>D. shibae </it>DFL12^T.</p> <p>Conclusion</p> <p>A genetic toolbox for members of the <it>Roseobacter </it>clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria.</p

Development of computational, mathematical and instrumental methods for the analysis of physiological processes in microbial populations

Die Fortschritte der modernen Mikrobiologie und die Verfügbarkeit neuer Hochdurchsatzmethoden liefern eine Fülle von Daten und Informationen. Durch geeignete Methoden und Modelle prozessiert, schaffen diese eine Basis für ein besseres Verstehen physiologischer Prozesse in mikrobiellen Populationen. Im ersten Teil dieser Arbeit wurde die Software eSOMet entwickelt. Sie ist ein Werkzeug zur Untersuchung metabolischer Profile mit dem Ziel diese ähnlichkeitsbasiert zu clustern. Da verschiedene Cluster unterschiedliche physiologische Zustände beschreiben, lassen sich damit diejenigen Metabolite identifizieren, deren Abundanzänderung für den Wechsel zwischen zwei Zuständen charakteristisch ist. Zum hochqualitativen Clustern wurde ein auf einer Hauptkomponentenanalyse basierter Rauschfilter und die Methode der emergenten selbstorganisierenden Karten implementiert. Am Beispiel verschiedener Kultivierungen von Corynbacterium glutamicum über mehrere Wachstumsphasen, konnte die Software erfolgreich evaluiert werden. Im zweiten Teil wurde ein spieltheoretisches Modell zur Untersuchung der Kulturheterogenität in mikrobiellen Populationen entwickelt. Als Heterogenitätskriterium wurde die Anzahl von Distribusomen definiert. Dies sind Zellbestandteile, deren Abundanz einen quantitativen Einfluss auf die Fitness der Subpopulation haben und die durch Zellteilung stochastisch weiterverteilt werden. Unter der Annahme, dass der Photosyntheseapparat von Dinoroseobacter shibae in solchen Distribusomen organisiert ist, konnte theoretisch gezeigt werden, dass sich unter fluktuierenden Umweltbedingungen oszillierend-stabile Subpopulationen ausbilden, die einen evolutionären Vorteil prägen. Um die theoretischen Ergebnisse des zweiten Teils experimentell zu verifizieren, wurde ein Kultivierungsverfahren im Mikrotitermaßstab entwickelt, welches vollautomatisiert durchgeführt eine in vivo Quantifizierung von Bacteriochlorophyll-a unter fluktuierenden Lichtbedingungen zuließ.The progress of modern microbiology and availability of novel high-throughput methods deliver a wealth of data and information. Processed by appropriate methods and models, they provide a basis for a better understanding of physiological processes in microbial populations. In the first part of this work the software eSOMet was developed. It is a tool for the investigation of metabolic profiles aiming to cluster those based on their similarity. Since distinct clusters are descriptors for different physiological states, this method allows the identification of metabolites, which changes of abundance are characteristic for the switch between two states. For high-quality clustering a principle component analysis based noise filter and the method of emergent selforganizing maps were implemented. The software could be successfully evaluated, using the example of different cultivations of Corynebacterium glutamicum over several growth phases. In the second part of this work, a game-theoretical model for the investigation of culture heterogeneity in microbial populations was developed. As heterogeneity criterion the number of distribusomes was defined. These are intracellular particles, which abundance influences the fitness of each subpopulation and which are distributed stochastically during cell division. Under the hypotheses, that the photosynthetic apparatus of Dinoroseobacter shibae is organized in such distribusomes it was shown in theory, that under fluctuating environmental conditions oscillating-stable fractions of subpopulations emerge, exhibiting an evolutionary advantage. In order to verify the theoretical results of the second part experimentally, a cultivation technique was developed at microtiter scale, which can be operated in a fully automated manner and allows for the in vivo quantification of Bchl-a under fluctuating light conditions

Microbial Communities Involved in Carbon Monoxide and Syngas Conversion to Biofuels and Chemicals

abstract: On average, our society generates ~0.5 ton of municipal solid waste per person annually. Biomass waste can be gasified to generate synthesis gas (syngas), a gas mixture consisting predominantly of CO, CO2, and H2. Syngas, rich in carbon and electrons, can fuel the metabolism of carboxidotrophs, anaerobic microorganisms that metabolize CO (a toxic pollutant) and produce biofuels (H2, ethanol) and commodity chemicals (acetate and other fatty acids). Despite the attempts for commercialization of syngas fermentation by several companies, the metabolic processes involved in CO and syngas metabolism are not well understood. This dissertation aims to contribute to the understanding of CO and syngas fermentation by uncovering key microorganisms and understanding their metabolism. For this, microbiology and molecular biology techniques were combined with analytical chemistry analyses and deep sequencing techniques. First, environments where CO is commonly detected, including the seafloor, volcanic sand, and sewage sludge, were explored to identify potential carboxidotrophs. Since carboxidotrophs from sludge consumed CO 1000 faster than those in nature, mesophilic sludge was used as inoculum to enrich for CO- and syngas- metabolizing microbes. Two carboxidotrophs were isolated from this culture: an acetate/ethanol-producer 99% phylogenetically similar to Acetobacterium wieringae and a novel H2-producer, Pleomorphomonas carboxidotrophicus sp. nov. Comparison of CO and syngas fermentation by the CO-enriched culture and the isolates suggested mixed-culture syngas fermentation as a better alternative to ferment CO-rich gases. Advantages of mixed cultures included complete consumption of H2 and CO2 (along with CO), flexibility under different syngas compositions, functional redundancy (for acetate production) and high ethanol production after providing a continuous supply of electrons. Lastly, dilute ethanol solutions, typical of syngas fermentation processes, were upgraded to medium-chain fatty acids (MCFA), biofuel precursors, through the continuous addition of CO. In these bioreactors, methanogens were inhibited and Peptostreptococcaceae and Lachnospiraceae spp. most likely partnered with carboxidotrophs for MCFA production. These results reveal novel microorganisms capable of effectively consuming an atmospheric pollutant, shed light on the interplay between syngas components, microbial communities, and metabolites produced, and support mixed-culture syngas fermentation for the production of a wide variety of biofuels and commodity chemicals.Dissertation/ThesisDoctoral Dissertation Civil, Environmental and Sustainable Engineering 201