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

180PSP018Screening for genes of Staphylococcus aureus that are involvedin the formation of persister cellsL. Mechler*, M. Zelder*, S. Lechner, M. Prax, R. BertramUniversity, IMIT, Microbial Genetics, Tübingen, GermanyPersisters are phenotypic variants of bacterial cells among a geneticallyidentical population. These slow- or non-growing (dormant) cells aretolerant to antibiotics and are formed both stochastically and in adaptationto adverse conditions. Persisters seem to be causative for the recalcitranceof chronic infections to antimicrobial therapy. Notably, the molecularmechanisms underlying this kind of dormancy largely remain unclearparticularly in bacteria beyond E. coli. We aimed at identifying genesgoverning the formation of persister cells in Staphylococcus aureus usingtwo different strategies.First, a screen for mutants exhibiting elevated persister levels was set up. 1to 3.5 kbp fragments of a genomic library of Staphylococcus aureusSA113 as well as S. aureus homologs of E. coli persister genes dnaJ, glpD,umuC and the toxin-antitoxin (TA) RNases yoeB-sa1 and yoeB-sa2 werecloned into plasmid pRAB11 for tetracycline inducible control. Uponexpression in SA113, mutants that show a reduced growth rate andenhanced antibiotic tolerance will be isolated. Sequencing of respectiveDNA fragments may thus reveal new or verify suspect S. aureus persister genes.A second approach aims at generating S. aureus strains with decreasedpersister levels. To this end, single and combinational deletion mutants ofS. aureus TA loci are constructed. These include the three verifiedchromosomally encoded systems mazEF, yefM-sa1/yoeB-sa1 and yefMsa2/yoeB-sa2,as well as two further putative TA-loci identified by insilico analysis. To remove resistance markers from newly generatedmutant strains we are establishing the use of the yeast derived Flp/FRTrecombination system in staphylococci. Obtained strains will be examinedfor persister formation and decreased antibiotic tolerance would supportthe hypothesis that TA-systems are crucial for persister formation instaphylococci.PSP019Will not be presented!PSP020Denitrification pathway is essential for complete functionalmagnetosome crystals for magnetic orientation inMagnetospirillum gryphiswaldenseY. Li, E. Katzmann*, S. Borg, D. SchülerLudwig-Maximilians-Universität München, Department 1, MikrobiologieAG-Schüler, Planegg-Martinsried, GermanyMagnetosomes are unique bacterial organelles used by magnetotacticbacteria (MTB) to orient in the Earth’s magnetic field. In the -proteobacterium M. gryphiswaldense (MSR-1) magnetosomes are crystalsof magnetite (Fe 3O 4) which are biomineralized within specific vesicles ofthe magnetosome membrane. Maximum magnetite synthesis occurs only atlow oxygen concentrations and in the presence of nitrate, suggesting apotential metabolic link between denitrification and magnetitebiomineralization. However, no genetic evidence has been available in vivo.Here we reconstructed a complete pathway of denitrification from thegenome of MSR-1, including gene functions for nitrate (nap), nitrite(nirS), nitric oxide (norCB), and nitrous oxide reduction (nosZ). Bycharacterizing deletion mutants of all genes, we showed that all proteinsare required for anaerobic growth. In addition, deletions of norCB,nirS andnap impaired magnetite synthesis. The loss of norCB caused shortermagnetosome chains in ammonium medium, suggesting that nitric oxidereduction is involved in magnetosome formation also in microaerobicaerobic respiration. Deletion of the nap operon resulted in fewer, smallerand irregular crystals not only during denitrification but also aerobicrespiration, probably due to disturbed redox balance for magnetitesynthesis. Magnetite induction experiments by iron addition innonmagnetic WT and nirS cells under reduced and oxidized conditionsrevealed that the, nitrite reductase NirS is likely involved in anaerobicmagnetosome formation by oxidizing ferrous to ferric iron. This processprobably takes places in the periplasm by providing electrons for nitritereduction, and ferric iron may subsequently be transported intomagnetosome vesicles for magnetite synthesis.Altogether, we provide evidence that the denitrification pathway has a keyrole for magnetite biomineralization by participating in redox reactions.This also shows that in addition to the various essential and accessoryfunctions encoded within the genomic magnetosome island, also genesoutside that region are involved in synthesis of functional magnetosomeparticles.PSP021A blueprint of organohalide respiration: Functional genomeanalysis of Sulfurospirillum multivoransT. Goris* 1 , T. Schubert 1 , T. Wubet 2 , M. Tarkka 2 , L. Adrian 3 , G. Diekert 11 Friedrich Schiller University, Institute of Microbiology, Department ofApplied and Ecological Microbiology, Jena, Germany2 Helmholtz Centre for Environmental Research - UFZ, Department of SoilEcology, Halle, Germany3 Helmholtz Centre for Environmental Research - UFZ, Department ofIsotope Biogeochemistry, Leipzig, GermanySome of the most frequently detected contaminants in groundwater arehalogenated organic compounds. Among them, tetrachloroethene (PCE) isthe most abundant one. Due to its inertness, PCE is not easily degradableand persistent under oxic conditions. However, several anaerobic bacteriaare able to couple the reductive dechlorination of PCE and of otherorganohalides to energy conservation via electron transportphosphorylation. Therefore, this process is often referred to asorganohalide respiration.Here, we present the functional analysis of the complete genome sequenceof Sulfurospirillum multivorans, an -proteobacterium capable ofdechlorinating tetrachloroethene (PCE) to dichloroethene. The latter can bereadily degraded to non-toxic compounds by aerobic microorganisms. Thehigh metabolic versatility of S. multivorans is reflected in one of thelargest genomes of the -proteobacteria, comprised of a single circularchromosome 3.1 Mbp in length and includes more than 3,200 open readingframes. Close to the region coding for the PCE dehalogenase, which is acorrinoid-containing Fe-S cluster enzyme, corrinoid biosynthesis genes arelocated, and surprisingly genes coding for an additional putative reductivedehalogenase. Furthermore, we identified genes coding for all componentsof an aerobic respiratory chain and the TCA cycle. Together with currentresults from growth experiments, the data point to the first known exampleof an organohalide respiring organism capable of thriving in microaerobicenvironments.In order to fill in the gaps in the understanding of anaerobic biologicaldehalogenation, this genome sequence of a gram-negative organohaliderespiring bacterium is a big step toward to the complete elucidation of anoutstanding way of microbial energy conservation.Acknowledgement: This work is supported by the DFG (research unit FOR1530) and the UFZ -Helmholtz centre for Environmental ResearchPSP022Genomic and transcriptomic insights into Allochromatiumvinosum DSM 180 T with special focus on genes involved indissimilatory sulfur metabolismT. Weissgerber* 1 , N. Dobler 2 , T. Polen 2 , C. Dahl 11 Universität Bonn, Institut für Mikrobiologie und Biotechnologie, Bonn,Germany2 Forschungszentrum Jülich, Institut für Bio- und Geowissenschaften IBG-1:Biotechnologie, Jülich, GermanyAnoxygenic purple sulfur bacteria like the GammaproteobacteriumAllochromatium vinosum, a member of the Chromatiaceae, flourishwherever light reaches sulfidic water layers or sediments and often occuras dense accumulations in conspicuous blooms in freshwater as well as inmarine aquatic ecosystems. Here they are major players in the reoxidationof sulfide produced by sulfate-reducing bacteria in deeper anoxic layers.The capability to oxidize reduced sulfur compounds is the centralmetabolic feature of A. vinosum during photolithoautotrophic growth.Light energy is used to transfer electrons from reduced sulfur compoundssuch as sulfide, polysulfide, thiosulfate, sulfur and sulfite to the level of themore highly reducing electron carriers NAD(P) + and ferredoxin forreductive carbon dioxide fixation.Here, we present a set of features of the complete genome (Acc:CP001896.1) of A. vinosum, the first member of the Chromatiaceae, forwhich a complete genome sequence is available. The genome consists of a3,526,903 bp chromosome and two plasmids of 102,242 bp and 39,929 bp,respectively, with a total number of 3,366 predicted genes. A globaltranscriptomic analysis was performed with a special focus on oxidativedissimilatory sulfur metabolism in A. vinosum. To this end, total RNA wasisolated after photolithoautotrophic growth on sulfide, thiosulfate, sulfur orsulfite as electron donor and compared to total RNA extracted fromcultures grown photoorganoheterotrophically on malate. Firstly, theseexperiments confirmed the increased expression of genes encodingproteins already known to be involved in oxidative sulfur metabolism.Among these are the dsr genes [1] including dsrAB for dissimilatory sulfitereductase and the sgp genes for the proteins of the sulfur globule envelope[2]. Secondly, we also detected a number of interesting candidate genesthat are highly upregulated in the presence of reduced sulfur compounds.Among these are several genes encoding potential sulfur relay proteinspredicted to reside in the cytoplasm. Notably, transcription of some genesappeared to be specifically increased on insoluble sulfur. One of thesegene products belongs to the lipocalin family of proteins. Members of thisBIOspektrum | Tagungsband 2012