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

101MEP038A new arylsulfate sulfotransferase involved in liponucleosideantibiotic biosynthesis in streptomycetesK. Eitel* 1 , L. Kaysser 2 , T. Tanino 3 , S. Siebenberg 1 , A. Matsuda 3 ,S. Ichikawa 3 , B. Gust 11 University of Tübingen, Pharmaceutical Institute, Tübingen, Germany2 Scripps Institution of Oceanography, La Jolla, United States3 Faculty of Pharmaceutical Science, Hokkaido, JapanSulfotransferases are involved in a variety of physiological processes andtypically use 3-phosphoadenosine 5-phosphosulfate (PAPS) as the sulfatedonor substrate. In contrast, microbial arylsulfate sulfotransferases(ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors.Yet, their genuine acceptor substrates are unknown. Here, we demonstratethat Cpz4 fromStreptomyces sp. MK730-62F2 is an ASST-typesulfotransferase responsible for the formation of sulfated liponucleosideantibiotics[1]. Gene deletion mutants showed that cpz4 is required for theproduction of sulfated caprazamycin derivatives.Cloning, overproduction, and purification of Cpz4 resulted in a 58-kDasoluble protein. The enzyme catalyzed the transfer of a sulfate group fromp-nitrophenol sulfate (Km 48.1 m, kcat 0.14 s1) and methylumbelliferone sulfate (Km 34.5 m, kcat 0.15 s1) onto phenol (Km 25.9and 29.7 mm, respectively). The Cpz4 reaction proceeds by a ping pongbi-bi mechanism. Several structural analogs of intermediates of thecaprazamycin biosynthetic pathway were synthesized and tested assubstrates of Cpz4. Des-N-methyl-acyl-caprazol was converted withhighest efficiency 100 times faster than phenol. The fatty acyl side chainand the uridyl moiety seem to be important for substrate recognition byCpz4. Liponucleosides, partially purified from various mutant strains werereadily sulfated by Cpz4 using p-nitrophenol sulfate. No product formationcould be observed with PAPS as the donor substrate. Sequence homologyof Cpz4 to the previously examined ASSTs is low. However, numerousorthologs are encoded in microbial genomes and represent interestingsubjects for future investigations.[1] L. Kaysser, K. Eitel, T. Tanino, S. Siebenberg, A. Matsuda, S. Ichikawa and B. Gust, J BiolChem.285(2010):12684-94.MEP039Tools for the analysis of metagenomic libraries regarding theproduction of secondary metabolites with biosurfactantpropertiesS. Thies* 1 , F. Rosenau 2 , S. Wilhelm 1 , K.-E. Jaeger 11 Heinrich-Heine-Universität , Institute for Molecular Enzyme Technology,Düsseldorf, Germany2 Universität Ulm, Institut für Pharmazeutische Biotechnologie, Ulm, GermanyMicrobiological produced compounds with tensidic properties(“biosurfactants”) may be useful alternatives for chemical synthesizedcompounds.Secondary metabolites like biosurfactants are synthesized in metabolicpathways with individual reactions catalysed by different enzymes. Inbacteria, the genes which encode enzymes involved in the same pathwayare often organized in gene clusters, meaning they are all localized in oneparticular region of the chromosome.In the case of biosurfactants, known gene cluster sizes are between ca.3.000 base pairs (bp) and up to 70.000 bp.Aim of this project is the construction of metagenomic libraries with DNAfragments containing clusters encoding enzymes of surfactant producingpathways. Constructing metagenomic libraries requires transfer of geneticinformation of certain habitats including non-cultivatable organisms whichcan exclusively live in those niches into a usable form by isolating theDNA directly from the environment and cloning it into appropriatevectors. By expression of the metagenomic DNA in suitable hosts,products of biosynthetic pathways like biosurfactants encoded by thisDNA can be identified. Since already the identification is done in wellestablishedand safe expression strains this method not only makes novelcompounds available but also ensures an option for recombinantproduction in these platform production strains. Promising habitats to findstrains producing tensidic molecules are fatty and oily environments likeslaughterhouses or tannery. Libraries containing DNA originated in suchhabitats will be screened for producing compounds with tensidic properties.At present construction of a novel expression vector for libraryconstruction is finished which allows the expression of gene clustersencoded in both directions of an inserted DNA fragment. Enabling a rapidworkflow as required for efficient work with the large libraries, we haveoptimized a recently developed fast screening method for biosurfactantproduction concerning our purposes.MEP040Anti-micobial activity of soil-living Bacillus species againsthuman pathogenic and sepsis-related bacteriaO. Makarewicz, M. Klinger*, M. PletzUniversitätsklinikum Jena, Sektion Klinische Infektiologie, Jena, GermanyObjectives: Soil living bacteria are known to produce compounds thatpromote plant growth and confer resistance to plant diseases caused bydifferent pathogens [1, 2]. The rhizosphere can be colonized bybiofilmformation by various species, thus anti-microbials ensure alsosurvival advantage against competing commensals. For example, B.amyloliquefaciensstrain FZB42 secrets at least 12 known antibiotics,which inhibit growth and destroy biofilms of other microorganisms andthat belong to different chemical classes: lipopetides, polyketides, smallpeptides [3]. The aim of our efforts is to scrren culture supernatants of soliliving Gram+ for novel substances with activity against biofilms of multidrugresistant major bacterial human pathogens involved into catheter- anddevice associated infections.Methods: We used supernatants of B. amyloliquefaciens(n=5), B. pumilus(n=1), B. licheniformis(n=1) and P. polymyxa (n=3) that were filtered,lyophilized and resuspended in 1/10 volume in sterilized water.Supernatants were used in disc diffusion tests against multi-drug resistantisolates of E. coli (n=3), K. pneumoniae (n=2), P. auruginosa(n=4), S.aureus (n=3), E. faecalis (n=2) and P. mirabilis (n=1) as indicator strains.A more detailed analysis of active compounds was performed usingbioautography based on thin layer chromatography.Results: Supernatants of P. polymyxa strainsexhibited strongest antimicrobialactivity against Gram+ and Gram- pathogens. B. amyloliquefaciensFZB 42 showed also high activities against all indicator strains. B. pumilus andB. licheniformis inhibited mainly growth of Gram+.Conclusion: Gram-positives soil living bacteria secrete a wide spectrum of bioactivesecondary metabolites, which can inhibit the growth of humanpathogens. Further experiments will concentrate on identification of particularsubstances antimicrobial activity and analyze their anti-biofilm activities.1. Verhagen, B.W., et al.,Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associatedwith induction and priming of defence responses in grapevine.J Exp Bot, 2010.61(1): p. 249-60.2. Ko, H.S., et al.,Biocontrol Ability of Lysobacter antibioticus HS124 Against Phytophthora Blight IsMediated by the Production of 4-Hydroxyphenylacetic Acid and Several Lytic Enzymes.Curr Microbiol,2009.3. Chen, X.H., et al.,Comparative analysis of the complete genome sequence of the plant growth-promotingbacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol, 2007.25(9): p. 1007-14.MEP041Identification of a gene participating in the sulphurmetabolism in Oenococcus oeniC. Knoll 1 , M. du Toit 2 , S. Schnell 3 , D. Rauhut* 4 , S. Irmler 51 Hochschule RheinMain, Fachbereich Geisenheim, Geisenheim, Germany2 University Stellenbosch, Institute for Wine Biotechnology, Stellenbosch, SouthAfrica3 Justus-Liebig-Universität Gießen, Institute for Applied Microbiology, Gießen,Germany4 Forschungsanstalt Geisenheim, Microbiology and Biochemistry, Geisenheim,Germany5 Forschungsanstalt Agroscope Liebefeld-Posieux ALP, Bern, SwitzerlandSulphur-containing compounds in wine have a high impact on wineflavour and quality. Recent studies demonstrated that Oenococcus oeni isable to produce, from methionine, different volatile sulphur compounds(VSC) (Pripis-Nicolau et al. 2004), but no specific enzymes have beenidentified and characterised so far.In this research work an enzyme that degrades sulphur-containing aminoacids was identified, heterologous expressed in Escherichia coliBL21(DE3) and biochemically characterised from two O. oeni strains ofoenological origins. The amplified PCR product consisted of 1140nucleotides encoding a deduced protein of 379 amino acids and was highlyconserved among the compared O. oeni strains.The enzyme has characteristics of a cystathionine-g-lyase (EC4.4.1.1), apyridoxal-5-phosphate-dependent enzyme catalyzing an a,g-eliminationreaction of l-cystathionine to produce l-cysteine, a-ketobutyrate andammonia. Moreover, it was able to catalyse an a,-elimination reactionsynthesizing homocysteine, pyruvate and ammonia from l-cystathionine.An elimination reaction of l-cysteine and dl-homocysteine was alsoefficiently catalysed by the enzyme, resulting in the formation of H 2S.Furthermore, the ability to demethiolate methionine into methanethiol, anunfavourable volatile sulphur substance, was shown.Climate change and specific vinification practices can result in wines withhigh alcohol concentrations (>13 % (v/v)). It could be demonstrated thatethanol contents up to 15 % (v/v) had no impact on the activity of thepurified enzymes. Furthermore, the enzymes were stable at temperaturessuitable for the wine production and storage. If l-cystathionine was used assubstrate, the enzyme activity was highest at pH 8.0. No activity wasobserved at a pH below 6.5. In, contrast, l-methionine was degraded at pH5.5 and 6.Therefore further work with natural substrates will be necessary todetermine its influence on the VSC production in wine (Knoll et al. 2011).BIOspektrum | Tagungsband 2012