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

133OTV011Large and frequent introns in the 16S rRNA genes of largesulfur bacteriaV. Salman*, R. Amann, D. Shub, H. Schulz-VogtMax Planck Institut für marine Mikrobiologie, Mikrobiologie, Bremen,GermanyThe gene encoding the small ribosomal subunit (16S/18S rDNA) serves asa prominent tool for the phylogenetic analysis and classification of livingorganisms owing to its high degree of conservation and its fundamentalfunction 1 . Nowadays, established methods to analyze this gene are takingadvantage of its conservation in size and nucleotide composition 2 . Wesequenced the 16S rRNA genes of not yet cultivated large sulfur bacteria,among them the largest known bacteriumThiomargarita namibiensis, andfound that the genes regularly contain numerous self-splicing introns ofvariable length. The 16S rRNA genes of these bacteria can thus beenlarged to up to 3.5 kb.Using a modified CARD-FISH approach we can show that the introns aretranscribed as part of the rRNA precursor, but they cannot be located in thenative ribosomes. Also, the introns show self-splicing abilities in invitroexperiments, i.e. they autonomously excise from RNA and mediatethe ligation of the two exons. These findings lead to the conclusion that theintrons are capable of independent removal during ribosome maturation,therefore minimizing negative impact on the host organism. Remarkably,introns have never been identified in bacterial 16S rRNA genes before,although being the most frequently sequenced gene today. This may becaused in part by a bias during the PCR amplification step discriminatingagainst longer homologues, as we can show experimentally as well. Thefact that introns were now located in the 16S rRNA genes in the largesulfur bacteria, and have also been found in the 23S rRNA genes of severalother bacteria 3,4 , implies that the presence of introns in the bacterial rRNAoperon is more common than previously recognized. Possibly, also othergroups of bacteria likewise have introns in their 16S rRNA genes, whichwould have profound implications for common methods in molecularecology - it may cause systematic biases and lead to the exclusion of theintron-containing fraction of a heterogeneous population. The generalimpact of this finding on the standard analysis of rRNA genes is apparent.1N. R. Pace, G. J. Olsen, C. R. Woese, Cell45 (1986) p. 325-326.2C. R. Woese, Microbiology Reviews51(1987) p. 221-271.3 R. Raghavan, S. R. Miller, L. D. Hicks, M. F. Minnick, Journal of Bacteriology189 (2007) p. 6572-6579.4 C. L. Nesbø, W. F. Doolittle, Proceedings of the National Academy of Science U S A100 (2003) p. 10806-10811.5 This study was funded by the Max Planck Society.OTV012Regulation of anaerobic respiratory pathways inDinoroseobacter shibaeS. Laaß*, J. Klein, D. Jahn, P. TielenTechnische Universität Braunschweig, Institut für Mikrobiologie,Braunschweig, GermanyDenitrification is part of the global nitrogen cycle and an importantmechanism of energy generation under anaerobic conditions.Dinoroseobacter shibae, a representative of the globally abundant marineRoseobacter clade, is used as a model organism to study the transcriptionalresponse to changing oxygen conditions in the presence of nitrate. Itsannotated 4.4 Mb genome sequence revealed clustered genes, which areinvolved in anaerobic respiratory energy metabolism with nitrate asalternative electron acceptor [1]. Interestingly, D. shibae contains theperiplasmic nitrate reductase Nap instead of the membrane bound Nar. D.shibae features nir, nor and nos operons in the vicinity of the nap operon.An unusual high number of Crp/Fnr-like regulators have been predicted:Beside one FnrL-homologue with a [4Fe-4S] 2+ -cluster, six Dnr-likeregulators are found. The genes encoding DnrD and DnrE are directlylocated between the nor- and nos-operon. We are interested in identifyinggene regulatory patterns after shifting from aerobic to anaerobicdenitrifying conditions. Therefore, we used continuous cultivation of D.shibae in a chemostat combined with time series microarray analysis. Wedetected anaerobic growth of D. shibae via denitrification. Transcriptomeanalysis revealed distinct patterns of gene expression in response tooxygen limitation. The change from aerobic to anaerobic growth showed asequential induction of gene clusters encoding the four reductases of thedenitrification machinery. Genes encoding Fnr/Crp-like regulators showeddifferent expression levels over time. In response to oxygen limitation, animmediate upregulation of universal stress proteins, fine-tuning of theelectron transport chain components, as well as the downregulation of thetranslational apparatus was observed. Furthermore, we predict a regulatorynetwork for the anaerobic respiratory pathway in D. shibae.[1] Wagner-Döbler et al.(2009), ISME J. 4: 61-77.OTV013Influence of subcellular antigen localization within different yeastgenera on the activation of ovalbumin-specific CD8 TlymphocytesS. Boschi Bazan 1 , G. Geginat 2 , T. Breinig 3 , M.J. Schmitt 1 , F. Breinig* 11 Universität des Saarlandes, Molekular- und Zellbiologie, Saarbrücken,Germany2 Universitätsklinikum Magdeburg, Klinische Mikrobiologie, Magdeburg,Germany3 Universität des Saarlandes, Informatik, Saarbrücken, GermanyYeasts of the genus Saccharomyces expressing recombinant antigens arecurrently evaluated as candidate T cell vaccines. We compared theinteraction kinetics between four biotechnologically relevant yeast genera(Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis and Pichia pastoris) and human dendritic cells. Further, we analyzedthe activation capacity of recombinant yeasts expressing ovalbumin(OVA) either intracellular, extracellular or surface-displayed by OVAspecificCD8 T lymphocytes. We found that the kinetic patterns of yeastuptake by phagocytic cells varied between the tested yeast genera and thatboth genus and subcellular OVA antigen localization influenced thestrength of T cell activation. In particular, in S. cerevisiae, a secretedantigen was less effectively delivered than its cytosolic variant, whereasmost efficient antigen delivery with P. pastoris was obtained by cellsurface bound antigen. Our data indicate that protein secretion might notbe an effective delivery pathway in yeast. [Bazan et al. (2011) Vaccine 29;8165]OTV014The quest for new oxidative catalysts: Expression ofmetagenomic membrane-bound dehydrogenases from aceticacid bacteria in Gluconobacter oxydansB. Peters*, M. Mientus, D. Kostner, W. Liebl, A. EhrenreichTechnische Universität München, Lehrstuhl für Mikrobiologie, Freising,GermanyAcetic acid bacteria are used in biotechnology due to their ability toincompletely oxidize a great variety of carbohydrates, alcohols and relatedcompounds. Many of these oxidations are unfeasible using organicchemistry. Because these reactions are mostly catalyzed by membranebounddehydrogenases, in a rapid, regio- and stereo-selective manner, thesubstrates do not have to be transported into the cytoplasm. Due to the factthat many acetic acid bacteria can not be cultivated in the laboratory weuse a metagenomic approach to indentify new membrane-bounddehydrogenases of potential value for biotechnology from a mother ofvinegar.The membrane-bound dehydrogenases are screened by sequence similarityfrom the metagenomic library and are functionally expressed in speciallydesigned Gluconobacter oxydans strains. In these strains all membranebounddehydrogenases were deleted using a clean deletion systemdeveloped by our group to avoid overlapping enzymatic specificities.Using specifically designed expression vectors we ensure functionalintegration in the membrane physiology of these organisms.In order to set up a high throughput assay to characterize the activity ofmembrane-bound dehydrogenases, we developed a whole cell system inmicrotiter-plates. The advantage of this system is a minimized cellpreparation together with the ability to compare many stains or substratesin one experiment. We used this approach to determine the in vivosubstrate spectrum of several membrane-bound dehydrogenases fromacetic acid bacteria for the first time.OTV015Growth phase dependent changes of the RNA degradingexosome in Sulfolobus solfataricusC. Witharana*, L. Hou, C. Lassek, V. Roppelt, G. Klug, E. Evguenieva-HackenbergJustus-Liebig-Universität Giessen, Institut für Mikrobiologie undMolekularbiologie, Gießen, GermanyWe are investigating the exosome of the hyperthermophilic and acidophilicarchaeonSulfolobus solfataricus(1).The archaeal exosome is a proteincomplex involved in the degradation and the posttranscriptional tailing ofRNA. The core of the complex is build of a phosphorolytically activehexameric ring of the subunits Rrp41 and Rrp42, to which a trimeric capof the RNA-binding proteins Rrp4 and/or Csl4 attaches (2). Rrp4 and Csl4confer different substrate specificity to the exosome (3). In addition tothese subunits, the archaeal DnaG protein is stably associated with theexosome (4). The majority of the protein complex including DnaG islocalized at the periphery of the cell and is detectable in the non-solublefraction (5). Here we show that DnaG directly interacts with Csl4 in theexosome, and that it differently influences the activity of complexes withhomotrimeric Rrp4- or Csl4-capsin vitro. We confirmed the existence ofBIOspektrum | Tagungsband 2012