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

55demonstrated that the Caulobacter cell cycle is composed of two separablecontrol modules [1]. One module centers on an essential DNA-bindingprotein called CtrA, which governs replicative asymmetry, polarmorphogenesis and cell division. The other module centers on thereplication initiator DnaA and dictates the periodicity of DNA replication,thereby acting as an intrinsic pacemaker of replication and the cell cycle.Although CtrA regulation is now understood in great detail, ourunderstanding of the mechanisms governing DnaA activity remainsincomplete. Using a combination of genetic and yeast two-hybrid screenswe have identified a novel regulator of DnaA, whose precise role in theregulation of DnaA and replication are currently under investigation.Dissecting the regulation of the cell cycle in Caulobacter andunderstanding how it relates to the cell cycles of other bacteria willultimately provide insight into how the bacterial cell cycle has evolved toallow cells to grow and proliferate in diverse environmental niches.[1] Jonas K, Chen YE, Laub MT. (2011). Modularity of the bacterial cell cycle enables independentspatial and temporal control of DNA replication. Current Biology. 21(13):1092-101.BDV002How to generate a protein gradient within a bacterial cell:dynamic localization cycle of the cell division regulator MipZD. Kiekebusch* 1,2 , K. Michie 3 , L.-O. Essen 4 , J. Löwe 3 , M. Thanbichler 1,21 Max Planck Institute for Terrestrial Microbiology, Max Planck ResearchGroup Prokaryotic Cell Biology, Marburg, Germany2 Philipps University , Department of Biology, Marburg, Germany3 Medical Research Council, Laboratory of Molecular Biology, Cambridge,United Kingdom4 Philipps University, Department of Chemistry, Marburg, GermanyIntracellular protein gradients play a critical role in the spatial organizationof both prokaryotic and eukaryotic cells, but in many cases themechanisms underlying their formation are still unclear. Recently, abipolar gradient of the P-loop ATPase MipZ was found to be required forproper division site placement in the differentiating bacterium Caulobactercrescentus.MipZ interacts with a kinetochore-like nucleoprotein complex formed bythe DNA partitioning protein ParB in proximity of the chromosomal originof replication. Upon entry into S-phase, the two newly duplicated originregions are partitioned and sequestered to opposite cell poles, giving rise toa bipolar distribution of MipZ with a defined concentration minimum atthe cell center. Acting as a direct inhibitor of divisome formation, MipZthus effectively confines cytokinesis to the midcell region.Based on the crystal structures of the apo and ATP-bound protein and bymeans of mutant variants of MipZ, we dissected the role of nucleotidebinding and hydrolysis in MipZ function. Gradient formation is found torely on a nucleotide-regulated alternation of MipZ between a monomericand dimeric form. MipZ monomers interact with ParB, which results inrecruitment of MipZ to the polar regions. Our results suggest that the polarParB complexes locally stimulate the formation of ATP-bound MipZdimers, the biological active form that inhibits FtsZ assembly. Moreover,dimers are retained near the cell poles through association withchromosomal DNA. Due to their intrinsic ATPase activity, dimerseventually dissociate into freely diffusible monomers that undergospontaneous nucleotide exchange and are recaptured by ParB.The MipZ gradient can thus be envisioned as an asymmetric distribution ofdimers that are released from a polar pool and slowly diffuse towards midcell.By virtue of the marked differences in the interaction networks anddiffusion rates of monomers and dimers, ATP hydrolysis promotesoscillation of MipZ between the polar ParB complexes and pole-distalregions of the nucleoid. The MipZ gradient thus represents the steady-statedistribution of molecules in a highly dynamic system, providing a generalmechanism for the establishment of protein gradients within the confinedspace of the bacterial cytoplasm.BDV003Regulation of cellular reversals in Myxococcus xanthusC. Kaimer*, D. ZusmanUniversity of California, Berkeley, Molecular and Cellular Biology,Berkeley, United StatesSocial behaviour patterns, such as predation or the formation of fruitingbodies in the soil bacterium Myxococcus xanthus, require the coordinatedmovement of cells. Myxococci lack flagella, but move by gliding on solidsurfaces using two genetically distinct mechanisms: social S-motilitymediates movement in groups while adventurous A-motility powersindividual cells.In both systems, coordinated movement is achieved by regulating thefrequency of cellular reversals. Reversals involve the inversion of thecell´s polarity axis, which is established by a pair of GTPase/GAP proteins(MglA and MglB) that localize to opposite cell poles. MglA and MglBswitch their position at reversal, resulting in the re-orientation of the S- andA-motility motors.The frequency of cell reversals is modulated by the Frz signallingpathway, which operates similar to the E. coli chemotaxis system. In thepresence of a chemoreceptor homologue FrzCD and a coupling proteinFrzA, phosphotransfer occurs from the FrzE histidine kinase to a responseregulator, FrzZ. We currently use genetic and biochemical approaches toidentify downstream targets of the response regulator FrzZ. Characterizingthe output of the pathway is essential to understand how the Frz systemcontrols the GTPase/GAP switch and times cell reversals.BDV004The cell wall amidase AmiC2 is pivotal for multicellulardevelopment in the cyanobacterium Nostoc punctiforme ATCC29133J. Lehner* 1 , Y. Zhang 2 , S. Berendt 1 , I. Maldener 1 , K. Forchhammer 11 Universität Tübingen, IMIT, Mikrobiologie/ Organismische Interaktionen,Tübingen, Germany2 Hertie-Institute, Clinical Brain Research, Tübingen, GermanyFilamentous cyanobacteria of the order Nostocales are primordialmulticellular organisms, a property widely considered unique toeukaryotes. Their filaments are composed of hundreds of mutuallydependent vegetative cells and, when deprived for a source of combinednitrogen, regularly spaced N 2-fixing heterocysts. Furthermore, specializedspore-like cells (akinetes) and motile filaments (hormogonia) differentiateunder certain environmental conditions. The cells of the filament exchangemetabolites and signaling molecules, but the structural basis for cellularcommunication within the filament remains elusive.Here we show that mutation of a single gene, encoding cell-wall amidaseAmiC2, completely changes the filamentous morphology of N.punctiforme and abrogates cell differentiation and intercellularcommunication. The mutant forms irregular clusters of twisted cellsconnected by aberrant septa. Rapid intercellular molecule exchange takesplace between cells of the wild-type filaments, but is completely abolishedin the mutant, and this blockage obstructs any cell-differentiation,indicating a fundamental importance of intercellular communication forcell-differentiation in N. punctiforme. AmiC2-GFP localizes in the cellwall implying that AmiC2 processes the newly synthesized septum into afunctional cell-cell communication structure during cell division.Ultrastructural analysis shows a contiguous murein sacculus withindividual cells connected by a single-layered septal cross-wall in themutant as well as in the wild type. AmiC2-GFP also accumulates in theregion of the polar neck during heterocyst differentiation and disappearsafter heterocyst maturation as well as in the septa of mature akinetes.Synchronously dividing cells of hormogonia accumulate AmiC2-GFP inthe septal cross walls. The AmiC2 protein could be expressed in E. coliand purified. It shows cell wall lytic activity and can complement thefilamentous phenotype of E. coli triple amidase mutants.From our studies we can conclude that the cell wall amidase AmiC2 of N.punctiforme is a novel morphogene required for cell-cell communication,cellular development and multicellularity in this cyanobacteriumBDV005Functional analysis of cytoskeletal proteins implicated inmagnetosome formation and cell division in MagnetospirillumgryphiswaldenseF.D. Müller*, O. Raschdorf, E. Katzmann, M. Messerer, D. SchülerLudwig-Maximilians-Universität München, Mikrobiologie, Planegg-Martinsried, GermanyMagnetotactic bacteria use magnetosomes to move along magnetic fieldlines. Magnetosomes are organelles which consist of membrane-enclosednanometer-sized magnetite crystals lined up along the cell axis. Thismagnetosome chain is located at midcell and split during cell division,whereupon magnetosomes are thought to re-localize from the new cellpoles to the new centres by an as yet unknown mechanism. Midcellinformation in bacteria is usually provided by the essential cell divisionprotein, FtsZ. Intriguingly, M. gryphiswaldense has two ftsZ homologs (agenuine ftsZ and ftsZm). ftsZm is co-located within the genomicmagnetosome island with other magnetosome genes including mamK,which encodes a further, actin-like cytoskeletal protein that polymerizesinto straight magnetosome filament structures. We analyzed the functionof these cytoskeletal elements likely implicated in the magnetosome chaindivision and segregation process.Fluorescence microscopy revealed that FtsZ Mgr and FtsZm co-localize atthe division plane in asymmetric spots opposite to the MamK filament.This asymmetry coincides with an asymmetric indentation and division ofM. gryphiswaldense cells which likely facilitates cleavage of themagnetosome chain owing to leverage. In contrast to previous observation,deletion of had no effect on magnetite crystal biomineralization butinfluenced the cell size of M. gryphiswaldense. To analyze the dynamics ofmagnetosome daughter chain segregation we performed fluorescence timelapse microscopy of growing cells. Our preliminary results suggest thatequal proportions of magnetosomes are rapidly removed from the divisionplane and become trapped at the centres of future daughter cells during thedivision process. Electron microscopy of division-inhibited cells suggestsBIOspektrum | Tagungsband 2012