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

published software package ARCIMBOLDO [3], we succeeded to solve thestructure by this de novo approach (Dayté Rodriguez, Isabel Usón-Finkenzeller, Instituto di Biología Molecular de Barcelona, Barcelona,Spain). In the crystal Cmi forms a dimer that is interlinked by a disulfidebridge. It is a highly charged protein with a surplus of negative chargespresumably responsible for interaction with Cma which contains a cluster ofpositive charges.[1] Gross, P., and V. Braun (1996): Mol. Gen. Genet. 251, 388-396.[2] Hullmann, J. et al (2008): Mol. Microbiol. 69, 926-937.[3] Rodríguez, D.D. et al (2009): Nature Meth. 6, 651-653.CBP040Dissecting the role of the seven chitin synthases ofNeurospora crassa in apical growth and septumformationR.A. Fajardo-Somera* 1 , R.W. Roberson 2 , S. Bartnicki-Garcia 1 ,M. Riquelme 11 Department of Microbiology, Center for Scientific Research and HigherEducation (CICESE), Ensenada, Mexico2 School of Life Sciences, Arizona State University, Tempe, USAFungal chitin synthases (CHS) are grouped into seven classes, four of them,III, V, VI and VII being exclusive of filamentous fungi. CHS classes V andVII have a myosin-like motor domain (MMD) at their amino terminus.Previous studies in Neurospora crassa showed that CHS-1, CHS-3, andCHS-6 tagged with GFP or mCherry accumulated at the core of the Spk, andalso at nascent septa. We endogenously tagged with gfp the remaining chitinsynthases genes, namely chs-2 (NCU05239.3), chs-4 (NCU09324.3), chs-5(NCU04352.3) and chs-7 (NCU04350.3) to study their distribution in livinghyphae of N. crassa. CHS-2, CHS-4, and CHS-7, appeared solely involvedin septum formation. As the septum ring developed, CHS-2-GFP movedcentripetally until it localized exclusively around the septal pore. CHS-5 waslocalized both at nascent septa and in the core of the Spk. We observed apartial colocalization of CHS-1-mCherry and CHS-5-GFP in the Spk. Totalinternal reflection fluorescence microscope (TIRFM) analysis revealedputative chitosomes containing CHS-5-GFP moving along wavy tracks.Collectively our results suggest that there are different populations ofchitosomes, each containing a class of CHS. Mutants with single genedeletions of chs-1, chs-3, chs-5, chs-6, or chs-7 grew slightly slower than theparental strain (FGSC#9718); only Δchs-6 displayed a marked reduction ingrowth. Both Δchs-5 and Δchs-7 strains produced less aerial hyphae andconidia. Currently, we are analyzing CHS activity and chitin content in allthe Knock Out mutant strains to determine the relative importance of eachCHS in cell wall biosynthesis.CBP041Neurosporacrassa class III chitin synthase 1 (CHS-1):subcellular distribution, vesicular trafficking andcytoskeleton associationsE. Sanchez-Leon* 1 , J. Verdin 1 , R.W. Roberson 2 , M. Freitag 3 , S. Bartnicki-Garcia 1 , M. Riquelme 11 Department of Microbiology, Center for Scientific Research and HigherEducation (CICESE), Ensenada, Mexico2 School of Life Sciences, Arizona State University, Tempe, USA3 Department of Biochemistry and Biophysics, Center for Genome Researchand Biocomputing, Oregon State University, Corvallis, USAApical growth in filamentous fungi is attained through different coordinatedcellular mechanisms that include cell polarity establishment andmaintenance. For cell wall expansion to occur at apical regions of growinghyphae, the orchestrated delivery of enzymes involved in carbohydratesynthesis is extremely important. In this study,by using different liveimaging techniques, we found that chitin synthase 1 (CHS-1), one of theseven putative chitin synthase of Neurosporacrassa, localizes at theSpitzenkörper (Spk) core, the apical cell surface and transiently toconstricting rings during septum development. Hyphae of heterokaryonstrains expressing CHS-1-GFP and CHS-3-GFP or CHS-6-GFP, exhibitedpartial colocalization of the three different chitin synthases, suggesting thateach CHS is contained in distinct chitosomal compartments. Total InternalReflection Microscopy (TIRFM) allowed us to observe the anterograde andretrograde traffic of rapidly moving CHS-1-GFP vesicles, some of themconverging at the Spk. The differential localization of CHS-1-GFP and GS-1-mChFP at the Spk, is the first evidence in living hyphaethat both proteinsare contained in different populations of vesicles, as predicted from earliertransmission electron micrographs. Using cytoskeleton inhibitors, weconcluded that microtubules are not essential for CHS-1 delivery to the Spkor nascent septa, whereas actin is necessary for the correct accumulation ofCHS-1 to the Spk.CBP042The morphogene AmiC2 is pivotal for multicellulardevelopment in the cyanobacterium Nostoc punctiformeJ. Lehner 1 , Y. Zhang 2 , S. Berendt 1 , T.M. Rasse 2 , K. Forchhammer 1 ,I. Maldener* 11 Institute for Microbiology and Infection Medicine, Eberhard-Karls-University, Tübingen, Germany2 Junior Research Group Synaptic Plasticity, Hertie Institute for ClinicalBrain Research, Eberhard-Karls-University, Tübingen, GermanyFilamentous cyanobacteria of the order Nostocales are primordialmulticellular organisms, a property widely considered unique to eukaryotes.Their filaments are composed of hundreds of mutually dependent vegetativecells and regularly spaced N 2-fixing heterocysts, exchanging metabolites andsignaling molecules. Furthermore, they may differentiate specialized sporelikecells and motile filaments. However, the structural basis for cellularcommunication within the filament remained elusive. Here we present thatmutation of a single gene, encoding cell-wall amidase AmiC2, completelychanges the morphology and abrogates cell differentiation and intercellularcommunication. Ultrastructural analysis revealed for the first time acontiguous peptidoglycan sacculus with individual cells connected by asingle-layered septal cross-wall. The mutant forms irregular clusters oftwisted cells connected by aberrant septa. Rapid intercellular moleculeexchange takes place in wild-type filaments, but is completely abolished inthe mutant, and this blockage obstructs any cell-differentiation, indicating afundamental importance of intercellular communication for celldifferentiationin Nostoc. AmiC2-GFP localizes in the cell wall with a focusin the cross walls of dividing cells, implying that AmiC2 processes thenewly synthesized septum into a functional cell-cell communicationstructure during cell division. AmiC2 thus can be considered as a novelmorphogene required for cell-cell communication, cellular development andmulticellularity.EMV001Anaerobic formate- and CO 2 -assimilating prokaryotictaxa in a methane-emitting fen soilS. Hunger*, O. Schmidt, M. Hilgarth, M.A. Horn, S. Kolb, H.L. DrakeDepartment of Ecological Microbiology, University of Bayreuth, Bayreuth,GermanyNatural wetlands such as fens and bogs contribute up to approximately 40%of the global biogenic emission of methane. Biopolymers in wetland soilsare anaerobically degraded via intermediary events that terminate in theemission of methane (i.e., ‘intermediary ecosystem metabolism’). Formateand CO 2 (together with H 2) are precursors of methanogenesis and have beenobserved to stimulate methanogenesis in anoxic microcosms of soil from theregional fen Schlöppnerbrunnen. However, active formate- and CO 2-utilizing methanogens of the fen remain unresolved. Active methanogens inanoxic fen soil microcosms were evaluated by stable isotobe probing ofmcrA/mrtA (encode for the alpha-subunit of methyl-CoM reductases I andII) and archaeal 16S rRNA genes. Bacterial taxa were also evaluated.Anoxic fen soil microcosms were incubated in the dark and periodicallypulsed with low concentrations of either [ 13 C]-formate or 13 CO 2. Theproduction of methane was stimulated by formate and CO 2; in contrast, onlyformate stimulated acetogenesis. 411 mcrA/mrtA sequences and 306archaeal 16S rRNA gene sequences were analyzed. 12 family-level 16SrRNA archaeal genotypes were detected, 7 of which had no isolated culturedrepresentatives. Methanocellaceae and Methanobacteriaceae were mainlylabeled by [ 13 C]-formate, whereas Methanosarcinaceae were mainly labeledby 13 CO 2, suggesting that formate-linked methanogenesis was mostlycatalyzed by fen soil-derived Methanocellaceae and Methanobacteriaceae,whereas CO 2-linked methanogenesis was mostly catalyzed by fen soilderivedMethanosarcinaceae. In total, 58 bacterial 16S rRNA family-levelgenotypes and 15 species-level fhs (encodes for formyltetrahydrofolatesynthetase) genotypes were detected, of which 29 of the bacterial 16S rRNAgenotypes and all 15 fhs genotypes were defined as novel. Two of the fhsgenotypes were affiliated with the acetogenic genera Sporomusa andMoorella. The collective results reinforce the likelihood thatspektrum | Tagungsband 2011