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

152OTP065The role of GvpM in gas vesicle formation of Halobacteriumsalinarum PHH1S. Tavlaridou*, F. PfeiferTU Darmstadt, Institut für Mikrobiologie und Genetik, Darmstadt, GermanyGas vesicles of Halobacterium salinarum PHH1 are proteinaceous, gasfilledstructures containing GvpA and GvpC as the major structuralproteins. The hydrophobic GvpA forms the ribbed structure of the gasvesicle wall that is stabilized by GvpC. Twelve additional genes areinvolved in gas vesicle formation arranged in two clusters gvpACNO andgvpDEFGHIJKLM (= p-vac region). The gvpFGHIJKLM transcriptappears earlier compared to gvpDE and gvpACNO mRNAs. GvpM is anessential protein for the gas vesicle formation but produced in smallamounts 1 . An alignment of GvpA and GvpM indicates sequencesimilarities, suggesting that GvpM might be a minor structural componentof gas vesicles.To gain further insights into the role of GvpM, we expressed the p-vacregion and an additional gvpM gene inHfx. volcanii transformants. Astrong reduction of gas vesicle formation was detected compared to cellsexpressing p-vac only, but in a few cells two or three extremely long gasvesicles were found. When GvpM was fused to GFP a strong aggregationof GvpM was observed in gvpM-gfp and p-vac+ gvpM-gfp transformants.It is possible that the aggregation of GvpM disturbs the gas vesicleformation. The aggregation of GvpM was also confirmed by Western analysis.In contrast to the strong reduction of the gas vesicle formation in p-vac+gvpM, transformants expressing p-vac +gvpGHIJKLM contained gasvesicles in normal amounts. These results suggested that additional geneproducts derived from gvpG-M counteract the aggregation of GvpM. Toidentify the gene(s) responsible for this effect, transformants containing p-vac +gvpM plus one other gvp gene were analyzed.Transformantsharboring p-vac+gvpMG did not produce gas vesicles, whereas theaddition of gvpMH, gvpMJ orgvpML led to a wild-type gas vesicleformation. From these results it appears that GvpJ, GvpH and GvpL areable to compensate the inhibitory effect of GvpM on gas vesicle formationin p-vac transformants.1 Offner et al., (2000)J Bacteriol 182:4328-4336OTP066Virus adsorption and elimination in the activated sludge of themunicipal wastewater treatment plant of Hannover-HerrenhausenK. Ulbricht*, K.-H. Rosenwinkel, S. WolterLeibniz Universität Hannover, Institut für Siedlungswasserwitschaft undAbfalltechnik Hannover, Hannover, GermanyThe safety of drinking water resources is actually one of the mostdiscussed issues in science. In this context the threat of waterborne diseaseoutbreaks caused by viruses must be particularly considered. The use ofbank filtrate for drinking water purpose carries the highest risks ofinfection because of the clarified, but still viruses containing wastewater inthe rivers (1-10 PFU/l at low and up to 10-100 PFU/l at highcontaminations) [1]. The most effective approach is to optimize virusreduction during the wastewater treatment process to eliminate the virusload before it is distributed by the water cycle. For this objective we haveto determine the elimination and adsorption processes of viruses in thewastewater treatment plant so that we can further on use this knowledgefor optimizing the processes within the limits of maintaining the treatmentperformance.In the current project we observed in batch experiments with activatedsludge the decreasing concentration of somatic coliphages infectingEscherichia coli strain WG5 and determined the dependency of adsorptionon the total solids content (TS). Furthermore, we also regarded if the virusload is temperature-dependent in the single treatment steps (primaryclarifier, activated sludge system, secondary clarifier) of the WWTPHannover-Herrenhausen (February and August 2011).The results of the batch experiments demonstrated that after ca. 30d ofincubation the elimination process ends, even though not all phages wereinactivated (decrease from 1,27 x 10 4 to 2,29 x 10 1 PFU/ml).Consequently, a total virus reduction cannot be achieved within a commonsludge age of 12-18d. Concerning the adsorption processes we found thatdoubling the TS from ca. 3,2 to 6,66 g/l only slightly speeds up theadsorption process. But the finally reached adsorption rates turned out tobe equal.Observing the WWTP indicated, that activated sludge can compensatevirus load fluctuations in the primary treatment step over longer periods. Incold season the efficiency of elimination within the WWTP is somewhatlower than in warm season. According to the batch tests the differing TS(February: 3 g/l, August: 4 g/l) have no influence onto the elimination. Butthe higher temperature in summer leads to increase of bacteria activity,which might be the reason for the better virus elimination.[1] Botzenhart K(2007) Viren im Trinkwasser, Bundesgesundheitsblatt 50: 296-301OTP067Hot Metagenomics - towards an archaeal expression host formetagenome analysisJ. Kort* 1 , A. Wagner 2 , S.V. Albers 2 , B. Siebers 11 University of Duisburg-Essen, Biofilm Centre, Molecular Enzymetechnologyand Biochemistry, Essen, Germany2 Max-Planck Institute for terrestrial Microbiology, Molecular Biology ofArchaea, Marburg, GermanyArchaea offer exciting potential for biotechnological applications, sincetheir proteins, so called “extremozymes”, are active under harshconditions. Unfortunately, the functional expression of many archaeal(hyper) thermophilic proteins in mesophilic or even thermophilic bacterialhosts is limited. Even more severe is the choice of expression hosts infunctional metagenomics. Since Archaea harbor a unique transcriptionmachinery, the use of bacterial expression systems might lead to a preselectionin current metagenomic approaches. The establishment of anexpression platform with a variety of host organisms, including Archaea,will help to capture the natural diversity.Sulfolobus acidocaldarius is a well characterized thermoacidophiliccrenarchaeon that grows optimally at 78°C and pH 2-3. It is geneticallytractable and a vector system for protein expression has been established [1].For the expression in S. acidocaldarius the promoter of the maltosebinding protein malE is employed. Extensive mutational analysis ofdifferent parts of the malE promoter including the TATA box, the BREsite and the promoter length resulted in a hybrid promoter that had 5 foldhigher expression levels than the wild type promoter. The insertion of theregulator that binds the malE promoter, termed MRP (maltose regulatoryprotein) into the optimized expression vector led to a more than 4 foldincrease of expression levels. First results about the expression of archaeal(gluco)amylases, that failed to be expressed in common bacterial andeucaryal expression systems, will be presented. Furthermore, preliminaryresults about the use of the vector for the expression of metagenomiclibraries from hot environments for identifying new and industriallyrelevant enzymes will be discussed.OTP068Microbial biofilm formation in photobioreactorsI. Krohn-Molt*, A. Pommerening-Röser, D. Hanelt, W.R. StreitUniversität Hamburg, Biozentrum Klein Flottbek, Mikrobiologie undBiotechnologie, Hamburg, GermanyStudies regarding the development of biofilms of microalgae and theinteraction between prokaryotic and eukaryotic microorganisms are verylimited, despite their importance for the development of photobioreactors.In the analyses presented here, the development of biofilm and thebacterial community of the microalgae Scenedesmus obliquus andChlorella vulgaris were examined in detail over a time period of threemonth in a reactor. The diversity and population dynamic of the bacteriawere examined through analyses with scanning electron microscope(SEM), fluorescence in-situ hybridization (FISH), denaturating gradientgel electrophoresis (DGGE) and 16S rRNA. Biomolecular analysesindicated that various populations of alpha- and betaproteobacteria(concerning the family of Comamonadaceae) as well as bacteroidetes (e.g.Pedobacter, Sediminibacterium, Flavobacterium and Bacteroidetes thathave not been cultivated yet) are associated with the microalgae examinedhere. However, the populations of alphaproteobacteria (e.g.Sphingomonas, Brevundimonas, Sinorhizobium, Arcicella andOchrobactrum) as well as the populations of bacteroidetes dominating.Altogether the diversity is rather limited. These results imply thatmetabolic performance of the bacterial populations is probably related andessential to the growth and stability of the algal culture. In addition, thecurrent work focuses on a detailed metagenome analysis of the algaebiofilm communities.OTP069The natural transformation machinery in Thermusthermophilus HB27: A pilus-independent DNA transportercomprising unique motor ATPase and secretin complexesR. Salzer* 1 , J. Burkhardt 1 , J. Vonck 2 , B. Averhoff 11 Molecular Microbiology & Bioernergetics, Institute for MolecularBiosciences, Goethe University, Frankfurt/Main, Germany, Germany2 Department of Structural Biology, Max-Planck Institute of Biophysics,Frankfurt/Main, Germany, GermanyTo get insights into the structure and function of DNA translocators wechose the thermophile T. thermophilus HB27 as model organism since itexhibits the highest natural transformation frequencies known to date. Agenome-wide genetic screen followed by mutant studies led to theidentification of 16 distinct competence proteins [1], several of them werefound to play a dual role in transformation and piliation. But the questionwhether the pilus structures itself are essential for DNA uptake was stillunanswered.BIOspektrum | Tagungsband 2012