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

about 600 bacterial proteins from only 10 6 cells in a time range from 1.5 to6.5 hours post-internalization.With this study we now wanted to extend this time window and monitor thelong-term adaptation of S. aureus RN1HG during survival within S9 humanlung epithelial cells over several days. We optimized our digestion protocol,because bacterial counts consistently decreased after a short term growthphase (up to 6 hours) finally reaching around 500 cfu per ml 6 days postinternalization. In order to quantify the changes of the protein compositionof internalized S. aureus, we added fully SILAC-labeled S. aureus controlcells as external standard to each time point after FACS-sorting, whichallowed the identification and quantitation of about 300 S. aureus proteinspost-internalization. In addition, small colony variants that appeared at latetime points after internalization were investigated.[1] Garzoni, C. and W.L. Kelley (2009): Staphylococcus aureus: new evidence for intracellularpersistence. Trends Microbiol, 17, 59-65.[2] Lowy, F.D. (1998): Staphylococcus aureus infection. N. Engl. J. Med, 339, 520-532.[3] Schmidt, F. (2010): Time resolved quantitative proteome profiling of host-pathogen interactions:The response of S. aureus RN1HG to internalisation by human airway epithelial cells. Proteomics, 10,2891-2911.[4] Ong, S.-E. et al (2002): Stable isotope labelling by amino acids in cell culture, SILAC, as a simpleand accurate approach to expression proteomics. Mol Cell Proteomics, 1, 376-386.MPP065Will not be presented!MPP066TAL effectors from Xanthomonas : a novel DNA-bindingdomain with programmable specificityJ. Boch*, H. Scholze, J. Streubel, M. Reschke, U. BonasDepartment of Genetics, Martin-Luther-University Halle-Wittenberg, Halle(Saale), GermanyPathogenicity of most plant pathogenic Xanthomonas spp. bacteria dependson the injection of effector proteins via a type III secretion system into plantcells. The translocated effectors manipulate cellular processes to the benefitof the pathogen. TAL (transcription activator-like) effectors fromXanthomonas spp. are important virulence factors and function astranscriptional activators in the plant cell nucleus. They directly bind totarget promoters via a novel DNA-binding domain and induce expression oftarget host genes. This domain is composed of tandem repeats of typically34-amino acids. Each repeat binds to a specific DNA base pair and repeatspecificities are determined by a simple two amino acid-code (termed RVD,repeat-variable diresidue). The array of repeats thus corresponds to aconsecutive target DNA sequence. The modular TAL repeat architectureenabled the construction of artificial TALs (ARTs) with novel repeatcombinations and target specificities. Recognition sequences of ARTs werepredicted and experimentally confirmed in a transient reporter system usingAgrobacterium-mediated expression in planta. The ARTs exhibitedpredicted specificities, indicating that DNA-targeting domains with novelpreferences can be generated. TAL repeats with different RVDs exist innature, but the DNA-specificity of only a few of them is known, so far. Wewill present novel repeat specificities that allow conclusions about the DNAbindingmechanism of TAL repeats. The use of TALs as programmable geneswitches will be shown. The programmable DNA-binding domaindemonstrates that TALs are versatile virulence factors for the pathogen andexceptional tools for biotechnology.NTV001Protein mobility in bacterial cytoplasmV. SourjikCenter for Molecular Biology (ZMBH), University of Heidelberg,Heidelberg, GermanyDevelopments in fluorescence microscopy led to tremendous advances inboth bacterial and eukaryotic cell biology in the last decades, but thequantitative potential of fluorescent microscopy still remains largelyunderappreciated. However, systematic quantitative approaches areabsolutely required to understand the complexity of biological systemsbeyond cartoon-type diagrams. The combination of quantitative fluorescenceimaging with other quantitative techniques and with computationalmodelling is thus going to be the next major frontier at the interface ofbiology and physics. This talk will focus on the application of quantitativeFRAP and time-lapse imaging to systematically study mobility of proteinsand protein complexes in the cytoplasm of Escherichia coli. The role ofprotein mobility in the controlled self-assembly and partitioning of proteincomplexes will be discussed.NTV002Hologram stacking with PICOLAY: How to get confocalmicroscopy for freeH. CypionkaInstitute for Chemistry and Biology of the Marine Environment, Carl vonOssietzky University, Oldenburg, GermanyA major issue of light microscopy is the low depth of focus, particularly athigh magnification. If images are taken as focus series (so-called z-stacks),one can use image processing software to extract sharp zones and combinethese to a single image with increased depth of focus. A depth mapindicating the z-positions of the sharp patches allows reconstructing theobject in its correct spatial dimensions. Normally, only the sharpest pixels inthe stack are selected while others are filtered out from the resulting image.Here I demonstrate the so-called hologram stacking with the freewareprogram PICOLAY (www.picolay.de, [1]). This can be used to display notonly the sharpest, but all pixels with a pre-defined minimum contrast orcolour. The program requires a single z-stack, only, and generatesstereoscopic 3D images for different observation methods (red-cyananaglyphs, observation with crossed or parallel eyes, rocking images). It isalso possible to freely rotate the objects and visualise structures that remainhidden during the normal stacking routine. The hologram-stacking techniqueis especially useful for multi-layered transparent objects such as biofilms ordiatoms, radiolaria etc., and can be used with various light-microscopictechniques, magnifications and illuminations (bright field, differentialinterference contrast, phase contrast, reflected-light or epifluorescencemicroscopy). Thus, one gets confocal microscopy for free, without beingrestricted to laser illumination and fluorescence images.Free download: www.picolay.de[1] Raap E. and H. Cypionka (2011): Vom Bilderstapel in die dritteDimension: 3D-Mikroaufnahmen mit PICOLAY. Mikrokosmos (in press).NTV003Studying fungal development: Utilization of laser capturemicrodissection and next-generation sequencingtechniquesI. Teichert*, M. Nowrousian, U. KückGeneral and Molecular Botany, Ruhr-University, Bochum, GermanyFungi are able to produce a number of different cell types and multicellularstructures during their life cycle. One prominent example is the formation offruiting bodies to propagate sexually. Our studies focused on the filamentousfungus Sordaria macrospora which produces fruiting bodies within sevendays under laboratory conditions. To identify regulators of sexualdevelopment, we have generated and characterized several sterile mutants bystandard molecular genetic approaches. Recently, next-generation (NGS)techniques have become available and have revolutionized the field ofgenomics / functional genomics. We employ NGS in different ways toidentify developmental genes in S. macrospora: First, we use NGS tosequence the genomes of yet uncharacterized sterile mutants that weregenerated by conventional mutagenesis. Mapping of sequence reads to therecently sequenced genome of the S. macrospora wild type andbioinformatics analysis is used to identify the respective mutation causingthe developmental defect. This strategy has already led to the identificationof a spore color and a developmental gene. Second, we apply laser capturemicrodissection (LCM) to separate vegetative and sexual structures.Subsequent RNA isolation from these structures followed by RNAamplification and RNA-Seq should enable us to identify genes specificallytranscribed in sexual structures. By this approach, we will generate geneexpression profiles that are much more accurate than those generated byconventional techniques that use a mixture of vegetative and sexual cellsharvested at different time points.spektrum | Tagungsband 2011