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

Bacillus subtilis. BiFC experiments show that RodA interacts with MreB,and with Mbl, the second MreB ortholog. Mbl in turn interacts with themembrane proteins MreC and MreD. Because RodA largely colocalizes withYFP-Mbl, our results indicate that MreB, Mbl, RodA, MreC and MreD forma large morphogenetic complex at and within the membrane. TIRFmicroscopy revealed highly dynamic localization kinetics of YFP-RodA focialong random paths. Contrarily, YFP-MreB filaments remodelled alongregular helical paths, showing that RodA molecules diffuse between helicalMreB filaments, but are not statically anchored. Consistent with this, RodAshowed diffusion-type kinetics in FRAP experiments. RodA also colocalizedwith fluorescently labelled vancomycin (Van-Fl) that marks sites of new cellwall synthesis. A partially functional RodA-mCherry fusion mislocalized tolarge clusters at irregular positions along the lateral cell wall, andconcomitantly changed the regular positioning of cell wall synthesis, as wellas cell shape, showing that the positioning of RodA influences thelocalization of new cell wall material and thereby cell morphology.CBP030Will not be presented!CBP031The scaffold protein Iqg1 plays an essential role duringcytokinesis in Ustilago maydisM. Wehr*, B. Sandrock, M. BölkerDepartment of Biology, Philipps-University, Marburg, GermanyCytokinesis is the process by which cells physically separate afterduplication and spatial segregation of the genetic material. Duringcytokinesis in Ustilago maydis two distinct septa were formed. Formation ofeach septa is initiated by the assembly of an CAR. This dynamic structureconsists of many different components for example F-actin, the myosin lightchain Cdc4 and the FCH-protein Cdc15. From other organisms it is known,that the IQGAP-proteins are also important components of this structure.To analyze the assembly of the actomysoin ring in U. maydis, we havecharacterized the homologous IQGAP protein Iqg1 (um10730) by genetic,cell biological and biochemical approaches. We will show that Iqg1 is anessential gene in U. maydis for haploid growth. In colocalisation studies ofIqg1 with F-actin, Cdc4 and Cdc15 Iqg1 was identified as an actomyosinring component.From other studies in our lab it is known, that the GTPase Cdc42 is a keyplayer in assembly of the second actomyosin ring. Deletion mutants ofcdc42 display a cytokinesis defect and cannot build the second actomyosinring. Interestingly, a similar phenotype was observed using mutants with aC-terminal GFP fusion of Iqg1. Using in vitro interaction assays, we couldshow that the Ras GTPase-activating protein related domain (GRD) of Iqg1bind to Cdc42. We propose that Iqg1 is an effector of Cdc42 during theassembly of the second actomyosin ring.CBP032Interactions between PTS transporters and thechemotaxis system in Escherichia coliK. Große*, S. Neumann, V. SourjikCenter for Molecular Biology, DKFZ-ZMBH Alliance, University ofHeidelberg, Heidelberg, GermanyThe phosphotransferase system (PTS) and the chemotaxis pathway of E.coli, which mediate uptake of and taxis towards carbohydrates respectively,are genetically and biochemically among the best studied bacterial systems.The crosstalk between both pathways is known to be important for taxis toPTS substrates, providing one of few known examples of direct interactionbetween nutrient transport and signalling. While signal processing by thecore of the chemotaxis pathway itself is largely understood, the mechanismsof PTS-mediated taxis, which results from concomitant perception ofsubstrates during their uptake, are largely unclear.Here, we investigate in vivo the interconnection among the PTS transportand taxis on the example of glucose, the preferred carbon source of E. coli.Our experiments showed that taxis towards low concentrations of glucose ismediated by membrane receptors, whereas taxis in the high concentrationrange requires glucose uptake through PTS transporters. Using intracellularpathway activity assay based on fluorescence resonance energy transfer(FRET), we demonstrated several intracellular interactions between PTS andchemotaxis proteins. Moreover, we quantitatively analyzed relativecontributions of the receptor-mediated and the PTS taxis towards glucose.We further found that adaptation in the PTS-mediated taxis depends on thereceptor methylation enzymes, suggesting that PTS-mediated signals affectreceptor activity. We propose a new model of signal exchange between bothsystems that unifies two types of chemotaxis.CBP033Deletion of the mamXY operon affects biomineralizationand chain alignment in Magnetospirillum gryphiswaldenseO. Raschdorf* 1 , F.D. Müller 1 , S. Ullrich 1 , E. Katzmann 1 , A. Lohße 1 ,M. Pósfai 2 , D. Schüler 11 Department Biologie I, Microbiology Research Field, Ludwig-Maximilians-University, , Munich, Germany2 Department of Earth and Environmental Sciences, University of Pannonia,Veszprém, HungaryMagnetospirillum gryphiswaldense employs magnetotaxis to find favorableenvironments in freshwater sediments by reducing the complex navigationin three dimensions to only one.Magnetotaxis is mediated by membrane-enveloped magnetite crystals calledmagnetosomes, which are arranged into a chain to provide a magnetic dipolemoment that passively aligns the cell to the earth’s magnetic field.Magnetosomes develop by invagination of the cytoplasmic membranefollowed by magnetite biomineralization.In M. gryphiswaldense almost all characterized genes that are involved inregulation of this complex process are clustered within a genomicmagnetosome island. Within this island four putative operons have beenidentified which are either essential for magnetosome formation or involvedin control of size and magnetic properties of the magnetite crystals. One ofthese operons is the mamXY operon, consisting of mamY, mamX, mamH-likeand intriguingly of ftsZm, a gene coding for a homolog of the FtsZ protein.Preliminary data from a mamXY operon deletion mutant suggest that thisoperon has an essential role in biomineralization as well as in magnetosomechain formation. Mutant cells display aberrant membrane vesicles.In addition to wildtype like magnetosomes, the mutant cells also containheterogeneous polycrystalline magnetite crystals and tiny crystal flakeswhich potentially do not consist of magnetite but rather of different ironoxides. This is indicating, that not only crystal size and shape are affectedbut also iron oxide composition itself is altered.To analyze the role of individual genes within the mamXY operon in detail,single gene deletion mutants are currently generated by a cre-lox basedmethod. Data will be presented showing the effects of loss of these genes onthe formation of magnetite crystals, vesicles and the structure of themagnetosome chain.CBP034Simkania negevensis replicates in the host endoplasmicreticulumA. Mehlitz*, G. Krohne, N. Weckesser, T. RudelBiocenter, Department of Microbiology, Julius-Maximilians-University,Würzburg, GermanySimkania negevensis is a Chlamydia-like emerging pathogen implicated inpulmonary diseases. Knowledge regarding its intracellular accommodationremains sparse. Questions: How is the replicative vacuole of the obligateintracellular bacterium S. negevensis organized and where is it locatedwithin the host cell. Results: We show that expansion of the S. negevensisvacuole within epithelial cells is accompanied by massive spatialreorganization of host mitochondria and endoplasmic reticulum (ER).Spatial reorganization was mitochondria- and ER- specific as the Golgiapparatus appeared intact and was positioned between the nucleus and the S.negevensis vacuole. Ultrastructural analysis and 3D reconstruction revealedthat S. negevensis forms one large vacuole located within the ER lumen.Location of the vacuole within the ER led to the formation of a so far notdescribed pathogen-containing triple membrane surrounded structure.Conclusion: Like Chlamydia, Simkania is entering and building a pathogencontaining vacuole within the host cell. Interestingly, the vacuoles show anunusual location within the cell. While the Simkania vacuole is locatedwithin the ER and thus pre-Golgi, the Chlamydia vacuole is a post golgistructure. Comparison of the Chlamydia and Simkania vacuole might shedlight on intracellular trafficking and vacuole accommodation within theChlamydiae.spektrum | Tagungsband 2011