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

100that the genes for AOH polyketide synthase and AOH-O-methyltransferase arein close proximity. Therefore identifying the alternariol-O-methyltransferasewill also reveal the responsible polyketide synthetase.Putative methyltransferases were also identified by BLAST-analysis in thegenome of the close relative A. brassicicola and the sequences were usedto clone several SAM dependent methyltransferases of Alternariaalternata. Three partial and one total sequence were cloned.With the active expression of the identified genes being not easy, thealternariol-O-methyltransferase of Alternaria alternata was alsocharacterized in crude extracts and partially purified. An SAM dependentactivity-test was developed to identify the enzyme. The products wereanalysed by HPLC. With the N-terminal sequence of the enzyme it shouldbe possible to determine the gene.1) Gatenbeck and Hermodsson (1965): Enzymic Sythesis of Aromatic Product Alternariol2) Stinson and Moreau (1986): Partial Purification and some Properties of an Alternariol-o-Methyltransferase fromAlternaria tenuis.3)http://www.vbi.vt.edu/archive/pdf/public_relations/annual_report/2009/ar2009-sci-07-lawrence.pdf4) Fetzner R., Lawrence C., Fischer R. (2011). Molecular analysis of secondary metabolite biosynthesis inAlternaria alternata. Biospektrum Special 2011: 128.5) Keller NP., Hohn TM. (1997). Metabolic pathway gene clusters in filamentous fungi. Fungal Geneticsand Biology 21: 17-29.MEP034The friulimicin producer Actinoplanes friuliensisN. Fischer*, N. Wagner, R. Biener, D. SchwartzUniversity of Applied Sciences Esslingen, Biotechnology, Esslingen,GermanyFriulimicin, a lipopeptide antibiotic produced by the rare actinomyceteActinoplanes friuliensis, is active against a broad range of multiresistantgram-positive bacteria such as methicillin-resistant Enterococcus sp.andStaphylococcus aureus (MRE, MRSA) strains.The complete biosynthetic gene cluster was characterized by sequenceanalysis and four different regulatory genes (regA, regB, regC and regD)were identified within the cluster (Müller et al., 2007).Knockout-mutants missing the regulatory genes regC/D showed nonproductionof friulimicin as well as deficiency in carotenoid pigmentsynthesis which indicates a pleiotropic mechanism of action of theencoded bacterial two component system.An in silico analysis of the A. friuliensis genome revealed the presence ofseveral fatty acid biosynthesis genes outside of the known biosyntheticgene cluster, that might be involved in biosynthesis of the lipid part of theantibiotic. Among others three putative FabH genes (-Ketoacyl-AcylCarrier Protein Synthase III) could be identified.To verify the role of these genes in antibiotic biosynthesis transcription analysisby RT Realtime-PCR as well as gene inactivation experiments are carried out.Moreover three so far unknown secondary metabolite NRPS- and one PKSgenecluster as well as genes responsible for carotenoid biosynthesis andflagella formation could be identified and are under further investigation.To study the formation of spore flagella the growth conditions forsporangia formation and sporulation were determined and analyzed byscanning electron microscopy and RT-Realtime PCR. Additionallydifferent methods for enrichment of spores were tested to improve andfacilitate the intergeneric conjugation procedure for A. friuliensis.MEP035Inhibition of quorum sensing in Gram-Negative bacteria by astaphylococcal compoundY.-Y. Chu* 1 , M. Nega 1 , M. Wölfle 2 , M.T. Nguyen 1 , S. Grond 2 , F. Götz 11 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin,Department of Microbial Genetics, Tuebingen, Germany2 Institut für Organische Chemie , Tuebingen, GermanyBacteria use signal molecules to regulate population density in a process ofbacterial communication called quorum sensing. This process plays criticalroles in regulating various physiological activities, including production ofantibiotics, secretion of virulence factors, formation of biofilms, swarmingmotility, bioluminescence, sporulation as well as symbiosis. Similarly, it isfound that various bacteria are able to secrete compounds for inhibiting,inactivating or stimulating quorum sensing signals in other bacteria. In ourprevious study on coinfection ofStaphylococcusandPseudomonasaeruginosa, we observed thatP. aeruginosacould repress the growth ofpathogenic staphylococcalspeciesbut not of nonpathogenicstaphylococcalspeciesby respiratory inhibitors [1]. Meanwhile, to oursurprise, some strains of the nonpathogenic staphylococcalspeciesexhibitunknown compound X to interrupt the function of quorum sensingcontrolledfactors in gram-negative bacteria, such as the red prodigiosinpigment inSerratia marcescens, the blue-green pyocyanininP.aeruginosaand bioluminescence inVibrio harveyi.Physical analysisusing XAD-16 resin and dialysis membrane demonstrated that themolecular weight of compound X is below 2 kDa. Moreover, compound Xresists alkaline and acid pH, high temperature and proteinase K treatment,which might exclude compound X as a normal peptide. However, themechanism of compound X expression is still unknown since it isindependent of the growth temperature, and oxygen concentration in themedium. In further study, not only purification and identification of thecompound X using high-performance liquid chromatography (HPLC) andmass spectrometry (MS) are essential. It also needs to indentify thecorresponding genes by transposon mutagenesis and cloning randomchromosomal DNA of compound producing staphylococcal stain into anonproducing strain.In the end, investigation of how compound X disruptsthe quorum sensing signaling system in gram-negative bacteria would bean important and interesting issue for new generation of antibiotics.1. Voggu L, Schlag S, Biswas R, Rosenstein R, Rausch C, Götz F.,J Bacteriol,2006,188(23),8079-86.MEP036Effect of gallidermin on biofilm of Staphylococcus aureus andStaphylococcus epidermidisJ. Saising* 1,2 , L. Dube 1 , A.-K. Ziebandt 1 , M. Nega 1 , S. Voravuthikunchai 2 ,F. Götz 11 University of Tuebingen, Microbial Genetics, Tuebingen, Germany2 Prince of Songkla University, Microbiology, Songkhla, ThailandStaphylococcus aureus and S. epidermidis are widely involved in minor tosevere infection. A major problem is the arising of highly virulent andmultiple resistant clones and the manifestation of persistent infections dueto biofilm-forming strains. Once a biofilm is formed during infection,particularly implant-associated infections, therapy is extremely difficultdue to the antibiotic resistance in a biofilm community. The objective ofthis study was to investigate the activity of gallidermin with respect toprevent biofilm formation and to kill staphylococci once a biofilm hasbeen formed. For planktonic grown S. aureus and S. epidermidis theminimal inhibitory concentration (MIC) and minimal bactericidalconcentration (MBC) values of gallidermin was in the order of 4-8 g/ml.This gallidermin concentrating is also sufficient to prevent biofilmformationof both species representatives. Also, the viability of 24 h and 5-day staphylococcal biofilm grown cells is significantly decreased aftertreated with gallidermin. We also investigated the effect of gallidermin onthe expression of biofilm-mediating genes such as major autolysin (atl)and PIA-synthesizing intercellular adhesin (ica). Northern blot analysisrevealed that in the presence of gallidermin the corresponding transcriptswere significantly decreased. Our finding indicates that galliderminefficiently prevents biofilm formation in staphylococci and represents agood candidate for treatment for appropriate therapy.MEP037the cyanobacterial toxin microcystin binds to proteins invivo and plays an essential role in oxidative stress response inMicrocystis aeruginosaS. Meissner* 1 , Y. Zilliges 2 , J.-C. Kehr 1 , M. Hagemann 3 , E. Dittmann 11 University of Potsdam, Department of Microbiology, Potsdam-Golm,Germany2 Humboldt University, Department of Molecular Ecology, Berlin, Germany3 University of Rostock, Department of Plant Physiology, Rostock, GermanyCyanobacteria produce a variety of secondary metabolites with yetunknown functions. Microcystin is one of the most intensely studiedsecondary metabolites due to its regular involvement in toxic freshwatercyanobacterial mass developments. Here we describe a new function ofmicrocystin acting on proteins of Microcystis aeruginosa PCC 7806,which indicates a putative involvement in physiological processes of theproducing organism.The phenotype of the microcystin deficient mcyB mutant showsincreased susceptibility towards high light conditions of above 300 E/ m 2· s when compared to the microcystin producing wild type. Microcystincovalently binds to Microcystis proteins in vivo and exposition to highlight strongly facilitates the binding. In vitro, blocking of free sulfhydrylgroups of proteins in mcyB mutant extracts disables the binding.Accordingly, microcystin most likely interacts with cysteinesof Microcystis proteins to form a stable thioether bond. One of the mostprominent binding partners of microcystin is the large subunit of thecarbon fixing enzyme RubisCO (RbcL). Interestingly, the binding ofmicrocystin to RbcL renders the enzyme less susceptible towardsproteolysis by the serine protease subtilisin. Comparative proteomicstudies revealed altered accumulation patterns of several Calvin cycleenzymes including RubisCO as a consequence to the loss of microcystinproduction.Altogether the findings outlined above strongly suggest an importantphysiological role of microcystin with regard to modifying the proteomeof Microcystis and increasing the capability of the cells to handleconditions triggering oxidative stress.Zilliges Y, Kehr J-C, Meissner S, Ishida K, Mikkat S, et al. (2011) The Cyanobacterial HepatotoxinMicrocystin Binds to Proteins and Increases the Fitness of Microcystis under Oxidative StressConditions. PLoS ONE 6(3): e17615. doi:10.1371/journal.pone.0017615BIOspektrum | Tagungsband 2012