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Doctoral School of
Molecular Biosciences and Biotechnology
3rd DocDay – NAWI Graz Doctoral School
Friday, 12th of February 2010
PLENARY LECTURES AND ORAL PRESENTATIONS
Abstracts in chronological order
Employing synthetic promoters for recombinant protein expression in the yeast Pichia pastoris
Ruth C.1 and Glieder A.1
1 Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010- Graz, Austria
Regulating gene expression on transcriptional level synthetic promoters are useful tools with various applications. The rising popularity of synthetic promoters or promoter libraries might be explained by the severity of common genetic methods; for example, gene knockouts or overexpression. Every protein behaves differently and thus, requires different adjustments in expression level or strategy. Promoter technology is a proven tool for identifying optimal promoter gene combinations and finetuning individual expression.
Interested in cis-motif discovery for synthetic promoter design and engineering, we performed a library approach for the methanol inducible promoter of the alcohol oxidase 1 gene (PAOX1) of the methylotrophic yeast Pichia pastoris. PAOX1 is tightly repressed in the presence of glucose, glycerol, and other carbon sources. The deletion of in silico predicted transcription factor binding sites produced differently regulated promoter variants of different strength. In addition we demonstrated the broad applicability of the generated promoter variants by employing commercial enzymes as reporters. For example, placing the horseradish peroxidase (HRP) gene under the control of a strong, depressed promoter variant caused increased volumetric activity. In contrast, placing the same promoter in control of porcine trypsinogen, an enzyme used for invitro processing of biopharmaceuticals, caused lower yields, but improved product quality due to a delay, or even prevention, of autoproteolytic product degradation. Further, employing a novel short synthetic promoter, generated by fusing an identified cis-acting sequence to a core promoter, enabled the production of significant amounts of trypsinogen, even without induction with methanol.
 C. Ruth, A. Glieder, ChemBioChem, 2010, accepted.
 F.S. Hartner, C. Ruth, D. Langenegger, S.N. Johnson, P. Hyka, G.P. Lin-Cereghino, J. Lin-Cereghino, K. Kovar, J.M. Cregg, A. Glieder, Nucleic Acids Res, 2008, 36, e76.
 C. Ruth, T. Zuellig, A. Mellitzer, R. Weis, V. Looser, K. Kovar, A. Glieder, Systems and Synthetic Biology, submitted.
iving little pesticides
Schmid F. and Berg G.
Institute of Environmental Biotechnology, Petersgasse 12/1, 8010 Graz
Corresponding author: Florian Schmid
Without disease management yield-oriented agriculture, which is necessary to provide world’s population with appropriate amounts of food, agriculture of crop plants is not feasible. The reason for that is that in order to ensure sufficient yields, systems of low biodiversity, monocultures, have been created. In these systems desired crop plants are free from pressure created by nutrient competitors, but due to the poorness of the biodiversity diseases easily can take over. Current practices countersteer this problem by use of pesticides with all its side effects reaching from soil contamination to enrichment of toxic compounds in food products and emergence of resistant pathogens.
Biological control or biocontrol is the application of microorganisms or mixtures of microorganisms in order to protect crop plants against diseases. Properties of such microorganisms are directed towards the pathogen by mechanisms like competition, production of enzymes, predation and parasitism or it can be directed towards the crop plants by direct plant growth promotion, improving availability of nutrients and inducing plant’s resistance towards pathogens. Successful application of biocontrol organisms requires the ability of the organisms to survive and to be active at the side of action. Therefore knowledge about the ecosystem, where the organism should be applied, and careful screening after and selection of biocontrol strains is necessary.
For the protection of grapevine against the grey mould disease caused by Botrytis cinerea high amounts of pesticides or, in organic farming, copper are used. We screened for potential microbial antagonists against this fungus and tested their application as biocontrol organisms. It was found, that a strain of Pantoea ananatis and one of Hanseniaspora uvarum showed promising results. Furthermore the biodiversity in conventionally and organically managed vineyards was compared using cultivation dependent and independent techniques. Surprisingly the overall diversity was found to be similar, but significant differences were found in the fungal population. Whereas in organically managed vineyards Aureobasidium pullulans was present in high abundances, Sporidiobolus pararoseus was found as a dominant species in conventionally managed vineyards. The reason for the enrichment of A. pullulans could be the resistance of strains of this species against copper and sulphur.
Results of studies about the efficiency of the application of biocontrol organisms show that sometimes biocontrol products can compete with traditional pesticides, but for adequate protection of crop plants a mixture of both methods will be necessary. In order to minimize the negative impact of pesticides and present farming practices on the environment much more knowledge will be needed to understand the complex functions of microorganisms within the ecosystem of cultivated land.
ubstrate recognition in bacterial type IV secretion pathways
Lang S.1, Gruber K.1, Mihajlovic S.1, Steinlechner S.1, Arnold R.2, Jehl A.2, Rattei T.2 and Zechner E.L.1
1Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/I, Graz, Austria, and 2Genome oriented Bioinformatics, Technische Universitaet Muenchen, Maximus-von-Imhof-Forum 3, Freising, Germany
Bacteria export macromolecules across cellular membranes via a variety of multi-component secretion systems. The Type IV secretion systems (T4SS) transport single proteins with signaling or virulence effects into targeted host cells. Between bacterial cells, T4SS are used to mobilize genetic material including genes for antibiotic resistance and virulence factors. Control at the initiation stage of secretion from a bacterial donor is the subject of our investigation. We report the molecular mapping of protein features crucial to proper recognition and uptake of export proteins by model systems of conjugative T4SS (cT4SS).
A genetics strategy identified two regions of the plasmid R1 conjugative DNA transesterase and helicase, TraI, which independently mediate T4 translocation of the protein. Comparison of the probable protein folding of these distinct regions, as well as those of a related substrate from plasmid F, with known macromolecular structures in the Protein Data Base matched an available structure of helicase RecD2 from Deinococcus radiodurans (PDB code 3e1s). Based on the sequence structure alignment, a cluster of conserved amino acids common to both regions (TSA and TSB) were identified: (G[E/D]R[L/M]R[V/F]T). A bioinformatics approach was taken to analyze conservation of the pattern among virulence- and cT4SS substrates, as well as the full proteome of Chlamydia trachomatis, a species without a T4SS. The Smith – Waterman pair-wise alignment algorithm as implemented in Jaligner was used with the BLOSUM62 substitution matrix. This analysis revealed conservation of the pattern among related and distant families of cT4SS, but not in known substrates of virulence systems. Single exchanges of amino acids within the defined TSA and TSB disrupted recognition or altered the specificity of substrate recognition among heterologous T4SS. Moreover disruption of the conserved pattern in an unrelated cT4SS substrate prevented transfer, thus confirming the general importance of this motif for function.
Current efforts to elucidate the mechanisms involved support the unexpected conclusion that substrate recognition is controlled not by the substrate receptor protein TraD directly, but instead by a pre-selection chaperone protein TraM. The data support a model where TraM mediates specific contacts with potential protein substrates and signals that information via physical interactions with the receptor protein. If this interpretation proves true, these findings define the role of the first known substrate recognition chaperone in bacterial T4 secretion.
ffect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae
Spanova M.1, Czabany T.1, Zellnig G.2, Leitner E.3, Hapala I.4 and Daum G.1
1 Institute of Biochemistry, Graz University of Technology, Graz, Austria;
2 Institute of Plant Sciences, University of Graz, Austria;
3 Institute of Analytical Chemistry and Food Technology, Graz University of Technology, Austria;
4 Institute for Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovak Republic
Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids and ubiquinons. In the yeast Saccharomyces cerevisiae the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report we show that a hem1∆ mutant accumulated a large amount of squalene which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1∆ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ) which is devoid of the classical storage lipids, triacylglycerols (TAG) and steryl esters (SE), and lacks lipid particles accumulated squalene at similar amounts as the hem1∆ mutant in wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested; and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.
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