Molecular Control of Extracellular DNA Release and Degradation in Shewanella oneidensis MR-1 Biofilms: The Role of Phages and Nucleases

Bakterien bilden unter natürlichen Bedingungen häufig oberflächen-assoziierte multizelluläre Gemeinschaften, welche allgemein als Biofilme bezeichnet werden. Die Bildung von Biofilmen ist ein komplexer und präzise regulierter Prozess, der es Bakterien ermöglicht, beinahe jede Art von Oberfläche zu besiedeln und dadurch physikalischen Stressfaktoren, Nährstoffmangel und Antibiotika standzuhalten. Des Weiteren kann oberflächenassoziiertes Wachstum die Virulenz von pathogenen Bakterien erhöhen und Umweltkeimen die Erschließung von Oberflächen als Nährstoff- und Energiequelle ermöglichen. Aus diesem Grund hat sich gezeigt, dass bakterielle Biofilmbildung von großer medizinischer, ökologischer und ökonomischer Relevanz ist. Ein wichtiger Bestandteil von Biofilmen ist die extrazelluläre polymere Matrix welche sich typischerweise aus Exopolysacchariden, Proteinen und extrazellulärer DNA (eDNA) zusammensetzt. Die Bedeutung der eDNA für Biofilme war lange unklar, jedoch konnte durch eine Reihe von Studien gezeigt werden, dass eDNA für die meisten Bakterienspezies, darunter Shewanella oneidensis MR-1, von essentieller Bedeutung für die strukturelle Entwicklung der Biofilme ist. Vielfach unbekannt sind jedoch Mechanismen, welche die Freisetzung von eDNA regulieren bzw. ausführen und solche, die an der Modulation und am Abbau (z.B. zur endogen induzierten Auflösung von Biofilmen oder zur Erschließung von eDNA als Nährstoffquelle) beteiligt sind. In der vorliegenden Arbeit wurde diese Mechanismen molekularbiologisch, mikroskopisch und biochemisch untersucht

Integrating Mass Spectrometry Based Proteomics and Bioinformatics Technologies for the Molecular Level Characterization of \u3cem\u3eShewanella oneidensis\u3c/em\u3e to Chromate Exposure

The research outlined in this dissertation involves the development and demonstration of a mass spectrometry-based proteomics approach to characterize the global level molecular response of Shewanella oneidensis MR-1 to chromate exposure. The proteomics approach is centered on a high performance technique of multidimensional on-line liquid chromatographic separations with subsequent tandem mass spectrometric detection. Since very complex proteome samples are digested into peptides and then directly measured by MS, this technique is termed shotgun proteomics. This approach affords the identification and quantification of complex mixtures by directly analyzing their proteolytic peptides and then using computational techniques to reassemble the protein information. The research goals for this dissertation project were two-fold: (1) enhancement of the experimental and computational methodologies to permit deeper and more confident proteome characterizations, and (2) demonstration of this optimized approach for the comprehensive investigation of the molecular level response of the bacterium S. oneidensis to chromate insult. To address research needs, we developed a single-tube lysis method for cell lysis-proteome digestion to enable investigations of small amounts of cellular biomass, and identified suitable bioinformatic approaches to mine post-translational modifications from proteome datasets. These advancements were then utilized to examine the molecular level response of S. oneidensis to chromate insult, which was accomplished by varying chromate concentrations, dosages, and time points. These measurements provided the first global proteome-level observation of the dynamic changes of S. oneidensis in response to chromate insult

Steering biogas performance by implementation of bioelectrochemical cell (BEC) technology

A new concept for integrating conventional anaerobic digester (AD) with bioelectrochemical cell (BEC) technology was investigated in the current study. The BEC technology can convert energy stored in organic matter directly into bioelectricity. Coupling AD with BEC could be a profitable approach that could lead to overcoming limiting factors in AD, such as hydrogen partial pressure and accumulation of volatile fatty acids, inhibiting the methanogenesis

Cross-talk between Dps proteins triggers manganese distribution as a defense strategy against oxidative stress

"Deinococcus radiodurans is a radiation resistant bacterium. For this reason, it has been a focus of several studies over the years. The aim has been to understand what makes this organism so resistant to different extreme conditions. Several protection mechanisms are present, such as enzymatic and non-enzymatic systems, namely Mn2+-Pi complexes. These mechanisms work synergistically, thereby conferring higher protection to this extraordinary organism.(...)

07. Environmental Implications of Francisella Tularensis Biofilms

https://digitalcommons.imsa.edu/class_of_2000/1007/thumbnail.jp

Environmental Implications of Francisella Tularensis Biofilms

Francisella tularensis survives in one of the widest environmental ranges of any pathogen. Numerous mammals and arthropod vectors are infected by this highly virulent organism. How this zoonotic pathogen persists outside of its many hosts remains unexplored. We aimed to examine how F. tularensis interacts with environmental surfaces, and hypothesized that biofilm formation may enable survival of this organism in nature. By understanding the role these surface-attached bacterial communities play in F. tularensis ecology, we hope to gain insight into the mechanisms of environmental persistence and transmission of this pathogen. We identify chitin as a potential non-host niche for F. tularensis in nature using genetic, microscopic, and biochemical techniques. This abundant polysaccharide supported F. tularensis biofilm formation in the absence of an exogenous carbon source. This interaction was dependent on putative chitinase enzymes which hydrolyze the glycosidic bonds that connect GlcNAc monomers. Using a genetic screen, we identified adherence factors, including FTN_0308 and FTN0714 that promote attachment to chitin and colonization of chitin surfaces. We propose that biofilm formation on chitin surfaces in nature enables nutrient scavenging in oligotrophic environments allowing this pathogen to replicate and seed disease transmission. We found that the effect of nutrient limitation on F. tularensis biofilm formation extended beyond chitin utilization. Genetic studies indicated that nutrient starvation triggers a biofilm stress response. We identified static growth and nutrient deprivation as cues for enhanced biofilm formation. Microarray expression studies identified genes highiy expressed under these conditions, including F. tularensis biofilm determinants. Expression of nutrient transporters further indicated that biofilm formation promotes environmental persistence. We finally examined statically grown F. tularensis microscopically to determine if altered morphology explained the enhanced biofilm phenotype of these cultures. We discovered a novel F. tularensis appendage conserved between subspecies and structurally homologous to the Caulobacter crescentus stalk. These structures were observed in association with surfaces during both biofilm formation and during intracellular infection. A genetic screen for mutants in stalk formation revealed that stalk biosynthetic components are essential. We predict this structure aids in environmental persistence by facilitating surface attachment and nutrient uptake. Through this collective work we define evidence that surface association via biofilm formation promotes survival during nutrient limitation

Regulation of Acetyl Phosphate-Dependent Acetylation and Identification of Novel Lysine Acetyltransferases in Escherichia Coli

Over billions of years, organisms have organized chemical reactions into metabolic pathways to sustain life. However, metabolic substrates can undergo many uncatalyzed, extra-metabolic reactions. Acetyl phosphate (AcP), an intermediate of the acetate fermentation pathway in E. coli, is one such metabolite that has been shown to non-enzymatically acetylate hundreds of proteins. This diverse set of targets suggests that acetylation could be a way for the cell to sense its nutritional status and regulate protein activity accordingly. However, how E. coli regulates acetylation, if at all, is unknown.Previous work showed that acetylation becomes pronounced in stationary phase cells. I determined that acetylation only increases once the culture enters stationary phase and continues to accumulate until carbon source depletion. Mass spectrometry indicated that the accumulation results from both additional lysine residues becoming acetylated and an increased ratio of acetylated to unacetylated isoforms. I performed anti-acetyllysine western blot analysis and metabolite measurements that suggested acetylation does not accumulate during exponential phase because 1) acetylated isoforms are diluted into nascent proteins and 2) glucose is not consumed until stationary phase. Additionally, I found that acetylation required rapid flux of carbon into the cell and through glycolysis. These data suggest that AcP-dependent acetylation is an unavoidable consequence of fermentation. I hypothesized that there is a physiological relevance to AcP-dependent acetylation such as utilization of acetylation as a carbon source or as protection against non-enzymatic protein damage, but I was unsuccessful in showing any effect.In addition to non-enzymatic acetylation, one lysine acetyltransferase (KAT), YfiQ, had been described in E. coli. I recognized that acetylation could occur without AcP or YfiQ. Indeed, I identified four proteins that produced acetylated bands in a strain lacking both mechanisms of acetylation. Variants of these proteins with conserved catalytic residues changed to alanine did not produce these acetylated bands, consistent with these proteins having KAT activity. One of these KATs, YiaC, inhibited migration in soft agar dependent on its KAT activity. To determine targets of these KATs, we determined proteins whose acetylation increased upon overexpresson of each of these proteins. Thus, this work opens new avenues of study to determine the regulatory potential of acetylation by these KATs

The self-organizing fractal theory as a universal discovery method: the phenomenon of life

A universal discovery method potentially applicable to all disciplines studying organizational phenomena has been developed. This method takes advantage of a new form of global symmetry, namely, scale-invariance of self-organizational dynamics of energy/matter at all levels of organizational hierarchy, from elementary particles through cells and organisms to the Universe as a whole. The method is based on an alternative conceptualization of physical reality postulating that the energy/matter comprising the Universe is far from equilibrium, that it exists as a flow, and that it develops via self-organization in accordance with the empirical laws of nonequilibrium thermodynamics. It is postulated that the energy/matter flowing through and comprising the Universe evolves as a multiscale, self-similar structure-process, i.e., as a self-organizing fractal. This means that certain organizational structures and processes are scale-invariant and are reproduced at all levels of the organizational hierarchy. Being a form of symmetry, scale-invariance naturally lends itself to a new discovery method that allows for the deduction of missing information by comparing scale-invariant organizational patterns across different levels of the organizational hierarchy

Computational approaches to complex biological networks

The need of understanding and modeling the biological networks is one of the raisons d'\ueatre and of the driving forces behind the emergence of Systems Biology. Because of its holistic approach and because of the widely different level of complexity of the networks, different mathematical methods have been developed during the years. Some of these computational methods are used in this thesis in order to investigate various properties of different biological systems. The first part deals with the prediction of the perturbation of cellular metabolism induced by drugs. Using Flux Balance Analysis to describe the reconstructed genome-wide metabolic networks, we consider the problem of identifying the most selective drug synergisms for given therapeutic targets. The second part of this thesis considers gene regulatory and large social networks as signed graphs (activation/deactivation or friendship/hostility are rephrased as positive/negative coupling between spins). Using the analogy with an Ising spin glass an analysis of the energy landscape and of the content of \u201cdisorder\u201d 'is carried out. Finally, the last part concerns the study of the spatial heterogeneity of the signaling pathway of rod photoreceptors. The electrophysiological data produced by our collaborators in the Neurobiology laboratory have been analyzed with various dynamical systems giving an insight into the process of ageing of photoreceptors and into the role diffusion in the pathway