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

144bacteria were resistant to acid, bile and digestive enzymes and were shownto lower the cholesterol levels in mice model. Bacterial cultures werecentrifuged and the pellets were washed (3 times) and were suspended inin AFB1 solution with final concentration of 1-1.5×10 10 CFU/ml. thebacterial solutions were incubated for 2 h at 37°C. The cell freesupernatants samples were analyzed with a reverse phase highperformanceliquid chromatography (HPLC) as well as the Enzyme LinkedImmunosorbent Assay (ELISA).Results: The results showed that the AFB1 binding capacity of strains wasstrain dependent. The strains were observed to possess variable AFB2-binding ability in the range from 8 to 63%. Most efficient binding of AFB1was observed byL.plantarumTD14, andL.caseiTD15. The differences inthe binding activities of AFB1 between the strains showed statisticalsignificance (p>0.05). Our results indicated the protective ability of theseindigenous probiotic strains against AFB1as well as their beneficial healtheffects. It is well documented that the AFB1 detoxification by thesebacteria involves sequestration by binding the toxin to the bacterial cellwall. These findings suggest that certain novel probiotic bacteria isolatedform Iranian traditional dairy products with high aflatoxin binding capacitycould be selected for detoxification of foods.OTP028Identification of the protoporphyrinogen IX oxidase inPseudomonas aeruginosaD. Zwerschke*, M. Jahn, D. JahnInst. f. Microbiologie, AG Jahn, Braunschweig, GermanyHeme is an important tetrapyrrole because of its function as a cofactor inseveral proteins which are linked to fundamental biological processes likerespiration, photosynthesis, the metabolism and transport of oxygen (Layeret al, 2010). The biosynthesis of heme is a well studied process,nevertheless there are bacteria which obviously lack one or more of theknown enzymes for this pathway and are still able to synthesize heme. Forat least three steps during the heme formation it is known that there existother enzymes responsible for catalysis (Boynton et al, 2011). One of theseenzymes is the protoporphyrinogen IX oxidase (PPO). PPO oxidizesprotoporphyrinogen IX to protoporphyrin IX which is the penultimate stepin the heme biosynthetic pathway (Layer et al, 2010). Until today noknown PPO-gene has been identified for Pseudomonas aeruginosa. Ourapproach was to isolate the oxygen-dependent PPO-gene from P.aeruginosa by complementation of an Escherichia coli PPO mutant with aP. aeruginosa ATCC 17933 gene library. UV/Vis and fluorescence spectrawere recorded via high pressure liquid chromatography to measure thelevel of heme in apparently positive clones. Complementary genes fromclones with high heme levels were sequenced. To investigate so obtainedputative PPO we will knock out these genes in P. aeruginosa andcomplement the phenotype with the E. coli PPO (hemG). Furthermore, wewill overproduce the putative P. aeruginosa PPO for their biochemicalcharacterization.Layer G., Reichelt J., Jahn D. and Heinz D. (2010) Structure and function of enzymes in hemebiosynthesis. Protein Sci. 19(6): 1137-1161Boynton T.O., Gerdes S., Craven S.H., Neidle E.L., Phillips J.D. and Dailey H.A. (2011) Discoveryof a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through ComparativeGenomic Analysis and Functional Complementation. Appl. and Environmental Microbiol. 77(14):4795-4801OTP029Isolation of Streptomyces integrative chromosomal elements byrolling-circle amplificationD. Heimlich*, N. Osipenkov, W. Wohlleben, G. MuthUniversität Tübingen, Mikrobiologie/Biotechnologie, Tübingen, GermanyThe order Actinomycetales consists of high G+C Gram-positive bacteriaof which many species form a branching mycelium by apical tip extension.Actinomycetes, in particular the genus Streptomyces, are the mostimportant source of biologically active microbial products, includingantibiotics. As antibiotic producers, the actinomycetes represent the naturalreservoir of resistance genes that are transferred to other bacteria byhorizontal gene transfer (HGT).The availability of genomic sequences of many actinomycetes revealed thepresence of multiple integrative chromosomal elements (ICE). ICE´s arecharacterized by their prophage-like mode of maintenance as part of thechromosome, and their ability to excise, to promote their transfer to a newhost, and to integrate into the host genome by site specific recombination.Since ICE´s normally disintegrate only prior to conjugation, which isregulated by unknown factors, such elements are very difficult to isolateby alkaline lysis.Here we show that it is possible to amplify novel ICE´s from differentStreptomyces strains using random hexamer primers and the Phi29 DNApolymerase. Sequence analysis of subcloned DNA fragments allows therapid characterization of the newly isolated Streptomyces ICE´s.The presented work was done as part of the “MikrobiologischesGroßpraktikum/Biotechnologie ( 1 ) and a bachelor thesis ( 2 ).OTP030A highly efficient Staphylococcus carnosus mutant selectionsystem based on suicidal bacteriocin activationB. Krismer*, M. Nega, G. Thumm, F. Götz, A. PeschelUniversity of Tuebingen, IMIT, Tuebingen, GermanyStrains from various staphylococcal species produce bacteriocin peptides,which are thought to play important roles in bacterial competition andoffer interesting biotechnological avenues. Many bacteriocins are secretedas inactive pre-peptides with subsequent activation by specific proteolyticcleavage. By deletion of the protease gene gdmP in Staphylococcusgallinarum Tü3928, which produces of the highly active lanthioninecontainingbacteriocin gallidermin (lantibiotic), a strain was createdproducing inactive pre-gallidermin. On this basis a new suicidal mutantselection system in the food-grade bacterium Staphylococcus carnosus wasdeveloped. Whereas pre-gallidermin was inactive against S. carnosus, itexerted potent bactericidal activity toward GdmP-secreting S. carnosusstrains. To take advantage of this effect gdmP was cloned in plasmidvectors used for random transposon mutagenesis or targeted allelicreplacement of chromosomal genes. Both mutagenesis strategies rely onrare recombination events and it has remained difficult and laborious toidentify mutants among a vast majority of bacterial clones that still containthe delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2enabled very fast, easy, and reliable identification of transposon or genereplacement mutants, respectively. Mutant selection in the presence of pregallidermincaused suicidal inactivation of all clones that had retained theplasmids and allowed only growth of plasmid cured mutants. Efficiency ofmutant identification was several magnitudes higher compared to standardscreening for the absence of plasmid-encoded antibiotic resistance markersand reached 100% specificity. Thus, the new pre-gallidermin based mutantselection system represents a substantial improvement of staphylococcalmutagenesis methodology.OTP031Reductive dechlorination in Desulfitobacterium hafniense Y51:Impact of vitamin B 12 on pceA gene stability and expressionA. Reinhold*, T. Schubert, G. DiekertFriedrich-Schiller-University, Institute for Microbiology, Department ofApplied and Ecological Microbiology, Jena, GermanyDesulfitobacterium hafniense Y51 is a strictly anaerobic, gram-positivebacterium, which is able to grow with aliphatic chlorinated compounds,such as tetrachloroethene (PCE), as terminal electron acceptors. PCE isreductively dechlorinated to cis-1,2-dichloroethene. The key enzyme is thePCE reductive dehalogenase, a corrinoid cofactor and iron-sulfur clustercontaining protein. All enzymes required for de novo corrinoid cofactorbiosynthesis are encoded in the genome of D. hafniense Y51 (1). The geneencoding the PCE reductive dehalogenase, pceA, is organized in thepceABCT gene cluster. The cluster is flanked by two almost identicalinsertion sequences including transposase genes. The excision of the pcegene cluster from the genome of D. hafniense Y51 can occur (2).In this study we investigated the impact of vitamin B 12 added to themedium on the transposition of the pceA gene. In the presence of thegrowth substrate PCE the pceA gene remains stable in D. hafniense Y51genome whether or not vitamin B 12 was added to the culture. When cellswere cultivated on fumarate instead of PCE and vitamin B 12 was omittedfrom the medium, the number of pceA genes per culture decreased rapidly.Interestingly, this effect is strongly delayed when external vitamin B 12 wasprovided (long-term effect).To acquire the data presented here, cells repeatedly grown on the differentmedia compositions were analysed for the number of pceA genes usingquantitative PCR (qPCR), for the pceA transcript level using reversetranscription quantitative PCR (RT-qPCR), for the PceA enzyme activityand the amount of PceA protein using specific antibodies. In parallel, theexpression of vitamin B 12 biosynthesis genes was examined. Based on theresults of this survey a positive effect of vitamin B 12 on the pceA genestability and expression in D. hafniense Y51 is discussed.(1) Nonaka H. et al. (2006) J Bacteriol 188, 2262-2274.(2) Futagami T. et al. (2006) Appl Microbiol Biotechnol 70, 720-728.OTP032TrxR system - A new target in the fight against MycobacteriumtuberculosisN. Rücker* 1 , O. Koch 2 , K. Heller 3 , F. Stuhlmann 3 , S. Schmitt 3 ,P.C. Khandavalli 3 , D. Schinzer 3 , L. Flohé 3 , P.M. Selzer 2 , F.-C. Bange 1 , T. Jäger 31 Medizinsche Hochschule Hannover, Institut für medizinische Mikrobiologie,Hannover, Germany2 Intervet/SP , Animal Health, Schwabenheim, Germany, Germany3 MOLISA GmbH, Magdeburg, Germany, GermanyMycobacterium tuberculosis (Mtb)depends on an efficient anti-oxidativesystem during infection. To maintain the survival, Mtb relies on theThioredoxin Reductase (TrxR) system, because it lacks a glutathioneBIOspektrum | Tagungsband 2012