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

94MEP007Identification and toxigenic potential of a cyanobacterialstrain (nostoc sp.)B. Nowruzi* 1 , R.A. Khavari-Nejad 1,2 , K. Sivonen 3 , B. Kazemi 4,5 , F. Najafi 1 ,T. Nejadsattari 21 Tarbiat Moallem University, Department of Biology, Faculty of Science,Tehran, Iran, Islamic Republic of2 Islamic Azad University, Department of Biology, Science and ResearchBranch, Tehran, Iran, Islamic Republic of3 University of Helsinki, Department of Applied Chemistry and Microbiology,Helsinki, Finland4 Shahid Beheshti University of Medical Sciences, Department of Biotechnology,Tehran, Iran, Islamic Republic of5 Shahid Beheshti University of Medical Sciences, Cellular and MolecularBiology Research Center, Tehran, Iran, Islamic Republic ofCyanobacteria are well known for their production of a multitude of highlytoxic and depsipeptides or alkaloids. Among the photosynthetic microorganisms,cyanobacteria belonging to the genus Nostoc are regarded as goodcandidates for producing biologically active secondary metabolites, whichare highly toxic to humans and other animals.The current scenario of toxicity has become more and more threatening andimportance in recent years due to increase in the rate of deaths in animalsespecially canine and cows. Toxin-producing cyanobacteria represent a healthhazard, and can cause death, mainly from liver damage, upon ingestion ofdrinking water infested with cyanobacterial supplement products.This prompted us to do an endeavor towards to molecular detection oftoxins, microcystin, anatoxin- a, and other bioactive compounds by PCRand LC-MS, in order to introducing the probably causative compound inincidents of fatal canine.Our molecular data, demonstrate that the studied strain contains nosF geneand most likely products of unusual amino acid 4-methylproline. Inaddition to validating the use of eight oligonucleotide primers set foridentification of potential of toxin/ bioactive compounds in Nostoc strain,this study also defines some chemical analyses, that will be useful asprobes for future studies of the synthesis of natural products in that strain.Result of ion chromatograms and MS 2 fragmentation patterns showed that,while, there were three different peptidic compound classes(anabaenopeptin, cryptophycin and nostocyclopeptides), there were notany sign from the presence of anatoxin- a, homoanatoxin-a, hassallidin andmicrocystin in that strain. Moreover, the biochemical assays have aimed todetection of the presence of antifungal effects in cell extract. Thephylogeny of the strain was also investigated by combination genetic andphenotypic relationships of the Nostoc strain.In spite of presence these compounds, especially the depsipeptidescryptophycins, with strong cytotoxic effect on the tubulin polymerization, thereis no evidence of overt neurotoxicity or histopathological changes indicative ofeffects on the brain and peripheral nerves were reported in the dogs or rats.The above findings indicate that cyanobacteria are a promising but stillunexplored natural resource possessing many bioactive compounds usefulfor the pharmaceutical, food and cosmetic industry. Of the new drugsapproved between 1983 and 1994, up to 80% of antibacterial andanticancer drugs were derived from natural products. Indeed, bioactivecompounds of algae are of special interest in the development of newenvironment harmless. The present study aims the preliminaryinvestigation of antimicrobial and toxicity evaluation of Nostoc. Thismerits further and more detailed investigations.MEP008Molecular mechanisms of rhamnolipid synthesis inPseudomonas aeruginosa during batch fermentationA. Schmidberger* 1 , M. Henkel 2 , U. Obst 1 , R. Hausmann 2 , T. Schwartz 11 Karlsruhe Institute of Technology, Institute of Functional Interfaces;Department of Interface Microbiology, Eggenstein-Leopoldshafen, Germany2 Karlsruhe Institute of Technology, Institute of Process Engineering in LifeSciences; Section II: Technical Biology, Karlsruhe, GermanyPseudomonas aeruginosa is a gram-negative, opportunistic humanpathogen that produces the biosurfactant rhamnolipid amongst others assecondary metabolites during stationary growth phase. The regulation ofrhamnolipid synthesis is tightly governed by a complex regulatory networkincluding bacterial quorum sensing systems as well as different sigma factors.Production of rhamnolipids is hence not solely dependent on cell density butalso nutrient availability and stress. The genes for mono- and di-rhamnolipidsynthesis, rhamnosyltransferases 1 and 2 respectively are encoded in onemutual operon which is under the direct control of the Rhl-quorum-sensingsystem and stationary phase sigma factor RpoS. The Rhl-quorum-sensingsystem in turn is controlled by the Las quorum-sensing system and nitrogenlimitation sigma factor RpoN. Additional fine-tuning of the regulatory networkis achieved by various external negative and positive regulators.Production of rhamnolipids by Pseudomonas aeruginosa PAO1 duringbatch fermentation under nitrogen limitation with sunflower oil as carbonsource was recently demonstrated [1] and the production capacity has beenevaluated [2]. However, the molecular regulatory network duringrhamnolipid batch fermentation is not yet fully elucidated on molecularregulation level.In this study we present gene expression data of the relevant systemsinvolved in the regulation of rhamnolipid production during small-scalebatch cultivation under different medium compositions and nutrientsupplies using SYBR Green mediated quantitative real-time PCR.Furthermore, the gene expression during the time course of a standard30L-batch fermentation is monitored.The aim of this project is the optimisation of rhamnolipid production underlarge-scale conditions for commercial production processes. Fullcomprehension of the molecular regulatory mechanisms behindrhamnolipid synthesis is the key to manipulating and improving therhamnolipid production capacities.1. Muller, M.M., et al.,Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production inbioreactor systems.Appl Microbiol Biotechnol, 2010.87(1): p. 167-74.2. Muller, M.M., et al.,Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosaPAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874.ApplMicrobiol Biotechnol, 2011.89(3): p. 585-92.MEP009Characterization of an antimicrobial substance produced byBacillus pseudomycoides DSM 12442S. Basi-Chiplau*, J. Dischinger, M. Josten, C. Szekat, H.-G. Sahl, G. BierbaumInstitute of Medical Microbiology Immunology and Parasitology,Microbiology, Bonn, GermanyLantibiotics are lanthionine containing antimicrobial peptides. Lantibioticspossess structural genes which encode inactive prepeptides. Duringmaturation, the prepeptide undergoes posttranslational modificationsincluding the introduction of rare amino acids as lanthionine andmethyllanthione as well as the proteolytic removal of the leader. The genecluster includes structural (lanA) and other genes which are involved inlantibiotic modification (lanM,lanB, lanC,lanP), regulation (lanR,lanK),export (lanT (P)) and immunity (lanEFG).Genomic data mining showed a new complete lantibiotic gene cluster inthe Gram-positive bacterium Bacillus pseudomycoidesDSM 12442. Anantimicrobial activity was detected only in an isopropanol extract of thecell pellet but not in the culture supernatant. In agar well diffusion assays,it showed activity against many Gram-positive bacteria, including bacilli,streptococci and staphylococci, whereas no activity was observed againstGram-negative bacteria. The antimicrobial substance was relatively stableat high temperature ( 100 0 C), low pH (< 7) and in organic solvents (e.g.acetone, ethanol, etc.). The partially purified substance was predicted tohave a mass of 2786.59 Da by MALDI-TOF analyis.To demonstrate the connection between the lantibiotic gene cluster and theantimicrobial activity,in vitrostudies and heterologous expressionoflanAandlanMwere conducted. Clones of both genes were constructed. Sofar, the LanM has been successfully expressed and purified. LanAexpression and purification is under progress. A factor Xa cleavage sitewas introduced into LanA, so that the leader peptide can be removed fromthe modified peptide to investigate its biological activity.MEP010Heterologous expression of synthetic lantibiotic libraries in S.carnosusS. Perconti* 1 , M. Urbanczyk 1 , P. Popella 1 , M. Nega 1 , B. Krismer 2 , M. Schlag 1 ,F. Götz 11 University of Tübingen, Microbial Genetics, Tübingen, Germany2 University of Tübingen, Medical Microbiology and Hygiene, Tübingen,GermanyMany gram-positive bacteria produce short peptides with antimicrobialactivity - so called “lantibiotics”. They are characterized by unusual aminoacids and lanthionine rings that are both introduced by posttranslationalmodifications. Lantibiotics primary act by binding to the cell wallprecursor Lipid II, thus inducing pores in the cytoplasmic membrane ofother gram-positive bacteria. All lantibiotics are synthesized as inactiveprecursors and subsequently activated through proteolytic cleavage byspecific proteases. The type A lantibiotic gallidermin, produced byStaphylococcus gallinarum, is considered for the treatment of acne(Propionibacterium acnes) and staphylococcal infections like mastitis.We introduced the relevant biosynthesis genes gdmBCDHTQ and thestructural gene gdmA on separate plasmids in S. carnosus TM300. Byusing bioactivity assays as well as HPLC- and MS-analysis, wedemonstrated that the modified S. carnosus is able to produce thegallidermin precursor that can be activated by the specific protease GdmP.This two-plasmid expression system is now used as a tool for theexpression of a synthetic gdmA-library in order to identify improvedgallidermin-derivatives. In a similar approach, other lantibiotics such asnisin can be produced in S. carnosus. With this efficient system, we expectto produce and identify a high variety of novel lantibiotics.BIOspektrum | Tagungsband 2012