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

153Here we report on structural and functional analyses of the AAA-ATPasePilF, a unique motor component and the secretin PilQ. Both were found tobe essential for natural transformation and piliation. PilF carries a uniqueN-terminal triplicated GSPII domain and a C-terminal tetracysteinmotifinvolved in zinc binding [2]. Mutant studies revealed that two of thecysteines are essential for Zn 2+ binding, piliation, twitching motility andadhesion, but not for natural transformation.Recently, we reported on the novel structure of a PilQ complex,comprising a stable cone and cup structure and six ring structures [3].Structural analyses of a set of PilQ deletion derivates in T. thermophilusHB27 identified 136 N-terminal residues, encoding an unusual fold as a ring building domain. Deletion of this domain had a dramaticeffect on piliation but did not abolish natural transformation.Taken together, these findings provide clear evidence that the pilusstructures are not essential for natural transformation.[1] Averhoff B. (2009) FEMS Microbiol. Rev. 33:611-626.[2] Rose I., Biukovi G., Aderhold P., Müller V., Grüber G., Averhoff B. (2011) Extremophiles15:191-202.[3] Burkhardt J., Vonck J., Averhoff B. (2011) J. Biol. Chem. 286:9977-9984.OTP070Construction of a Bifunctional Cellulase-Xylanase fromThermophilic MicroorganismsM. Rizk*, S. Elleuche, G. AntranikianTechnische Universität Hamburg-Harburg, Technische Mikrobiologie,Hamburg, GermanyPlant cell walls contain complex polymers and polysaccharides, such ascellulose and hemicellulose. The hydrolysis of these compounds has beenshown to be of relevant importance for the industry. Enzymes required forthis catalysis are extensively used in different industrial fields rangingfrom the textile industry to food processing and biofuel production. Anumber of separate bacterial enzymes work in tandem to efficiently digestpolysaccharides, through the hydrolysis of cellulose and hemicellulose.Xylanases hydrolyze -1,4 glycosidic linkages of hemicellulose, whereascellulases catalyze random cleavage of the cellulose chain. Few bacteriaare able to form multi-component enzyme complexes, known ascellulosomes, while others have separate enzymes or even isozymesworking in synergy. Such complexes and processes can be mimicked inlaboratories, owing to a number of different molecular and genetictechniques. Several methods, including end-to-end fusion have beenshown to generate bi-functional enzyme constructs.The aim of this study is to generate bi-functional enzyme variants foroptimized polysaccharide degradation, by fusing the genes encoding forcellulase and xylanase. A linker, composed of 8 amino acids, is addedbetween the two genes, which can lead to increased stability andflexibility. Here we report the construction of the bi-functional enzymesand their characterization regarding synergestic effects.OTP071Ornithine aminotransferase (rocD) is essential for optimalgrowth with arginine as single nitrogen source inMycobacterium smegmatisA. Hampel* 1,2 , B. Beckmann 2 , F.M. Gutzki 2 , D. Tsikas 2 , F.-C. Bange 11 MH Hannover, Institute of Microbiology, Hannover, Germany2 MH Hannover, Institute of Clinical Pharmacology, Hannover, GermanyPreviously, we studied arginine metabolism under strictly anaerobicconditions, when mycobacteria are unable to replicate but persist instead.However, in the presence of oxygen, mycobacteria show robust growth,even when arginine is present as the only source of nitrogen. Themolecular mechanisms for this metabolic activity are unknown. RocD,encoding the ornithine aminotransferase, is part of the arginase pathway,and converts ornithine to glutamate which is subsequently assimilated intocentral metabolic pathways. A rocD mutant of Bacillus subtilis is not ableto utilize arginine as a source of nitrogen.To investigate the role of ornithine aminotransferase in mycobacteria, arocD mutant in Mycobacterium smegmatis (Msmeg) was generated andtested for growth in minimal medium with arginine as a single source ofnitrogen. In addition the intra- and extracellular ornithine concentrationwas measured by gas chromatography mass spectrometry (GC-MS).The rocD mutant of Msmeg had a growth defect on arginine, suggestingthat rocD is essential for arginine assimilation. The mutant also showed anintra- and extracellular accumulation of ornithine, the substrate for theornithine aminotransferase.However, we observed residual growth of the mutant on arginine,indicating that in mycobacteria utilization of arginine is more complexthan expected. At present we perform experiments to further definearginine metabolism in Msmeg.OTP072Transport and removal of bacteriophages in saturated sandcolumns under oxic and anoxic conditionsA. Frohnert* 1 , S. Apelt 2 , S. Klitzke 2 , H.-C. Selinka 1 , A. Reuchsel 1 ,I. Chorus 2 , R. Szewzyk 11 Umweltbundesamt, FG II 1.4, Berlin, Germany2 Umweltbundesamt, FG II 3.3, Berlin, GermanyTo protect groundwater as a drinking water resource againstmicrobiological contamination protection zones are installed. Whiletravelling through these zones concentrations of potential pathogens shalldecline to levels that pose no risks to human health. The removal duringthe subsurface passage is influenced by physicochemical conditions, e.g.oxygen concentration. The survival of microorganisms is affected by theamount of oxygen. In addition, depending on whether dissolved oxygen ispresent or not, mineral phases with different adsorption properties can bepresent. In studies examining the transport of virus particles, the RNAbacteriophage MS2 and the DNA bacteriophage X174 are often usedbecause they resemble human viruses in structure and size. Moreover, theirdetection is much easier and cheaper to accomplish than that of humanviruses. Experiments in glass columns (length 55 cm, inner diameter 7.3cm) filled with medium grained sand were conducted. Different mobilephases either containing dissolved oxygen or being oxygen-free werespiked with bacteriophages MS2 and X174 and pumped through thesecolumns from bottom to top at a filter velocity of about 1m/d. At theeffluent physicochemical parameters were measured, and samples foranalysing the bacteriophages by plaque assay were taken. Bacteriophagebreakthrough curves were compared to breakthrough curves of NaCl, usedas a conservative tracer. Both were analysed by one-dimensional models ofhydrogeological transport. Total eliminations of bacteriophages weredetermined by calculating the differences between the input and recoveredamounts of viruses. In all experiments, the RNA bacteriophage MS2 waseliminated more efficiently than the DNA bacteriophage X174.Compared to experiments with oxygen-free water, a higher elimination ofviruses was observed in oxic water. In connection with batch experimentsthe data suggest that differences in the inactivation rate coefficients areimportant to explain the results. Our results will contribute to a betterunderstanding of the transport of viruses through oxic and anoxic zones inthe subsurface.OTP073Construction of a Xanthomonas sp. 35Y rubber oxygenase(RoxA) deletion mutant and improvement of a homologousexpression system for RoxA muteinsN. Hambsch*, J. Birke, D. JendrossekUniversität Stuttgart, Institut für Mikrobiologie, Stuttgart, GermanyXanthomonas sp. 35Y is the so far only known Gram-negative bacteriumcapable to degrade natural rubber (polyisoprene) and to use rubberdegradation products as the sole source of carbon and energy. The primaryattack of the carbon backbone of polyisoprene is catalyzed by a novel typeof an extracellular diheme dioxygenase (rubber oxygenase RoxA) [1-3].To investigate the unknown RoxA cleavage mechanism, structure-functionanalysis of RoxA muteins is necessary. Unfortunately, heterologousexpression of RoxA was not possible, neither in Escherichia coli, Bacillussubtilis nor in Pseudomonas putida. Therefore, a homologous RoxAexpression system was established in the host strain Xanthomonas sp. 35Y[4]. However, it turned out that expression of a roxA copy from a broadhost range plasmid (with rhamnose-dependent promoter) transferred to theXanthomonas strain could not be obtained immediately. Only afterspontaneous integration of the plasmid (after weeks up to months andseveral transfers on solid media) into the chromosome, stable rhamnosedependentRoxA expression was obtained. Thus, a roxA deletion mutantwas constructed using sucrose counter selection with sacB. To improve theefficiency of integration of the expression plasmid into the chromosome,the phage PhiC31 integration system was applied. Using this system, wesucceeded in rapid and reproducible integration of roxA copies into theXanthomonas sp. chromosome. Wild type RoxA and first RoxA muteinswere successfully expressed. High yields of recombinant wild type RoxA( 1 mg/L culture) were reproducibly obtained within 2-3 days ofcultivation in the presence of rhamnose. Purified recombinant RoxA wasactive, its activity could not be distinguished from RoxA that had beenpurified from Xanthomonas sp. wild type.[1] Braaz, R., P. Fischer, D. Jendrossek (2004). AEM 70(12): 7388-7395.[2] Braaz, R., W. Armbruster, D. Jendrossek (2005). AEM 71(5): 2473-2478.[3] Schmitt, G., G. Seiffert, P. M. H. Kroneck,R. Braaz and D. Jendrossek (2010). Microbiology156: 2537-2548[4] Hambsch, N., G. Schmitt and D. Jendrossek (2010). JAM 109: 1067-1075BIOspektrum | Tagungsband 2012