thesis
Computational genomics of hyperthermophiles
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Abstract
With the ever increasing number of completely sequenced prokaryotic genomes and the subsequent use of functional genomics tools, e.g. DNA microarray and proteomics, computational data analysis and the integration of microbial and molecular data is inevitable. This thesis describes the computational analyses on (hyper)thermophilic archaeal and bacterial genomes with a particular emphasis on carbohydrate metabolic pathways and their regulation. These analyses were integrated with wet-lab functional genomics data and results from classical molecular biology and microbial physiology experiments. The research was conducted on the archaea Sulfolobus solfataricus, Pyrococcus furiosus, T. kodakaraensis and the hydrogen producing bacterium Caldicellulosiruptor saccharolyticus. The reconstruction of the central carbohydrate metabolism in the thermo-acidophile S. solfataricus was carried out by a combination of genome sequence, whole transcriptome and proteome analyses. Only slight differences in the mRNA and the protein expression levels were shown when S. solfataricus was grown on peptides vs. glucose. However, the breakdown of D-arabinose vs. D-glucose revealed a complete novel pathway in the domain of Archaea. Similar catabolic pathways were identified in other prokaryotes and therefore a comprehensive genomic reconstruction was carried out on the pentose utilizing pathways in Archaea and, additionally, the results were compared to Bacteria and Eukarya. A computational promoter analysis of the glycolytic genes in the anaerobic species of the order Thermococcales (P. furiosus and T. kodakaraensis) indicated a clear cis-regulatory element that putatively controls all the genes of the glucose and starch degrading pathways. A comparative genomic analysis of the hyperthermophilic Thermococcales species led to the discovery of a putative transcriptional regulator that is probably involved in regulation of the entire regulon. The complete genome sequence of the extremely thermophilic Caldicellulosiruptor saccharolyticus revealed a circular genome of 2,970,275 base pairs that encodes 2679 putative proteins. The central carbohydrate pathways of C. saccharolyticus were studied in detail and the pathways for producing biohydrogen from plant cell wall material were unraveled. Subsequently, a whole transcriptome analysis of C. saccharolyticus grown on different monosaccharides showed a tight transcriptional regulation of these pathways, without glucose-based catabolite repression. C. saccharolyticus is therefore a good candidate to produce molecular hydrogen from biomass feedstock. The new insights into how prokaryotic genomes, genes and their encoded proteins function, as described in this thesis, can be applied on hyperthermophilic proteins and strains for use in and improvement of industrial processes. <br/