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

57differentiation and organelle biogenesis in prokaryotic systems. To becomedivided and segregated faithfully during cytokinesis, the magnetosomechain has to be properly positioned, cleaved and separated againstintrachain magnetostatic forces. Here we demonstrate that magnetotacticbacteria use dedicated mechanisms to control the position and division ofthe magnetosome chain, thus maintaining magnetic orientation throughoutdivisional cycle. Using electron and time-lapse microscopy ofsynchronized cells of Magnetospirillum gryphiswaldense, we demonstratethat magnetosome chains undergo a dynamic pole-to-midcell translocationduring cytokinesis. Nascent chains were recruited to division sites also indivision-inhibited cells, but not in a mamK mutant, indicating an activemechanism depending upon the actin-like cytoskeletal magnetosomefilament. Cryo-electron tomography revealed that both the magnetosomechain and the magnetosome filament are split into halves by asymmetricseptation and unidirectional indentation, which we interpret in terms of aspecific adaptation required to overcome the magnetostatic interactionsbetween separating daughter chains. Our study demonstrates thatmagnetosome division and segregation is coordinated with cytokinesis andresembles partitioning mechanisms of other organelles andmacromolecular complexes in bacteria.BDP003Differentiation of bacterial spores by Fourier transform infraredspectroscopy (FTIR) and chemometrical data treatmentH. Brandl*, A. Brandes AmmannUniversity of Zürich, Environmental Sciences, Zurich, SwitzerlandFourier transform infrared spectroscopy (FTIR) has been used as analyticaltool in chemistry for many years to elucidate chemical structures. Inaddition, FTIR can also be applied as a rapid and non-invasive method todetect and identify microorganisms. The specific and fingerprint-likespectra allow - under optimal conditions - discrimination down to thespecies level. The aim of this study was to develop a fast and reproduciblenon-molecular method to differentiate Bacillus spores originating fromdifferent species as well as to identify spores in a simple matrix, such asthe clay mineral, bentonite. We investigated spores from pure cultures ofseven different Bacillus species by FTIR in reflection or transmissionmode followed by chemometrical data treatment. All species investigated(B. atrophaeus, B. brevis, B. circulans, B. lentus, B. megaterium, B.subtilis, B. thuringiensis) are typical aerobic soil-borne spore formers. Tosimulate soil, mixtures of bentonite and spores of B. megaterium at variouswt/wt ratios were included in the study. Both hierarchical cluster analysisand principal component analysis of the spectra along withmultidimensional scaling allowed the discrimination of different speciesand spore-matrix-mixtures. Our results show that FTIR spectroscopy is afast method for species-level discrimination of Bacillus spores. Sporeswere still detectable in the presence of the clay mineral bentonite. Even atenfold excess of bentonite (corresponding to 2.1 x 10exp10 colonyforming units per gram of mineral matrix) still resulted in an unambiguousidentification of B. megaterium spores.BDP004Functional complementation of large operon deletions withinthe magnetosome Island of Magnetospirillum gryphiswaldenseI. Kolinko* 1 , C. Jogler 2 , Y. Zhang 3 , R. Müller 4 , D. Schüler 11 LMU München, AG Schüler, Institut für Mikrobiologie, Planegg-Martinsried, Germany2 Harvard Medical School, Armenise Building, Boston, United Kingdom3 Gene Bridges GmbH, BioInnovationsZentrum, Dresden, Germany4 Saarland University, Department of Pharmaceutical Biotechnology,Saarbrücken, GermanyThe magnetotactic bacterium Magnetospirillum gryphiswaldense producesintracellular organelles, the magnetosomes, which consist of magnetitecrystals surrounded by a magnetosome membrane. Their uniform sizes andunique magnetic properties make them highly attractive forbiotechnological and medical applications. Most of the genes controllingmagnetosome formation have been identified within a genomicmagnetosome island (MAI) of 115 kb. By mutational analysis, themamAB, mamGFDC, mms6 and mamXY operons, which have sizesbetween 2 and 17 kb and which comprise 30 genes in total, wereimplicated in the synthesis of properly sized and shaped magnetosomes.However, complementation of operon mutants has proven difficult due tothe requirement to clone, transfer and express large genomic fragments.Complementation of smaller regions up to 5 kb (mamGFDC, mamXY) wasaccomplished by conjugational transfer of replicative plasmids, resulting instable in trans expression and reconstitution of wildtype phenotypes. Forcloning of larger fragments, comprising for example the large mamABoperon encoding 17 magnetosome genes, we used recombinogeniccloning. Conjugational transfer of replicative vectors harboring this region,however, revealed high instability of the plasmids and resulted in partialdegradation of cloned genomic fragments, probably due to toxic effects ofmulticopy expression of encoded magnetosome membrane proteins.Therefore, alternative strategies, such as expression in RecA - backgroundstrains, use of inducible expression systems, and chromosomal insertionare currently investigated for stable expression. Eventually, cloning andfunctional expression of entire large operons from M. gryphiswaldensemight be also useful for future metabolic engineering of the magnetosomesynthesis pathway.BDP005A small acid soluble spore protein is essential for germinationof Clostridium acetobutylicum sporesD. Wetzel*, H. Janssen, R.-J. FischerUniversity of Rostock, Division of Microbiology, Rostock, GermanyClostridium acetobutylicum is a potent solvent producer and in recentyears it has become a model organism for the understanding of themolecular biology of non-pathogenic clostridia. We are interested in theevents during the cell cycle of C. acetobutylicum. Here, we focus on resultsdealing with aspects of the resistance and germination capability of its spores.Alpha/beta-type small acid soluble spore proteins (SASP) are usuallylocated in the core of the endospores. They carry important functions likethe protection of spore DNA against damage due to desiccation, heat orchemical agents. Furthermore, during germination fast degradation ofSASPs by germination specific proteases provide an important amino acidpool for the development of the new vegetative cell.In the genome of C. acetobutylicum five open reading frames are expectedto encode SASP-like proteins [1]. To unravel the individual functions ofthese proteins we generated specific knock out mutants using the ClosTronTechnology by insertional inactivation based on the selective retargetinggroup II intron [2]. Analysis of the phenotypes using transmission electronmicroscopy revealed the production of morphological intact spores.However, sporulation assays [3] proved that the individual germinationcapabilities of the mutant strains were affected to different levels. Mostinterestingly, one SASP was essential for germination which could berestored by a plasmid-based complementation of the gene knock out.[1] Nölling et al., 2001, J Bacteriol. 183:4823-4838[2] Heap et al., 2007, J Microbiol Methods 70:452-464[3] Burns et al., 2010, J Bacteriol. 192:657-664BDP006Spore formation in Clostridium acetobutylicum ATCC 824depends on granulose synthesisK. Zimmermann*, R.-J. FischerUniversität Rostock, Biowissenschaften/Mikrobiologie, Rostock, GermanyThe transition phase of growth of the Gram-positive, spore-forminganaerobe Clostridium acetobutylicum is characterized by severalmorphological changes. At the beginning swollen and cigar shaped cells,clostridial stages, are formed. In the cells, a polymeric carbohydrate,granulose is accumulated in the form of granules. Granulose is expected tobe a energy- and carbon storage, necessary as a prerequisite for sporulation.We proved that a single glycogen synthase (GlgA) in the genome of C.acetobutylicum plays a crucial role in the biosynthesis of granulose. AglgA insertion mutant (ClosTron ® technology, [1]) was unable toaccumulate granulose and did not form endospores. Results of thephenotypic characterisation are presented. This data includes colonymorphology, cell differentiation and sporulation assays ofglgA-mutantcells in comparison to the wildtype.Detailed comparative TEM studies revealed that even prespore formationin the mutant strain seemed to be blocked at a very early stage. Molecularanalysis confirmed the correct insertion into the target gene and a negativeinfluence on granulose-gene specific mRNA formation. However,transcription of the master regulator of sporulation spo0A seemed not to beaffected (RT-PCR). Almost every gene of the granulose metabolism wasinfluenced, whereas first evidence could be gained, that missing granuloseaffects degradation by a feed-back mechanism.[1] Heapet al.,2007, J Microbiol Methods 70:452-464BDP007Monitoring of population dynamics of Corynebacteriumglutamicum by multiparameter flow cytometryA. Neumeyer*, M. Bott, J. FrunzkeForschungszentrum Jülich, Biotechnologie 1, Jülich, GermanyCorynebacterium glutamicum is a Gram-positive soil bacterium that isused as an industrial amino acid producer. For strain analysis and processmonitoring usually average data resulting from the analysis of bulks ofcells are provided for key parameters such as growth rate, productivity,and viability. However, several studies of the last decades revealed thateven isogenic bacterial populations may exhibit significant cell-to-cellvariation due to differences in microenvironment, cell age, cell cycle orstochastic effects on gene expression. In this context, fluorescenceactivatedcell sorting (FACS) allows a rapid and efficient insight intocomplex phenotypes and allows high-throughput analysis at the single celllevel.BIOspektrum | Tagungsband 2012