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

156OTP084The Use of GFP-GvpE fusions to characterize the interaction ofthe two regulatory proteins GvpD and GvpE of HalobacteriumsalinarumI. Schmidt*, F. PfeiferTU Darmstadt, Biology, Darmstadt, GermanyGas vesicle formation in Halobacterium salinarum involves fourteen gvpgenes arranged in two oppositely oriented gene clusters gvpACNO andgvpDEFGHIJKLM. The expression of these genes is regulated by twoendogenous regulatory proteins, GvpD which is involved in repression andthe transcriptional activator GvpE. Both proteins are able to interact andthis interaction results in the lack of GvpE in Haloferax volcanii D + Etransformants [1, 2]. To quantify the reduction of the amount of GvpE inthe presence of GvpD, an N-terminal fusion of GFP to GvpE wasconstructed. The activating function of the GFP-E fusion protein wasshown in P A-bgaH transformants using the -galactosidase activity ofBgaH as reporter, since wild-type GvpE (E WT) or the GFP fusion GFP-Eresulted in similar BgaH activities. The amount of GFP-E was thenquantified by fluorescence measurements in the absence or presence ofGvpD WT or GvpD mutants. GvpD mutant D 3-AAA acts as a superrepressor,whereas two other GvpD mutants, D Mut1 and D Mut6, lack the repressingfunction. The fluorescence level determined for the GFP-E transformantwas set 100%, the fluorescence of GFP-E + D WT was reduced to 40% intransformants, whereas the superrepressor D 3-AAA reduced the fluorescenceof GFP-E to 20%. No reduction of fluorescence was observed in GFP-Etransformants carrying the defective mutant proteins D Mut1 or D Mut6. TheGFP-E reporter system was also used to study the effect of various GvpEmutants that lost their activating function. The GvpE mutants E ARA andE K104A incurred mutations in one of the two putative DNA binding regionsand is completely abolishing the activator function. D WT and also thesuperrepressor mutant D 3-AAA reduced the fluorescence of both GvpEmutants to 50% and 40%, respectively. Further studies on GvpE and GvpDmutants are under way, to search and identify contact sites in bothregulatory proteins that are important for the reduction of GvpE.(1) Zimmermann, P. & Pfeifer, F. (2003). Mol. Mircobiol. 49(3): 783-794(2) Scheuch, S., Marschaus, L., Sartorius-Neef, S., Pfeifer, F., (2008).ArchMicrobiol190: 333-339OTP085Enhanced FMN-binding fluorescent proteinsM. Wingen* 1 , T. Drepper 1 , S. Hausmann 2 , J. Potzkei 1 , K.-E. Jaeger 31 Heinrich Heine University Düsseldorf, Institute of Molecular EnzymeTechnology / WG Drepper, Jülich, Germany2 Evocatal GmbH, Düsseldorf, Germany3 Heinrich Heine University Düsseldorf, Institute of Molecular EnzymeTechnology, Jülich, GermanyFluorescent proteins (FP) like the green fluorescent protein (GFP) and itsvariants are widely used in vivo reporters to study protein expression,localization and interaction [1]. Flavin-mononucleotide (FMN)-bindingFPs (FbFPs) are a new class of fluorescent reporters, which are derivedfrom bacterial ‘Light Oxygen Voltage’ (LOV) photoreceptor domains. Incontrast to the well-established FPs of the GFP-family, FbFPs do notrequire molecular oxygen for the development of their fluorescence signaland are therefore suitable reporter proteins for fluorescence imaging underaerobic, as well as under anaerobic conditions [2]. Applicability of FbFPshas so far been demonstrated for various anaerobic bacteria [2-4] and yeast[5]. Their independence of molecular oxygen also enables them tooutperform GFP-like proteins as quantitativein vivoreal-time reporters [6].We are now conducting directed evolution experiments, in order to furtherenhance the fluorescence properties of FbFPs. Here, we report on thedevelopment of novel enhanced FbFP derivatives exhibiting improvedbrightness or blue-shifted absorption and fluorescence spectra.1. Chudakov DM, Lukyanov S, Lukyanov KA:Fluorescent proteins as a toolkit for in vivo imaging.TrendsBiotechnol2005,23(12):605-613.2. Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gärtner W, JaegerKE:Reporter proteins for in vivo fluorescence without oxygen.Nat Biotechnol2007,25(4):443-445.3. Choi CH, Deguzman JV, Lamont RJ, Yilmaz Ö:Genetic Transformation of an Obligate Anaerobe, P.gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction.PLoSOne2011,6(4):e18499.4. Lobo LA, Smith CJ, Rocha ER:Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) asreporter for gene expression in the anaerobe Bacteroides fragilis.FEMS Microbiol Lett2011.5. Tielker D, Eichhof I, Jaeger KE, Ernst JF:Flavin mononucleotide-based fluorescent protein as an oxygenindependentreporter in Candida albicans and Saccharomyces cerevisiae.Eukaryot Cell2009,8(6):913-915.6. Drepper T, Huber R, Heck A, Circolone F, Hillmer AK, Büchs J, Jaeger KE:Flavin mononucleotidebasedfluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivoreal-time reporters.Appl Environ Microbiol2010,76(17):5990-5994.OTP086Interaction of engineered anorganic nanoparticles with bacterialbiofilmsA. Grün*, M. Madzgalla, W. ManzInstitute for Integrated Natural Sciences, University Koblenz-Landau,Department of Biology, Koblenz, GermanyIn the last decade engineered inorganic nanoparticles (EINP) have beenbrought to the market in large quantities partly used as agents withantibacterial properties (e.g. Ag-EINP). Today, the resulting ambientconcentration of Ag nanoparticles in river water is estimated in a quitebroad range from 0.01 g/l up to 300 g/l [1, 2]. Based on a potentialpollution rate of EINP, bacterial biofilms in the environment will certainlybe encountered as well. These Biofilm communities ensure essentialecosystem functions of lakes and rivers (e.g. self purification) and provideimportant ecosystem related services for drinking water reservoirs,recreational centres, and biodiversity resources [3]. The biofilms displaythe locality where adsorption of organic and inorganic matter does occur,and microbially mediated degradation, metabolism and mineralisation takeplace. Even though numerous studies have been dealt with biologicaleffects of Ag nanoparticles already, knowledge of the ecotoxicology ofEINP to bacteria is mostly limited to studies on single bacterial species inliquid cultures [4].To investigate interactions of these widely used EINP at the aquaticterrestrialinterface, the DFG research unit INTERNANO was approvedvery recently. The study presented here is part of the preliminary work ofthe respective subproject BIOFILMS. To get first insights into the mutualeffects of EINP on structure and function of biofilms, both monospeciesbiofilms and native biofilms obtained from the river Rhine wereinvestigated after exposure to different concentrations of Ag nanoparticles.Aquabacterium citratiphilum was used as model organism to generatebiofilms under laboratory conditions applying commercial drip flowbiofilm reactors (BioSurface Technologies, Inc.). By using fluorescentdyes SYBR® Green and epifluorescence digital imaging we obtainedinformation regarding the amount of adhered biomass. Application ofLIVE/DEAD®BacLight Bacterial Viability Kit showed the relativeamount of bacteria with uncompromised membrane integrity and thereforegave an estimate of the active total biofilm volume to surface area. Fromthese results, effective concentration of Ag nanoparticles on the adheredmicrobial biomass can be estimated for the model system investigated.[1] Boxall, A. et al (2008): Current and future predicted environmental exposure to engineered nanoparticles.In. Report to Defra.[2] Blaser, S.A. et al (2008): Estimation of cumulative aquatic exposure and risk due to silver: Contributionof nano-functionalized plastics and textiles. Sci. Tot. Environm., 390, 396-409.[3] Gerbersdorf, S.U. et al (2011): Anthropogenic pollutants affect ecosystem services of freshwatersediments: the need for a “triad plus x“ approach. Journal Soils Sedimentsdoi 10.1007/s11368-011-0373-0.[4] Pal, S. et al (2007): Does the antibacterial activity of silver nanoparticles depend on the shape of thenanoparticle? A study of the gram-negative bacterium Escherichia coli. Applied EnvironmentalMicrobiology73, 1712-1720.OTP087Construction of an expression system based on mannitol PTSin Bacillus subtilis and its regulationK. Morabbi Heravi*, J. AltenbuchnerInstitut für Industrielle Genetik, Universität Stuttgart, Stuttgart, GermanyRegulation of the mannitol utilization system (mtl operon) in B. subtiliswas studied in order to construct an expression system. The mtl operonconsists of mtlA (encoding the phosphoenolpyruvate-dependentphosphotransferase system (PTS) enzyme IICB Mtl ), mtlF (encoding thePTS enzyme IIA Mtl ), and mtlD (encoding the mannitol 1-phosphatedehydrogenase). The mtlAFD operon is activated by MtlR (encoding bymtlR), which is a transcriptional activator containing so-called PTSregulatory domains (PRDs) as well as EIIB Gat and EIIA Mtl domains.Principally, the phosphorylation state of the domains of such activatorsregulates its function. In other words, phosphorylation of PRDII activates aPRD containing activator, while phosphorylation of other domainsdeactivates it. In this study, the promoters of mtlAFD operon (P mtlA) andmtlR (P mtlR) were fused to lacZ as a reporter gene. Measurement of -galactosidase indicated that the P mtlA and P mtlR were induced by mannitol,whereas glucose repressed their activities. Using primer extension method,transcription start sites as well as -10 and -35 boxes were identifiedindicating a A -like structure of P mtlA and P mtlR. Specific regulation of P mtlAand P mtlR were investigated by deletion of mtlAF, mtlD and mtlR ormutation of mtlR to mtlR-H342D (as a phosphorylated PRDII mimic).Here, it was observed that the deletion of EIICB Mtl and EIIA Mtl componentsand MtlR-H342D mutation resulted in constitutive expression of P mtlA andP mtlR, while deletion of mtlR strongly reduced the promoter activity.Subsequently, the effect of carbon catabolite repression (CCR) wasinvestigated wherein generaltrans andcis components of CcpA-dependentCCR, and ptsG (encoding glucose transporter)were deleted or mutated.Altogether, the results indicated that glucose repression was mainly causedby an inhibition of MtlR by PtsG, while CcpA-dependent CCRcomponents exhibited minor effects. Consequently, we assume that thephosphorylation state of PRDII domain (H342) plays the main role inglucose repression of mannitol system.OTP088Self-inducible Bacillus subtilis expression systemM. Wenzel*, J. AltenbuchnerInstitut für Industrielle Genetik, Universität Stuttgart, Stuttgart, GermanyHigh product yields and low costs are two of the main objectives of aneconomic production process. Hence, we developed a novel technicallyBIOspektrum | Tagungsband 2012