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

181family have been reported to participate in transport of insoluble substratesin Eubacteria [3].[1] Dahl, C. et al., 2005. J. Bacteriol. 187, 1392-1404[2] Prange, A. et al., 2004. Arch. Microbiol. 182, 165-174[3] Bishop, R. E. et al., 2006. Bacterial Lipocalins: Origin, Structure, and Function. In: Akerström,B., Borregaard, N., Flower, D. R., Salier, J.-P., editors. Lipocalins. Austin (TX): Landes BiosciencePSP023Genetic evidence for a second anaerobic monoterpeneactivatingenzyme in Castellaniella defragransF. Lüddeke, M. Grünberg, R. Marmulla, J. Harder*Max-Planck-Institut für marine Mikrobiologie, Mikrobiologie, Bremen,GermanyMonoterpenes are natural compounds with an annual emission rate of0.127 - 0.480 Gt C into the atmosphere, thus nearly reaching methaneemission rates. The huge production rate is reflected in a frequentutilization of monoterpenes by bacteria. Most probable number studiesrevealed that each denitrifying bacterium in forest soil had the capacity. Inactivated sludge, one of 150 denitrifiers can grow on monoterpenes. Thebiochemistry of anaerobic monoterpene utilization is currently investigatedwith Castellaniella defragrans, a betaproteobacterium. The anaerobicmonoterpene degradation of Castellaniella defragrans exhibits uniqueenzyme activities, but is still not fully elucidated. Deletion mutants werecreated lacking the gene for the linalool dehydratase-isomerase (ldi) aswell as for both ldi and geraniol dehydrogenase (ged). These enzymescatalyze in vitro reactions of the anaerobic -myrcene metabolism, thehydration of myrcene to geraniol and the geraniol oxidation. In thedeletion mutants, the genes were absent on the genomic as well as thetranscriptomic level without causing polar effect on the adjacent ORFs.The physiological characterization exhibited a substrate-dependentphenotype. The activity of the linalool dehydratase-isomerase was requiredfor growth on -myrcene, an acyclic monoterpene, but not on cyclicmonoterpenes, i.e. -phellandrene or limonene utilization proceededwithout the presence of the linalool dehydratase-isomerase. This indicatesa second enzyme system in Castellaniella defragrans that activatesunsaturated hydrocarbons with cyclic structure.F.Lüddeke and J.Harder (2011) Enantiospecific (S)-(+)-linalool formation from -myrcene bylinalool dehydratase-isomerase. Zeitschrift für Naturforschung 66c, 409-412D.Brodkorb, M.Gottschall, R.Marmulla, F.Lüddeke and J.Harder (2010) Linalool dehydrataseisomerase,a bifunctional enzyme in the anaerobic degradation of monoterpenes Journal ofBiological Chemistry 285, 30436-30442J.Harder, U.Heyen, C.Probian, S.Foß (2000) Anaerobic utilization of essential oils by denitrifyingbacteria. Biodegradation 11, 55-63PSP024Toxin-Antitoxin Systems in Staphylococcus equorumC.F. Schuster* 1 , J.-H. Park 2 , N. Nolle 1 , M. Prax 1 , A. Herbig 3 , K. Nieselt 3 ,M. Inouye 2 , R. Bertram 11 Universität Tübingen, Microbial Genetics, Tübingen, Germany2 Robert Wood Johnson Medical School, Center for Advanced Biotechnology andMedicine, Department of Biochemistry, Piscataway, NJ, USA, United States3 Universität Tübingen, Integrative Transcriptomics, Center for Bioinformatics,Tübingen, GermanyChromosomally encoded Toxin-Antitoxin (TA) systems are assumed toplay an important role in physiological adaptation of bacteria toenvironmental stresses. In Staphylococcus aureus three such TA systemshave been characterized so far: One encoded by the mazEF sa locus and twoby the paralogous yefM sa/yoeB sa genes. S. equorum, a food industryrelevant, nonpathogenic organism has recently been sequenced and iscurrently being annotated. The goal of this work was to identify andcharacterize putative TA systems in S. equorum. An in silico analysisyielded a mazEF homologue with a high similarity to its S. aureuscounterpart, including co-localization with the sigB locus and twoyefM/yoeB systems. mazF se from S. equorum was cloned into ananhydrotetracycline (ATc) inducible vector and used to transform E. coliDH5. Induction of mazF se led to a ten-fold reduction of the OD 578 valuesand to a severe growth inhibition on solid media. Even more strikingly, thenumber of CFUs was about 100-fold decreased compared to uninducedcells. For further characterization, the mazEF se transcription start wasmapped via 5’-RACE and MazFse-(His) 6 was purified through affinitychromatography. MazFse was incubated in vitro with MS2 phage RNAand the resulting fragments analyzed via primer extension. Findingssuggest the same target sequence as elucidated for the S. aureus MazFhomologue: 5’ U^ACAU 3’. This recognition sequence is overrepresentedin some genes’ mRNAs, most notably in rsbV, encoding an anti-anti-sigmafactor of B , possibly regulating sigB expression. In addition, the putativeTA toxin genes yoeB se1/2 were cloned into arabinose inducible E. colivectors. Overexpression of these genes leads to a growth defect and furtherwork to characterize these candidates is in progress. Based upon theseobservations, the inspected loci in S. equorum are highly indicative ofencoding three functional TA systems.PSP025Will not be presented!PSP026Growth by Anaerobic Sulphur Dismutation in ThermophilicArchaea and BacteriaD. Petrasch*, A. KletzinTU Darmstadt, Institut für Mikrobiologie und Genetik, Darmstadt, GermanyAnaerobic dismutation (disproportionation) of elemental sulphur forenergy conservation is not well known. A few mesophilic bacteriaincluding Desulfocapsa sulfoexigens were identified so far [1]. Theproducts are hydrogen sulphide and sulphate. Here we show that severalcocultures of different novel thermophilic microorganisms grow bychemolithoautotrophic sulphur dismutation.We collected environmental samples from hot springs (60° to 90° C) onthe island of São Miguel (Açores). Enrichment cultures were incubated ina minimal salt medium with elemental sulphur as energy source and inserum bottles with defined gas phases (aerobic, CO 2/H 2, or CO 2) toestablish sulphur-oxidising, reducing or dismutating conditions,respectively.We obtained one sulphur-oxidising culture (Acidianus brierleyi) plus onecoculture (similar to Thermus scotoductus, Alicyclobacillus). Two sulphurreducingcultures (Acidianus brierleyi, Thermoplasma acidophilum) andone sulphur-reducing coculture (Thermoanaerobacter sulfurophilus,Thermoanaerobacter brockii) were discovered. Furthermore we got twococultures, which grew by sulphur dismutation under CO 2 atmosphere.The sulphur-dismutating cultures grew to cell densities of 5 x 10 7 ml -1within 7 days. The maximal H 2S production was 860 M in 7 days. One ofthese cultures was microscopically homogeneous and grew at 60 °C andpH 1.5. 16S rDNA sequencing showed that it was 99% identical toThermoplasma acidophilum. The second culture grew at 80°C and pH 4and showed a mixture of a rod-shaped and a coccoid microorganism. The16S rDNA presumably of the rod-shaped microorganisms was 97%identical to Vulcanisaeta distributa. The coccoid microorganism could notbe assigned phylogenetically.In a second approach an Acidianus ambivalens / Sulfurisphaera MC1coculture was grown autotrophically at 70-80 C at pH 2-3 under CO 2atmosphere and elemental sulphur as energy source. The coculture showeda stable growth with a doubling time of 120 h and a maximal cell densityof 1 x 10 8 ml -1 . The results suggest that anaerobic sulphur dismutation is acommon mechanism of energy conservation in habitats of volcanic origin,where sulphur is abundant and anaerobic habitats occur frequently.[1] Finster, K. et al.(1998). Appl Environ Microbiol 64, 119-125.PSP027Biotechnological production of 1,3-propanediol (1,3-PD):Overexpression of 1,3-PD operon and stabilization of 1,3-PDdehydrogenase from Colombian Clostridium sp.S. Flüchter* 1 , J. Montoya 1 , D. Montoya 2 , P. Dürre 1 , B. Schiel-Bengelsdorf 11 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,Germany2 Institute of Biotechnology, Universidad Nacional de Colombia, Bogota,ColombiaThe non-pathogen Colombian strains Clostridium sp. IBUN 13A andIBUN 158B are able to produce 1,3-propanediol (1,3-PD) from rawglycerol. With the aim of improving the 1,3-PD yield of these strains forindustrial purposes, the genes involved in the reductive pathway leadingfrom glycerol to 1,3-PD were analyzed. The three genes dhaB1, dhaB2 anddhaT are located next to each other on the genome of the strain IBUN 13A,lacking promoter or terminator sequences in between them. This suggeststhat they are coexpressed as an operon (1,3-PD operon). In order to verifythat they belong to the same transcriptional unit, the correspondingpromoterless region was cloned in the vector pJet1.2 (Fermentas) under thecontrol of a T 7 promoter. The resulting plasmid was transformed in E. coliBL21(DE3), where overexpression of the cloned genes can be inducedwith IPTG for Northern blot analysis of the resulting RNA.The product of the dhaT gene from IBUN 13A, a 1,3-PD dehydrogenase,was characterized after cloning in the overexpression vector pET-28a(+)(Novagen ® ). From this plasmid, the DhaT enzyme was overproduced andanaerobically purified through Ni-NTA-agarose (Qiagen) and PD-10 (GEHealthcare) columns. The size of the active enzyme was determined bynative PAGE with the help of the NativeMark protein standard(Invitrogen), using polyacrylamide gels with concentrations in the range of6-18 %. Activity of the enzyme was determined with 1,3-PDconcentrations from 0 to 300 mM. In this way, the K m value was calculatedand a linear region of the activity curve was found, which is used for 1,3-PD measurement in an enzymatic test recently established (Franz et al.,2011). To improve the stability of the isolated DhaT enzyme, alyophilisation protocol was developed, which allows activity preservationfor up to 70 days.BIOspektrum | Tagungsband 2012