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Konstantinidis, Konstantinos (2008): Genome-Wide Proteomics and Quantitative Analyses on Halophilic Archaea. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. With genome-wide shotgun proteomics, 886 soluble proteins (929 proteins in total) of the theoretical Natronomonas pharaonis soluble proteome consisting of 2187 proteins have been confidentially identified by MS/MS. By comparing the identified proteins of Natronomonas pharaonis with homologues of other organisms, both extreme diversity between halophiles and occasional extraordinary sequence conservation to proteins from unrelated species were observed, substantiating genetic exchange between organisms that are evolutionary nearly unrelated to cope with several extreme conditions. Alternative and largely overlapping open reading frames (called overprinting) could not be identified in the genome of neither Natronomonas pharaonis nor Halobacterium salinarum, leading to the conclusion that in halophiles, not more than one protein can be produced from the same genomic sequence stretch. In the second part of this work, analyses on both the transcriptional and translational level have been performed on the halophilic archaeon Halobacterium salinarum, to gain insights into its lifestyle changes leading to cell response when challenged by heat shock. Thereby, quantitative proteomic data obtained from two different approaches regarding the labeling method (ICPL; SILAC), the fractionation of the protein or peptide mixtures (2DE; 1DE-LC), the mass spectrometric analysis (MALDI-TOF/TOF; ESI Q-TOF), and the choice of the growth medium (complex; synthetic) were integrated with data from whole-genome DNA microarrays, real-time quantitative PCR (RTqPCR), and Northern analyses. A number of genes congruently displayed substantial induction after heat shock on both the transcript and protein level as in the case of the thermosome, two AAA-type ATPases, a Dps-like ferritin protein (DpsA), a hsp5-type molecular chaperone, and the transcription initiation factor tfbB. In contrast, the dnaK operon (hsp70) did not exhibit any significant upregulation in either of the approaches. Some genes encoding enzymes of the TCA cycle, of pathways flowing into the latter, and of pathways leading to pyrimidine synthesis, were only translationally induced. Finally, differential transcriptional induction of the transcription initiation factors tfbB and tfbA, determined by RTqPCR, led to the conclusion that they may regulate genes by reciprocal action. The multiplicity of proteomics and transcriptomics methods are complementing one another, covering a bigger area on the one hand, but also confirming some unexpected findings.