Research Projects - Friedhelm Pfeiffer
HaloLex allows the central management, integration, curation, and web-based visualization of genomic and other -omics data for any given microorganism.It enables the integrated analysis of genome-wide proteomic results with the underlying genomic data. Application of homology-based methods to the published genome of Haloarcula marismortui permitted a major improvement of gene prediction.
The genome of the the halophilic archaeon Halobacterium salinarum strain R1 was sequenced and a high-quality gene set was established. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1 (previously considered to represent a distinct species). From genome-wide mobility data for insertion sequences we conclude that strains R1 and NRC-1 originate from the same natural isolate and differ by evolution in the laboratory.
The genome of the halophilic and alkaliphilic archaeaon Natronomonas pharaonis was sequenced. A high degree of nutritional self-sufficiency was predicted and confirmed by growth in a minimal medium. The respiratory chain was analyzed in silico and experimentally. Unlike other alkaliphiles, Nmn. pharaonis uses protons as coupling ion between respiratory chain and ATP synthase. Secretome analysis predicts many extracellular proteins with alkaline-resistant lipid anchors, which are predominantly exported through the twin-arginine pathway.
The genome of the square halophilic archaeon Haloquadratum walsbyi strain HBSQ001 was sequenced in cooperation with Henk Bolhuis. Haloquadratum dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), are amongst the most outstanding features. Haloquadratum has a GC content which is much lower than in other halophilic archaea, and one of the lowest coding densities known for prokaryotes.
The genome of the moderately halophilic bacterium Halobacillus halophilus was sequenced in cooperation with Volker Mueller and is currently being annotated. The emerging genome sequence triggered analysis of chloride-induced gene expression and the usage of glutamine and glutamate as compatible solutes.
read paper abstract (glutamine/glutamate biosynthesis)... read paper abstract (autoinducer-2-producing protein LuxS)...A large-scale survey of N-terminal protein maturation in archaea is based on 873 proteomically identified N-terminal peptides from Halobacterium salinarum and Natronomonas pharaonis. Our analyses show that protein maturation involves methionine cleavage and N-terminal acetylation. Contrary to previous assumptions, we found that N-terminal acetylation is common in archaea. N-terminal maturation primarily depends on the residue following the ini-Met and reveals an archaea-specific N-acetylation pattern. A large fraction of the integral membrane proteins does not contain a cleavable signal sequence but show N-terminal protein maturation equivalent to that of cytosolic proteins.
In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. Metabolic diversity was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species.
Brz (bacteriorhodopsin-regulating zinc finger protein) is a regulator for the bop gene coding for bacteriorhodopsin. Brz is a small CPxCG-related zinc finger protein, encoded directly upstream of the bop gene. A large decrease of bop mRNA levels is caused by deletion of the brz gene and by site-directed mutagenesis of Cys and His residues in the zinc finger motif. In silico genome analysis revealed a large family of similar small zinc finger motif proteins in archaea and - to a lesser extent - in bacteria. The study was initiated after identification of the Brz protein in the small proteome project.
In close cooperation between the proteomics and bioinformatics project groups, we analyzed the proteome of Halobacterium salinarum and could identify ca 70% (1984) of the proteins. Detailed analyses allowed a major increase in the quality of the gene set for this organisms with its very GC-rich genome. A special focus was on proteomic identification of N-terminal peptides and on difficult sub-proteomes: the membrane proteome and the small proteome.
read paper abstract (membrane proteome)... read paper abstract (cytosolic proteome)... read paper abstract (quantitative profiling of membrane proteome)... read paper abstract (low molecular weight proteome)... read paper abstract (N-terminal peptides)...In a shotgun approach, one-third of the Natronomonas proteins could be identified (929 proteins). About 60% of the proteins involved in metabolism and genetic information processing are covered in this set. We did not find any indication of "ORF overprinting" (i.e. the usage of distinct reading frames in the same genome region).
The department developed a whole-genome DNA microarray based on the Halobacterium salinarum strain R1 genome sequence. This allowed to measure genome wide transcriptional changes for cells growing under aerobic and anaerobic/phototrophic conditions. We found that the transcriptional changes were relatively weak, though significant.
The bioinformatics group provided a genome-scale metabolic reconstruction for the halophilic archaeon Halobacterium salinarum. The resulting metabolic network is the basis for subsequent computational analyses. Experimental metabolic data were obtain for growth of Halobacterium in a defined medium with 15 amino acids. Dynamic simulations in silico were compared with the experimental data and allowed to predict internal fluxes. All of the supplied amino acids were simultaneously degraded, unexpectedly including five which are essential. Cells maximized energy production even at the cost of longer term concerns such as growth prospects.
A recent phototaxis model of Halobacterium salinarum, which is symmetrical in the clockwise and counter-clockwise rotations of the motor, explained all considered experimental data on spontaneous switching and response time to repellent or attractant light stimuli. However, the model could not explain the behaviour of a mutant which always moves forward and does not respond to light. The introduction of asymmetry in the motor switch model allowed to explain the mutant behaviour Thus, those experimental data that could previously be explained only by ad hoc assumptions are now obtained 'naturally' from the revised model.
rss
Top
Print