VAAM-Jahrestagung 2012 18.–21. März in Tübingen

60BDP016The paryphoplasm of Planctomycetes is a highly derivedperiplasmM. Krehenbrink* 1 , R. Stamboliyska²1 University of Oxford, Biochemistry, Oxford, United Kingdom²Ludwig-Miximillians-Universität, Department of Evolutionary Biology,Munich, GermanyPlanctomycetes are bacteria with an unusually high degree of intracellularcompartmentalization. Although the extent of compartmentalization varies,the cell content of all planctomycetes is differentiated into at least a centralriboplasm containing the genomic DNA and ribosomes, and an extensiveperipheral compartment termed the paryphoplasm. Uniquely in bacteria,endocytotic protein uptake and membrane trafficking has been observed inthe paryphoplasm of Gemmata obscuriglobus. As the division of thecellular contents into paryphoplasm and riboplasm is reminiscent of thedivision of the cell contents of Gram-negative bacteria into a centralcytoplasm and a peripheral periplasm, the genome sequence of the modelplanctomycete Planctomyces limnophilus was examined for the presenceof proteins involved in the maintenance and functioning of the Gramnegativeperiplasm and outer membrane. The P. limnophilus genome wasfound to encode a large number of proteins typical for the periplasm andthe outer membrane, including the outer membrane insertion proteinBamA and outer membrane components of pili and flagella. Fewhomologs of Gram-negative protein secretion systems were found, andvery few proteins were found in the culture supernatant. In contrast, ~22%of all encoded proteins were predicted to carry a Sec signal peptide, whichcorresponds well with 20-30% of all proteins targeted to the periplasm in atypical Gram-negative bacterium. A comparison of these proteins with theperiplasmic proteins of Gram-negative bacteria also revealed substantialfunctional overlap between the two sets. We propose that theparyphoplasm is derived from a modified and greatly expanded periplasmand discuss the role of this cellular compartment in the lifestyle of thisgroup of organisms.BDP017Lipid specificity of a bacterial dynamin-like proteinP. Sawant* 1 , M. Bramkamp 21 University of Cologne, IGSDHD, Biochemistry, Köln, Germany2 University of Cologne, Cologne, GermanyMembrane fusion and fission are rapid, dynamic processes that occur ineukaryotic and prokaryotic cells to facilitate generation and transport ofvesicles, induce membrane trafficking, maintain cell shape and size.Proteins of dynamin superfamily play an important role in maintenance ofmembrane dynamics. This protein family includes members like classicaldynamins, dynamin-related proteins and guanylate-binding proteins oratlastins. Dynamin GTPases demonstrate functions such as vesiclescission, division of organelles, cytokinesis and microbial resistance.DynA is a 136 KDa GTPase in Bacillus subtilis. Its structure isremarkable, as it seems to have developed from a fusion event betweentwo molecules thus consisting of two separate GTPase and dynamin-likesubunits. On account of sequence homology to other bacterial andeukaryotic dynamins, similar biochemical properties such as GTPhydrolysis and membrane fusion, DynA is classified as a member of thedynamin superfamily. It is a bacterial dynamin-like protein (BDLP) whosefunction is reasonably parallel to eukaryotic mitofusins, involved inmitochondrial outer membrane fusion. Mitofusins mediate nucleotidedependentfusion whereas DynA shows nucleotide-independent membranetethering and fusion in vitro. Our recent in vitro data has shown DynA tomediate nucleotide-independent fusion of vesicles generated fromphosphatidylglycerol (PG) and cardiolipin (CA). Vesicle tethering but notfusion was observed with other lipids tested so far which is suggestive ofDynA’s affinity for PG and CA phospholipids. Currently we determine theamino acid positions in DynA that mediate such lipid specificity. Thismight allow identifying DynA’s target on bacterial membrane. Overall aimof this project is revealing the function and actual mechanism of DynA inbacteria. B. subtilis DynA seems like a promising BDLP candidate due tothe well characterised molecular biology of its host organism and theunique structural features of the molecule. Biochemical and cell biologicalcharacterisation of DynA using the simple B. subtilis may providemechanistic implications in particular for the mitochondrial membranedynamics as well as other dynamin-like proteins (DLPs).BDP018The mamXY operon is involved in controlling magnetiteformation and magnetosome chain positioning inMagnetospirillum gryphiswaldenseO. Raschdorf* 1 , F. Müller 1 , E. Katzmann 1 , M. Pósfai 2 , D. Schüler 11 Ludwig-Maximillians-Universität München, Department Biologie I -Mikrobiologie, Martinsried, Germany2 University of Pannonia, Department of Earth and EnvironmentalSciences, Veszprém, Hungary, GermanyMagnetotactic bacteria (MTB) use intracellular chains of membraneenvelopedmagnetite crystals, called magnetosomes, to orientate alongmagnetic fields. The sequential steps of magnetosome synthesis involveintracellular differentiation and include vesicle formation, magnetitenucleation and mineralization as well as magnetosome chain alignmentand are subject to tight genetic regulation. Most of the genes implicated inmagnetosome formation are organized in four operons that are clusteredwithin a genomic magnetosome island. Despite of recent progress incharacterization of these genes, the function of the mamXY operon has notbeen well investigated so far. To close this gap, we created unmarkeddeletions of all four individual genes within this operon and analyzed thephenotype of the mutants. The mamH-like gene encodes for a uniquemembrane-spanning protein affiliated to the group of MFS transporters butfused to a putative ferric reductase-like domain. The mamH-like mutantforms magnetite crystals with heterogenic size, structure and cellulardistribution. The mutant also displays a delay in production offerrimagnetic magnetosomes. A similar phenotype was observed upondeletion of mamX, indicating a function in the same cellularbiomineralization process. Deletion of the MTB-specific mamY genehowever, did not influence mineralization but led to mislocalization ofmagnetosome chains. Fluorescence microscopy revealed that MamYlocalizes as a filamentous structure coinciding with the expected positionof the magnetosome chain. The protein may therefore directly participatein targeting magnetosomes to their assigned position by an as yet unknownmechanism. Unexpectedly, deletion of ftsZm, coding for a truncatedhomolog of the major cell division protein FtsZ, did not show any obviouscell division phenotype, and in contrast to previous reports also nobiomineralization defects. In conclusion, our data suggests that the proteinsencoded within the mamXY operon play a major role in magnetosomebiomineralization and chain positioning.BDP019Mapping the interaction surfaces of the bacterial cell divisionregulator MipZB. He* 1,2 , M. Thanbichler 1,21 Max Planck Institute for Terrestrial Microbiology, Prokaryotic CellBiology, Marburg, Germany2 Philipps University, Department of Biology, Marburg, GermanyProper positioning of the cell division site in Caulobacter crescentus isregulated by the ATPase MipZ, which forms bipolar gradients within thecell, thus restricting assembly of the cytokinetic FtsZ ring to the midcellregion. Gradient formation is driven by a dynamic localization cycle thatinvolves the alternation of MipZ between a monomeric and dimeric statewith distinct interaction patterns and diffusion rates. This cycle depends onthe oscillation of MipZ between non-specific chromosomal DNA and apolarly localized complex of the chromosome partitioning protein ParB.To map the surface regions that mediate the interaction of MipZ with FtsZ,ParB and DNA, we systematically exchanged surface-exposed residuesusing alanine-scanning mutagenesis. Analyzing the subcellular distributionof the mutant proteins as well as their ability to support division siteplacement, we identified three clusters of residues each of which is likelyresponsible for contacting one of the interacting proteins. Notably, theDNA-binding pocket of the MipZ dimer is composed of residues from bothdimer subunits. Moreover, it was found to be located opposite the putativeFtsZ-binding region, consistent with the previous finding that the regulatoryeffect of MipZ is specific for its dimeric form and involves contacts with bothDNA and FtsZ. These results provide the first detailed analysis of theinteraction determinants of MipZ and yield new insights into the mechanismsthat underly the function of this unique regulatory system.BDP020Bactofilins: polar landmarks in Myxococcus xanthusL. Lin* 1,2 , A. Harms 3 , J. Kahnt 3 , L. Søgaard-Andersen 3 , M. Thanbichler 1,21 Max Planck Institute for Terrestrial Microbiology, Prokaryotic CellBiology, Marburg, Germany2 Philipps University, Department of Biology, Marburg, Germany3 Max Planck Institute for Terrestrial Microbiology, Department ofEcophysiology, Marburg, GermanyBacteria, similar to eukaryotes, possess cytoskeletons that are involved inthe temporal and spatial organization of various cellular processesincluding cell division, cell morphogenesis, cell polarity, as well as DNABIOspektrum | Tagungsband 2012