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

236of these proteins are putative mechano-sensitive channels, of which twowere mutated and further analyzed concerning their role in ectoine efflux.SSP034The ZIP (ZRT/IRT protein family) member ZupT fromCupriavidus metallidurans CH34 has pleiotropic effects on zinchomeostasisM. Herzberg*, D.H. NiesMartin-Luther-Universität Halle-Wittenberg, Molekulare Mikrobiologie,Halle (Saale), GermanyThe well-studied heavy-metal resistant bacteria Cupriavidus metalliduransharbors a network of metal efflux systems, which allow survival in heavymetalpolluted environments. These efflux systems remove in a “worrylater” scenario surplus cytoplasmic metal cations that were previouslyimported into the cell by a variety of highly redundant metal uptakesystems. To understand the contribution of these metal uptake systems tometal resistance, a systematic deletion analysis of the genes zupT, pitA,corA 1, corA 2, corA 3, zntB, hoxN, mgtA and mgtB was performed.Expression of the genes for all of these transporters was down-regulated byincreasing zinc concentration while that of zupT was up-regulated by zincstarvation. ZupT was required for import of zinc at conditions of zincstarvation. The zupT deletion strain produced the largest and zinccontainingsubunit of the DNA-dependent RNA polymerase, RpoC (betaprime) in excess, and accumulated this protein in inclusion bodies,indicating disturbance of zinc homeostasis, although growth of the mutantstrain was not impaired. Additionally, plasmid-bound expression of theczcCBA genes encoding the Czc transenvelope efflux protein complex ledto disappearance of CzcA in various zupT-containing mutant strains. Thisall indicated a central role of ZupT in zinc homeostasis.SSP035BapA is required for biofilm formation in poor-phosphatemedium and modifies the structure of the biofilm produced bySalmonella enterica sv. TyphimuriumB. Haßing, A. Felipe-López*, M. HenselUniversität Osnabrück, Abt. Mikrobiologie, Osnabrück, GermanySalmonella Pathogenicity Island 9 (SPI9) encodes a type 1 secretionsystem (T1SSs) and its substrate BapA. BapA was associated with biofilmformation but its role during host infection remains unknown. In order tofind out the expression conditions and its role in biofilm formation,luciferase reporter and mutant strains of the bap-operon as well as csgDBAmutant strains were created by lambda-red recombination using asbackground the NCTC 12023 (WT) strain. Western Blot (WB) andimmunofluorescence (IF) were performed using a rabbit antibody anti-BapA, kindly provided by Dr. Lasa, Spain. Strains were incubated eitherwith rigorous shaking or in static conditions for 72 h at 37° or 30° C. Asphosphate concentration was described to induce the biofilm formation,PCN with 0.4, 1 and 25 mM PO 4 -3 was used in addition to LB. Biofilmformation was evaluated with crystal violet in 96 well plates. Bacterialpellets and their supernatant were taken from 2 to 72 h at several timeintervals for WB and expression kinetics. Luciferase activity demonstratedthat in static conditions bapA and bapD was around 10-fold higher at 30°C than at 37° C in static conditions after 8 h in WT background. bapAdeficientstrain was produced 4.0-fold less biofilm, as evaluated by crystalviolet, in PCN 0.4 or 1.0 mM PO 4 -3 than in LB or PCN 25mM PO 4 -3 inwhich the mutation did not have any effect. Biofilm formation on glassslides revealed several cluster patterns.bapA-decifient strains formedcolumnar clusters after 96 h which were 10-fold larger than those formedat the same time by the WT strain. Secretion of BapA in WT strain wasobserved after 120h of static incubation in LB. BapA-positive bacterialcells showed a decreased signal of GFP, which was used as marker forbacterial cells, in contrast to those bacterial cells without BapA. BapApsoitivecells were also featured by forming isolated groups of bacteriaconsisting of approx. 10 cells. These results showed that BapA is requiredfor biofilm formation under restrictive low phosphate conditions and thatthe secretion of BapA is associated with the architecture of the biofilm.Current work is on progress to understand how BapA can modify thearchitecture of the biofilm and what regulation mechanism controls theexpression and secretion of BapA.SSP036Molecular approaches to determine the diversity of humanadenoviruses present in sewage-contaminated waterN. Hartmann*, M. Dartscht, H.-C. Selinka, R. SzewzykUmweltbundesamt, II 1.4 Microbiological Risks, Berlin, GermanyHuman adenoviruses (hAdVs) are promising candidates for monitoringviral health risk from environmental water sites. Relatively harmless butcommon these DNA viruses persist continuously within the population andare routinely detected in polluted surface and wastewater. The 58 currentlyknown serotypes, classified into seven subgenera (subgenus A to G) on thebasis of biochemical and biophysical properties, cause a wide range ofinfections with manifold clinical manifestations such as gastroenteritis,conjunctivitis and respiratory diseases. Different serotypes were reportedwith different frequencies, showing a prevalence of enteric adenoviruses inwater samples, though other serotypes were occasionally detected. TheUBA stream and pond simulation system (FSA) allows for tracking ofviruses in sewage-contaminated surface water under definedenvironmental conditions. Adenoviruses do not display strong seasonalfluctuations, but the prevalence of certain serotypes in sewage may changeover time, both for epidemiological and virus stability reasons. Thereforethe diversity patterns of human adenoviruses from sewage contaminatedwater were investigated during four long time-experiments carried out inthe FSA from 2009 to 2010 using different molecular biological methods,including denaturing gradient gel electropheresis (DGGE) adapted for thedetection of human adenoviruses by our group. Additionally,representatives from every subgenus were characterized regarding theirstability within the water used. According to our results, human adenovirusserotype 41 was the most prominent adenovirus detected in the samples.Since quantification was connected to PCR amplification the meltingpoints of adenoviral qRT-PCR products were also determined, promisingyet another method for rapid diversity investigations. The results indicateapplicability of the approaches for other virus groups, including humannorovirus genotype analysis from sewage samples and may support thesearch for viral indicators.SSP037Missing protection of DNA by Dps does not enhance toxicity ofmetallic copper in Escherichia coliC. GrosseUni Halle, Mikrobiologie, Halle, GermanyEscherichia coli protects itself from toxic copper ions by several systems.The cytoplasmic membrane localized, coppertransporting P-type ATPaseCopA extrude Cu(I) out of the cytoplasm (1). The copper efflux systemCusCFBA as well as the multicopper oxidase CueO detoxify theperiplamic space from excess copper. The cytoplasmic factor Dps which isabundant in the stationary phase, seems to be involved in copperhomeostasis too (2). Dps (DNA binding protein of starved cells) is able tobind the DNA to protect it from oxidative damage (3). Recent studies ofsurvival of E. coli on metallic copper leads to the assumption that killingof the bacteria is proceeds by membrane damage, cell death and DNAdamage (4). Another study contrary concluded the DNA as the main targetof copper toxicity followed by rapid DNA degradation and cell death (5).The role of Dps in copper toxicity mechanisms in E. coli was determinedby growth experiments under the influence of ionic copper as well as onmetallic copper surfaces.(1) Rensing C, Grass G. 2003. FEMS Microbiol Rev 27:197-213(2) Thieme D, Grass G. 2010. Microbiol Res 165:108-115(3) Ilari A, Ceci P. 2002. J Biol Chem 277:37619-23(4) Espirito Santo C, et al. 2011. Appl Environ Microbiol 77:794-802(5) Warnes SL, Green SM, Michels HT, Keevil CW. 2010. Appl Environ Microbiol 76:5390-5401SSP038Cloning, expression and purification of extracellular serineprotease Esp, a biofilm-degrading enzyme, from StaphylococcusepidermidisK. Okuda 1 , S. Sugimoto 1 , T. Iwase 1 , F. Sato 2 , A. Tajima 1 , H. Shinji 1 ,Y. Mizunoe* 11 The Jikei University School of Medicine, Department of Bacteriology, Tokyo,Japan2 The Jikei University School of Medicine, Division of Infectious Diseaseand Control, Tokyo, JapanStaphylococcus epidermidis Esp, an extracellular serine protease, inhibitsStaphylococcus aureus biofilm formation and nasal colonization. Tofurther expand the biotechnological applications of Esp, we developed ahighly efficient and economic method for the purification of recombinantEsp based on a Brevibacillus choshinensis expression-secretion system.Theespgene was fused with the N-terminal Sec-dependent signal sequenceof the B. choshinensis cell wall protein and a C-terminal hexa-histidine-taggene. The recombinant Esp was expressed and secreted into the optimizedmedium as an immature form and subsequently activated by thermolysin.The mature Esp was easily purified by a single purification step usingnickel affinity chromatography and showed proteolytic activity as well asS. aureus biofilm destruction activity. The purification yield of thedeveloped extracellular production system was 5 mg recombinant matureEsp per 20-ml culture, which was much higher than that of an intracellularproduction system in Escherichia coli (3 mg recombinant Esp per 1-lculture). Our findings will be a powerful tool for the production andpurification of recombinant Esp and also applicable to a large variety ofrecombinant proteins used for basic researches and biotechnologicalapplications.BIOspektrum | Tagungsband 2012