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

protein interactions are changed by phosphorylation to cause NPCdisassembly. Additionally we are interested in defining how certain NPCproteins play roles at the NPC during interphase and at chromatin duringmitosis. We are employing single-step affinity purification and MassSpectrometry analysis of NPC sub-complexes from G2 and mitotic cells toidentify NPC proteins and to define how these complexes change duringmitosis. Using this approach we have defined previously undefined NPC andnuclear envelope proteins and have established that some of these newlydefined proteins help facilitate mitotic progression. Importantly we find thatthe mitotic behaviour of the NPC can be mimicked by ectopic induction ofNIMA kinase activity which promotes the relocation of specific NPCproteins from the NPC onto chromatin. These data provide direct evidencethat protein phosphorylation driven by the NIMA kinase regulates manyaspects of mitotic nuclear structure.(Supported by the National Institutes of Health)ISV07The general stress response, biofilm formation and cyclicdi-GMPsignaling in Escherichia coliR. HenggeFaculty of Biology, Department of Microbiology, Free University, Berlin,GermanyThe ubiquitous bacterial signaling molecule cyclic-di-GMP, which isproduced and degraded by diguanylate cyclases (carrying GGDEF domains)and specific phosphodiesterases (EAL domains), respectively, regulatestransitions between the motile-planktonic and sedentary biofilm "life-styles"[1]. c-di-GMP controls a variety of targets, including transcription and theactivities of enzymes and complex cellular structures. Many bacterialspecies possess many GGDEF/EAL proteins (29 in E.coli), which has leadto the concept of temporal and functional sequestration of c-di-GMP controlmodules [1]. Some GGDEF/EAL domain proteins (four in E.coli) havedegenerate GGDEF/EAL motifs, are enzymatically inactive and can act bydirect macromolecular interactions.In E.coli, c-di-GMP signaling is tightly integrated with the general stressresponse, as many GGDEF/EAL genes are regulated by RpoS [3].Moreover, c-di-GMP-dependent down-regulation of motility and inductionof biofilm-associated functions such as the production of (auto)adhesivecurli fimbriae occur during entry into stationary phase and require RpoS [2].The talk will cover (i) the molecular mechanism of switching from motilityto adhesion, which is based on a mutual inhibition of the FlhDC/motility andRpoS/CsgD/curli control cascades involving c-di-GMP signaling, and (ii)the molecular function of a locally acting c-di-GMP control module thatregulates the transcription of the curli control gene csgD. Taken together,these and other studies [4] have also generated a novel general concept ofthe evolution of complex bacterial second messenger signaling [1].[1] Hengge, R. (2009): Principles of cyclic-di-GMP signalling. Nature Rev. Microbiol. 7:263-273.[2] Pesavento, C. et al (2008): Inverse regulatory coordination of motility and curli-mediated adhesionin Escherichia coli. Genes Dev. 22:2434-2446.[3] Sommerfeldt, N. et al (2009): Gene expression patterns and differential input into curli fimbriaeregulation of all GGDEF/EAL domain proteins in Escherichia coli. Microbiology 155:1318-1331.[4] Tschowri, N. et al (2009): The BLUF-EAL protein YcgF acts as a direct anti-repressor in a bluelight response of E.coli. Genes Dev. 23:522-534.ISV08Driving up the pressure: genetic and cellular responses ofBacillus subtilis to osmotic stressE. BremerDepartment of Biology, Laboratory for Microbiology, Philipps-UniversityMarburg, GermanyThe soil-dwelling bacterium Bacillus subtilis inhabits an ecological nichesubjected to frequent changes in osmotic and saline conditions that arecaused by rainfall and desiccation. Such changes elicit water fluxes acrossthe cytoplasmic membrane and can drive up turgor under hypo-osmoticconditions to such an extent that the cell will rupture, or under hyperosmoticconditions cause the dehydration of the cytoplasm, a reduction inturgor and eventually growth arrest and cell death. Proteome and genomewidetranscriptional profiling studies have highlighted the complexity andmultifaceted nature of the osmotic stress response systems of B. subtilis.However, it is beyond doubt that an effective water management by the cellis the cornerstone of its acclimatization to either sudden or sustained rises inthe environmental osmolarity and the osmotic downshift that inevitably willfollow hyperosmotic growth conditions [1]. The accumulation and expulsionof ions and compatible solutes play key roles in these cellular osmoticadjustment processes. I will discuss the nature of the systems responsible forion fluxes in osmotically challenged B. subtilis cells and highlight thecentral role of the compatible solutes proline and glycine betaine in theacclimatization of the B. subtilis cell to sustained high salinity growthconditions.Funding for our studies on cellular stress responses to changes osmolarity inB. subtilis is provided by the LOEWE program of the State of Hesse(SynMicro; Marburg) and a grant from the BMBF through the BaCell-SysMO2 consortium.[1] Bremer, E. and R. Krämer (2010): The BCCT family of carriers: from physiology to crystalstructure. 78:13-34.ISV09Tailor-made cell factories for a sustainable bio-economyC. WittmannInstitute of Biochemical Engineering, University of Technology,Braunschweig, GermanyThe shortage of fossil resources and global warming are major drivers for abio-based economy, basing the production of bulk and fine chemicals, biopolymersas innovative plastics and biofuels on renewable resources. In theheart of this development are efficient biocatalysts, which provide thedesired product at high yield and titer and open novel applications. Thecreation of such tailor-made cell factories requires the right combination oftargeted genetic modifications, not an easy task taking the several thousandsof genes into account which typically form a microbial genome. Novelconcepts now open a design-based strain optimization on the basis of highlyvital wild types. These combine systems wide omics analysis andcomputational modeling of metabolic networks as genome scale towardsdetailed understanding of the underlying metabolism as basis of knowledgebased optimization. Key targets hereby comprise the utilization ofalternative raw materials, the reduction of by-product formation as well ashigh titer, yield and productivity for the compound of interest. Design-basedsystems metabolic engineering will be demonstrated for the feed amino acidL-lysine with a world market of about 1.000.000 tons per year, the novelbio-polyamide building block diaminopentane as well as the platformchemical succinic acid. Integrated into the development of efficient fedbatchbioprocesses the created cell factories enable novel industrialapplications.Becker J, Zelder O, Häfner S, Schröder H, Wittmann C (2011) From zero to hero - design-basedsystems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng.In press.Buschke N, Schröder H, Wittmann C (2011) Metabolic engineering of Corynebacterium glutamicumfor Production of 1,5-diaminopentane from hemicellulose. Biotechnol. J. In press.Kohlstedt M, Becker J, Wittmann C (2010) Metabolic fluxes and beyond - systems biologyunderstanding and engineering of microbial metabolism.Appl. Microbiol. Biotechnol. 88:1065-1075.Kind S, Jeong WK, Schröder H, Wittmann C (2010) Systems-wide metabolic pathway engineering inCorynebacterium glutamicum for bio-based production of diaminopentane. Met Eng 12: 341-351.ISV10Genome-wide aspects of cellulase regulation inTrichoderma reeseiC. P. KubicekVienna University of Technology, Vienna, AustriaMost of the industrial production of enzymes for plant biomass hydrolysistowards biofuel production is performed with mutants of the fungusTrichoderma reesei (the anamorph of the tropical ascomycete Hypocreajecorina). Consequently, this fungus meanwhile serves as the model systemfor the molecular understanding of cellulase gene expression and secretionof the encoded cellulase proteins. The recent complete sequencing of the T.reesei genome (Martinez et al. 2008. Nature Biotechnol) enabled to studythese biochemical events on a genome wide scale. Analysis of the T. reeseitranscriptome during cellulase induction has led to the identification andfunctional characterization of new genes relevant to this process. In addition,I will demonstrate a regulation of cellulase and hemicellulase formation atthe chromatin level. The results open new avenues for strain improvementtowards further improvement of T. reesei strains.spektrum | Tagungsband 2011