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The past decades are characterized by various efforts to provide complete sequence information of genomes regarding various organisms. The availability of full genome data triggered the development of multiplex high-throughput assays allowing simultaneous measurement of transcripts, proteins and metabolites. With genome information and profiling technologies now in hand a highly parallel experimental biology is offering opportunities to explore and discover novel principles governing biological systems. Understanding biological complexity through modelling cellular systems represents the driving force which today allows shifting from a component-centric focus to integrative and systems level investigations. The emerging field of systems biology integrates discovery and hypothesis-driven science to provide comprehensive knowledge via computational models of biological systems. Within the context of evolving systems biology, investigations were made in large-scale computational analyses on transcript co-response data through selected prokaryotic and plant model organisms. CSB.DB - a comprehensive systems-biology database - (http://csbdb.mpimp-golm.mpg.de/) was initiated to provide public and open access to the results of biostatistical analyses in conjunction with additional biological knowledge. The database tool CSB.DB enables potential users to infer hypothesis about functional interrelation of genes of interest and may serve as future basis for more sophisticated means of elucidating gene function. The co-response concept and the CSB.DB database tool were successfully applied to predict operons in Escherichia coli by using the chromosomal distance and transcriptional co-responses. Moreover, examples were shown which indicate that transcriptional co-response analysis allows identification of differential promoter activities under different experimental conditions. The co-response concept was successfully transferred to complex organisms with the focus on the eukaryotic plant model organism Arabidopsis thaliana. The investigations made enabled the discovery of novel genes regarding particular physiological processes and beyond, allowed annotation of gene functions which cannot be accessed by sequence homology. GMD - the Golm Metabolome Database - was initiated and implemented in CSB.DB to integrated metabolite information and metabolite profiles. This novel module will allow addressing complex biological questions towards transcriptional interrelation and extent the recent systems level quest towards phenotyping.
Diet is a major force influencing the intestinal microbiota. This is obvious from drastic changes in microbiota composition after a dietary alteration. Due to the complexity of the commensal microbiota and the high inter-individual variability, little is known about the bacterial response at the cellular level. The objective of this work was to identify mechanisms that enable gut bacteria to adapt to dietary factors. For this purpose, germ-free mice monoassociated with the commensal Escherichia coli K-12 strain MG1655 were fed three different diets over three weeks: a diet rich in starch, a diet rich in non-digestible lactose and a diet rich in casein. Two dimensional gel electrophoresis and electrospray tandem mass spectrometry were applied to identify differentially expressed proteins of E. coli recovered from small intestine and caecum of mice fed the lactose or casein diets in comparison with those of mice fed the starch diet. Selected differentially expressed bacterial proteins were characterised in vitro for their possible roles in bacterial adaptation to the various diets. Proteins belonging to the oxidative stress regulon oxyR such as alkyl hydroperoxide reductase subunit F (AhpF), DNA protection during starvation protein (Dps) and ferric uptake regulatory protein (Fur), which are required for E. coli’s oxidative stress response, were upregulated in E. coli of mice fed the lactose-rich diet. Reporter gene analysis revealed that not only oxidative stress but also carbohydrate-induced osmotic stress led to the OxyR-dependent expression of ahpCF and dps. Moreover, the growth of E. coli mutants lacking the ahpCF or oxyR genes was impaired in the presence of non-digestible sucrose. This indicates that some OxyR-dependent proteins are crucial for the adaptation of E. coli to osmotic stress conditions. In addition, the function of two so far poorly characterised E. coli proteins was analysed: 2 deoxy-D gluconate 3 dehydrogenase (KduD) was upregulated in intestinal E. coli of mice fed the lactose-rich diet and this enzyme and 5 keto 4 deoxyuronate isomerase (KduI) were downregulated on the casein-rich diet. Reporter gene analysis identified galacturonate and glucuronate as inducers of the kduD and kduI gene expression. Moreover, KduI was shown to facilitate the breakdown of these hexuronates, which are normally degraded by uronate isomerase (UxaC), altronate oxidoreductase (UxaB), altronate dehydratase (UxaA), mannonate oxidoreductase (UxuB) and mannonate dehydratase (UxuA), whose expression was repressed by osmotic stress. The growth of kduID-deficient E. coli on galacturonate or glucuronate was impaired in the presence of osmotic stress, suggesting KduI and KduD to compensate for the function of the regular hexuronate degrading enzymes under such conditions. This indicates a novel function of KduI and KduD in E. coli’s hexuronate metabolism. Promotion of the intracellular formation of hexuronates by lactose connects these in vitro observations with the induction of KduD on the lactose-rich diet. Taken together, this study demonstrates the crucial influence of osmotic stress on the gene expression of E. coli enzymes involved in stress response and metabolic processes. Therefore, the adaptation to diet-induced osmotic stress is a possible key factor for bacterial colonisation of the intestinal environment.
With populations growing worldwide and climate change threatening food production there is an urgent need to find ways to ensure food security. Increasing carbon fixation rate in plants is a promising approach to boost crop yields. The carbon-fixing enzyme Rubisco catalyzes, beside the carboxylation reaction, also an oxygenation reaction that generates glycolate-2P, which needs to be recycled via a metabolic route termed photorespiration. Photorespiration dissipates energy and most importantly releases previously fixed CO2, thus significantly lowering carbon fixation rate and yield. Engineering plants to omit photorespiratory CO2 release is the goal of the FutureAgriculture consortium and this thesis is part of this collaboration. The consortium aims to establish alternative glycolate-2P recycling routes that do not release CO2. Ultimately, they are expected to increase carbon fixation rates and crop yields. Natural and novel reactions, which require enzyme engineering, were considered in the pathway design process. Here I describe the engineering of two pathways, the arabinose-5P and the erythrulose shunt. They were designed to recycle glycolate-2P via glycolaldehyde into a sugar phosphate and thereby reassimilate glycolate-2P to the Calvin cycle. I used Escherichia coli gene deletion strains to validate and characterize the activity of both synthetic shunts. The strains’ auxotrophies can be alleviated by the activity of the synthetic route, thus providing a direct way to select for pathway activity. I introduced all pathway components to these dedicated selection strains and discovered inhibitions, limitations and metabolic cross talk interfering with pathway activity. After resolving these issues, I was able to show the in vivo activity of all pathway components and combine them into functional modules.. Specifically, I demonstrate the activity of a new-to-nature module of glycolate reduction to glycolaldehyde. Also, I successfully show a new glycolaldehyde assimilation route via arabinose-5P to ribulose-5P. In addition, all necessary enzymes for glycolaldehyde assimilation via L-erythrulose were shown to be active and an L-threitol assimilation route via L-erythrulose was established in E. coli. On their own, these findings demonstrate the power of using an easily engineerable microbe to test novel pathways; combined, they will form the basis for implementing photorespiration bypasses in plants.