VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

incompatible form seemed to compensate each other. We therefore considerguanidino-ectoine a new member of incompatible solutes characterized by aguanidinium group [4].[1] Courtenay, E.S. et al (2001): Thermodynamics of interaction of urea and guanidinium salts wothprotein surface: relationship between solute effect on protein processes and changes in wateraccessiblesurface area. Protein Sci. 10: 2485-2497.[2] Mountain, R.D. and D. Thirumalai (2004): Alterations in water structure induced by guanidiniumand sodium salts. J Phys Chem B 108: 19711-19716.[3] Rishi, V. et al (1998): Role of non-compatible osmolytes in the stabilization of proteins duringheat stress. Biochem J 329: 137-143.[4] Galinski, E.A. et al (2008): Zwitterionische Guanidinium-verbindungen als selektiveantimikrobielle Wirkstoffe. DE 10 2008 009 591 A1, 15.02.2008; PCT/EP 2009/001075.Here we present kinetic and regulation studies of H. elongata and C.salexigens aspartokinases and compare them with our previous findings forthe P. stutzeri enzymes [6]. These results provide a first glance into theimportance of different aspartokinase constellations in ectoine biosynthesis.[1] Bestvater, T. et al (2008): Saline Systems. 4:12.[2] Curien, G. et al (2008): Plant Physiol Biochem. 46:325-339.[3] Lo, C. et al (2009): Microbiol Mol Biol Rev. 73:594-651.[4] Schwibbert, K. et al (2010): Env Microbiol. Epub ahead of print DOI: 10.1111/j.1462-2920.2010.02336.x[5] Seip, B. (2010): Appl Env Microbiol. accepted for publication.[6] Weissgerber, T. et al (2010): VAAM annual meeting.SRP021Etherlipid biosynthesis in myxobacteriaW. Lorenzen* 1 , M.W. Ring 2 , H.B. Bode 11 Institute for Molecular Bio Science, Goethe-University, Frankfurt,Germany2 Department of Pharmaceutical Biotechnology, Saarland University,Saarbrücken, GermanyMyxobacteria are Gram-negative, motile, soil-dwelling δ-proteobacteriawhich are known for their complex life cycle including the formation ofcomplex spore-containing fruiting bodies as well as their richness in diversesecondary metabolites formed by partly unique biosynthetic pathways.Many of these compounds exhibit antibacterial, antifungal or cytotoxicbiological activities, which makes them interesting as drug candidates).Non-ribosomal [1] peptide synthethases (NRPS) and polyketide synthases(PKS) as well as NRPS/PKS hybrids are prevalently involved in thesynthesis of many of these secondary metabolites.A detailed analysis of Myxococcus xanthus cells exposed to various stressesshowed that the formation of lipid vesicles is a common response of thosecells towards environmental adversities [2] whereas starvation induced lipidvesicles proved to contain substantial amounts of unusual branched chainfatty acid-derived ether lipids. Those ether lipids make up at least one thirdof all lipids found in mature myxospores [3].When investigating the biosynthesis of these ether lipids we discovered agene encoding a multifunctional PKS/NRPS-like enzyme, designated aselbD, which is part of a five gene operon. This operon can be found in allmyxobacteria sequenced so far. After its inactivation, a strong reduction ofether lipid formation in M. xanthus and a complete loss of ether lipidformation in S. aurantiaca under vegetative growth and starvationconditions was observed. Additionally, the speed of fruiting body formationis being affected in the respective mutants.Therefore we cloned, heterologously expressed and purified ElbD in order toinvestigate the function of this protein by the means of in vitro activityassays and MALDI-TOF MS.[1] Weissman, K. J. and R. Muller (2010): Nat. Prod. Rep. 27, 1276-1295.[2] Hoiczyk, E. et al (2009): Mol Microbiol 74, 497-517.[3] Ring, M. W. et al (2006): J. Biol. Chem. 281, 36691-36700.SRP022Significance of aspartokinases for the regulation ofectoine biosynthesisS. Höfs*, M. KurzInstitute for Microbiology und Biotechnology, Friedrich-WestphalianWilhelms-University, Bonn, GermanyThe biosynthetic pathway of ectoine-type compatible solutes is an offbranchof the metabolic network leading to the aspartate family amino acids.Lysine, methionine, threonine and isoleucine share the precursors aspartylphosphate and aspartic acid semialdehyde as starting point in theirbiosynthesis. A strict feedback regulation of the aspartokinases [2] wasreported to cause a metabolic bottle-neck in heterologous production ofectoines, when using ectoine biosynthetic genes from Marinococcushalophilus [1].As we learn from latest reports [5] these findings are not valid for allheterologous production systems. Interestingly, some of the ectoinebiosynthesis gene clusters have an aspartokinase in close proximity (e.g. inPseudomonas stutzeri, [5]) which indicates a possible evolutionary andfunctional correlation [3] while others have not [4]. In addition, someectoine producers have multiple enzymes of the aspartokinase type, whereasin others only one gene encodes for this function (e.g. Halomonas elongataand Chromohalobacter salexigens).SRP023Solvent accessible surface characteristics of compatiblesolutes for the prediction of their protein-stabilizingpotentialA. Korsten*, E.A. GalinskiInstitute for Microbiology und Biotechnology, Friedrich-WestphalianWilhelms-University, Bonn, GermanyCompatible solutes are small osmolytes that influence the equilibrium of theprotein folding reaction (U ↔N). They raise the free energy of the unfoldedstate (U) and, in doing so, stabilize the native conformation (N) [1]. Fromthe work of the Bolen group it is known that such solutes operatepredominantly on the protein backbone, and that the backbone transfer freeenergy (Δ G tr) from water to osmolyte solution is a good predictor for acompatible solute´s relative strenght as a protein stabilizer [2]. Structures ofcompatible solutes can differ considerably, it is therefore a challenge topredict their stabilizing potential from structural properties. Recently, Streetet al. [3] tried to correlate structural properties of solutes and proposed aminimal model in which the fractional polar surface area served as anindication for Δ G tr and hence the solute´s stabilizing power (the smaller therelative polar surface the better).In this work, the concept by Street et al. [3] is put to the test by applying it tothe natural ectoine-type compatible solutes, ectoine and 5-hydroxy-ectoine,the latter distinctly more polar than the former, as well as to the hithertounavailable peptide-type compatible solute N-acetylglutaminyl glutamine-1-amide (NAGGN). NAGGN is characterized by an unusually large fractionalpolar surface area, which according to the polar surface concept shouldresult in week stabilizing properties. The characterization of solute surfaceproperties serves as a tool to enlarge the knowledge of interactionmechanisms between compatible solutes and biomolecules.[1] DaCosta, M.S. et al (1998): An Overview of the Role and Diversity of Compatible Solutes inBacteria and Archaea. Book: Advances in Biochemical Engineering/Biotechnology, Vol. 61,Springer-Verlag Berlin Heidelberg.[2] Liu, Y. and D.W. Bolen (1995): The peptide backbone plays a dominant role in proteinstabilization by naturally occurring osmolytes. Biochemistry 34: 12884-12891.[3] Street, T.O. et al (2006): A molecular mechanism for osmolyte-induced protein stability. PNAS,Vol. 103, No. 38, pp. 13997-14002.SRP024A carbon monoxide dehydrogenase-similar protein inClostridium acetobutylicumM. Wietzke* 1,2 , N. Quosdorf 1,2,3 , O. Riebe 1,2,3 , H. Bahl 1,2,31 Department of Microbiology,University of Rostock, Rostock, Germany2 Department of Biology, Universtiy of Rostock, Rostock, Germany3 Department of Biology, Universtiy of Rostock, Rostock, GermanyA machinery for the scavenging of molecular O 2 and its reactive derivatives(ROS) is essential for the survival of strict anaerobic bacteria under aerobiccondition. Previous studies with Clostridium acetobutylicum proved a robustdefense mechanism under oxidative stress which leads to a protection ofmultiple cellular processes. The analysis of the transcriptome revealed ahighly induced expression of an open reading frame cac0116 after exposureto air (1). The deduced amino acid sequence of cac0116 is annotated as acarbon monoxide dehydrogenase (CODH) and shows similarities to therespective homologue from Carboxydothermus hydrogenoformans, a typeIV CODH. It has been speculated that this enzyme plays an important role inthe oxidative stress response of this organism, e. g., by providing electronsfrom CO to rubrerythrin to reduce H 2O 2 to water (2).To analyze the role of cac0116 in C. acetobutylicum, we constructed a strainwith altered expression of this gene. The CODH knock-out mutant ishypersensitive to oxygen, leading to a decreased ability to cope with O 2.Furthermore, the putative role of CODH in the electron transfer chain fromNADH + H + or H 2 to O 2 and ROS will be elucidated using the purifiedspektrum | Tagungsband 2011