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

140membrane permeability of 390Lh -1 m -1 bar -1 . One operation cycle consistedof 20min of filtration and a backwash of 20sec. Samples of fouledmembranes were investigated after one, three and six cycles of filtration.The biofouling was analyzed by confocal laser scanning microscopy(CLSM) after simultaneous staining. The bacteria in the fouling werestained with DAPI specific to nucleic acids and different fluorescentlabeled lectins specific to polysaccharides of the EPS.Confocal laser microscopy showed that biofouling on the membrane was acomposition of heterogeneous colonization of bacteria and extra cellularpolymeric substances (EPS) containing, N-acetylglucosamine, N-acetylgalactoseamine and L fucose. The detection of the bacteria and thelocation of the polysaccharides could be related to the biofoulingaccumulation. Our investigations assume, that at first polysaccharides ofthe influent adsorbed to the membrane surface and serve as layer for thedevelopment of a conditioning film. Backwashing was able to remove cellsfrom the membrane, but was unable to remove adsorbed substances of theconditioning film.OTP010Evaluation of analytical sensitivity and specificity of thebiothreat assay for clinical Bacillus anthracis diagnostics bythe PLEX-ID SystemM. Hanczaruk* 1 , B. Thoma 1 , M. Antwerpen 1 , S. Schmoldt 1 , C. Tiemann 2 ,D. Knoop 2 , A. Hartmann 2 , L. Zöller 1 , G. Grass 11 Bundeswehr Institute of Microbiology, Munich, Germany2 LABCON-OWL GmbH, Bad Salzuflen, GermanyBacillus anthracis causes a clinical condition known as Anthrax diseaseand the bacterium is placed top on the list of biological agents potentiallyto be used in bioterrorism and biological warfare.B. anthracis belongs tothe B. cereus group spp., which are genetically closely related. Forinstance, plasmids similar to B. anthracis pXO1 and pXO2 can also befound in B. cereus. These plasmids are of paramount importance forvirulence of the bacilli. pXO1 codes for the toxins edema- and lethal-factoralong with protective antigen needed for toxin delivery into host cells.pXO2 is required for capsule formation enabling evasion of host immuneresponse. The PLEX-ID System is a technique based on PCR andElectrospray-Ionization Mass Spectrometry (ESI-MS) providing the exactbase-composition of (partial) gene amplificates. As part of a so-called“biothreat assay” species specific primer sets were developed enabling thedetection of 46 viral and bacterial biothreat pathogens.Herein, closely related organisms can be differentiated in a single run on amultiplex-assay-base aiming at reliable and fast identification of unknownsamples by (subspecies-) specific base pair signatures. B. anthracisdetection, for example, is achieved via two B. anthracis specificchromosomal and one pXO1- and pXO2-plasmid specific targets. Toevaluate this “biothreat assay” we tested its analytical specificity (crossreactivity)and analytical sensitivity [limit of detection (LoD)]. For this, weanalyzed a panel of B. anthracis (plasmid positive and negative) strainsand Bacillus spp. isolates closely related to B. anthracis. Included in thisstudy were also other organisms representing the resident flora of clinicalmatrices and various matrices relevant in clinical B. anthracis diagnostics.The LoD was as low as 5 genome copies per l from culture and 5 to 10genome copies from clinical matrices such as EDTA blood. Takentogether, the PLEX-ID technique allows for the reliable identification ofB. anthracis (plasmid positive and negative strains) and the discriminationfrom other B. cereus-group bacteria (incl. plasmid positive strains) withinacceptable clinical sensitivity.OTP011Seeking novel Hydrogenases from a hydrothermal ventenrichment cultureM. Hansen*, M. PernerUniversity of Hamburg, Molecular Biology of Microbial Consortia,Hamburg, GermanyA culture enriched with diffuse fluids taken at the hydrothermal ventSisters Peak (5° S on the Mid-Atlantic Ridge) grows autotrophically onartificial seawater supplemented with hydrogen. Analyses of amplified 16SrRNA genes revealed the presence of species commonly not known toutilize hydrogen as electron donor, namely the AlphaproteobacteriumThalassospira sp. and the Gammaproteobacteria Thiomicrospiracrunogena, Pseudomonas pachastrellae and Alteromonas macleodii.Fluorescence in situ hybridization with specific probes designed to targeteach species individually demonstrated little community shifts in theculture within 4 weeks. The relative abundance of T. crunogena variedbetween 40-71% and of Thalassospira sp. between 25-40%, respectively.Relative abundances of A. macleodii and P. pachastrellae were between2% and 10%. We also performed hydrogen consumption measurementswith the enrichments, which clearly illustrated the active uptake ofhydrogen. The uptake hydrogenase activity of membrane associatedproteins from the mixed culture was 0.253 ± 0.079 mol H 2*min -1 *mg -1 ,contrasting the low uptake activity for the soluble proteins (0.023 ± 0.132mol H 2*min -1 *mg -1 ). Conclusively, hydrogenases are being expressedand are active in this culture. Since we have not been able to assign thehydrogenases to one of the species in the enrichment culture we arecurrently pursuing 2 strategies: (i) Native PAGE and an in-gelhydrogenase activity assay in combination with sequencing of activeprotein bands and (ii) investigation of the isolated species with respect togrowth with hydrogen, uptake hydrogenase activities and hydrogenconsumption.OTP012Three-dimensional obstacles for bacterial surface motilityN. Kouzel*, C. MeelBiocenter/Institute of Theoretical Physics, AG Prof. Dr. Maier, Cologne,GermanyMany bacterial species live at surfaces. For surface colonization they havedeveloped mechanisms which allow them to move while remainingattached to surfaces. The most ubiquitous mode of surface motility ismediated by type IV pili. These polymeric cell appendages mediatemotility through cycles of pilus polymerization, adhesion, anddepolymerization. Natural adhesion surfaces, including mammalian hostcells, are not flat. It is unknown, however, how the topography of a surfaceinfluences bacterial surface motility. Here, we show that the roundNeisseria gonorrhoeae (gonococcus) was preferentially reflected frombarriers with a depth of 1 m but not by lower barriers. Gonococcalmotility was confined to grooves whose dimensions were on the order ofthe size of the bacteria and the dynamics of movement was in agreementwith a tug-of-war model. Likewise, the motility of the rod-likeMyxococcus xanthus (myxococcus) was confined to grooves. In summary,the data demonstrate that surface-motile bacteria can sense the topographyof the surface and that their movements are guided by microscopicelevations.Meel, C., Kouzel, N., Oldewurtel, E.R., Maier, B. Three-dimensionalobstacles for bacterial surface motility, Small, accepted.OTP013Recombinant production of genetically modified S-layerproteins in different expression systems*F. Lederer 1 , S. Kutschke 1 , K. Pollmann 21 Helmholtz-Zentrum Dresden, Institute of Radiochemistry, Biophysics Division,Dresden, Germany2 Helmholtz-Zentrum Dresden, Helmholtz Institute of Freiberg, Dresden,GermanySurface layer (S-layer) are proteins which cover the outermost of manyprokaryotes and are probably the basic and oldest forms of bacterialenvelope. These proteins are mostly composed of protein and glycoproteinmonomers and have the ability to self-assemble into two-dimensionalarrays on interfaces. Several characteristics like their work as molecularsieve, as virulence factor or the protection of the cell from toxic heavymetal ions make S-layer proteins interesting for their usage asultrafiltration membranes, drug microcontainers, filter materials orpatterning structures in nanotechnology.Heterologous expression of S-layer proteins is not simple and depends onthe used vector and the expression system. Equally the S-layer protein size,genetic specifics, and the existence of adapted signal peptides influence theexpression. To enable an efficient and economical protein productionprotein secretion is the most favoured method.In this work we describe the recombinant production of different S-layervariants and characterize the differences of the used protein expressionsystems.We used four different S-layer genes of Lysinibacillus sphaericus JG-A12,Bacillus spec. JG-B12 and Lactobacillus acidophilus and expressed theirproteins in Escherichia coli, Pichia pastoris and Lactococcus lactis. Someof these proteins were genetically modified to adapt the construct to theused S-layer expression system.Our work identified Lactococcus lactis as the best expression system forthe used S-layer genes.OTP014Biological applications for nano-mechanical detectionofmolecular recognitionM. Leisner* 1 , A. Mader 1 , K. Gruber 1 , R. Castelli 2 , P.H. Seeberger 2 , J. Raedler 11 LMU, Physik, Munich, Germany2 Freie Universitaet Berlin, Biology, Chemistry and Pharmacy, Berlin, GermanyAdvances in carbohydrate sequencing technologies have revealed thetremendous complexity of the glycome. Understanding the biologicalfunction of carbohydrates requires the identification and quantification ofcarbohydrate interactions with biomolecules. The increasing importance ofcarbohydrate-based sensors able to specifically detect sugar bindingmolecules or cells, has been shown for medical diagnostics and drugscreening. Our biosensor with a self-assembled manno side based sensingBIOspektrum | Tagungsband 2012