VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

NTP0083D chemical and elemental imaging of the purple sulfurbacterium Allochromatium vinosum by STXM spectrotomographyA. Prange* 1,2,3 , J. Wang 4 , J. Hormes 3,4 , A. Hitchcock 4 , C. Dahl 5 ,C. Karunakaran 4 , B. Franz 11 Microbiology and Food Hygiene, Niederrhein University of AppliedSciences, Mönchengladbach, Germany2 Institute for Microbiology and Virology, University of Witten/Herdecke,Witten, Germany3 CAMD, Louisiana State University, Baton Rouge, LA, USA4 Canadian Light Source, Saskatoon, Canada, Canada5 Institute for Microbiology and Biotechnology,Friedrich-WestphalianWilhelms-University, Bonn, GermanyThe scanning transmission X-ray microscope (STXM) at the Canadian LightSource (covering 130 - 2500 eV) images the structure and quantitativedistributions (maps) of chemical components for a wide range of samples athigh spatial resolution (~30 nm). Recently, STXM spectro-tomography wasdeveloped to enable morphological visualization and quantitative chemicalmapping in 3D. In this proof-of-principle experiment, spatial distributions ofcalcite, protein, and polysaccharide in the sulfur-oxidizing bacteriumAllochromatium vinosum (cultivated in Pfennigs medium) were determinedby STXM spectro-tomography at the C 1s and Ca 2p edges. The 3Dchemical mapping shows that the sulfur globules are located inside thebacteria with a strong spatial correlation with calcite and polysaccharide,suggesting an influence of the organic components onto the formation of thesulfur and calcite deposits (resulting form the medium). In future, this newand innovative technique will allow more detailed insight into the cellularstructure and will enhance our knowledge on sulfur globule formation andsulfur utilization by A. vinosum.NTP009Global transcription changes upon nutrient limitation inSynechococcus sp. strain PCC 7002M. Ludwig* 1 , Z. Liu 1 , C.A. Praul 2 , D.A. Bryant 11 Department of Biochemistry and Molecular Biology, Pennsylvania StateUniversity, University Park, USA2 Huck Institutes for the Life Sciences, Pennsylvania State University,University Park, USAGlobal transcription analysis in Synechococcus sp. PCC 7002 wasperformed by high throughput cDNA sequencing using the SOLiD-3sequencing platform. Transcripts were detected for nearly all of the 3,241annotated ORFs of the model cyanobacterium Synechococcus sp. PCC 7002,with a dynamic range spanning more than five orders of magnitude. RNAwas isolated from cells grown under limitation for five major nutrients: CO 2,nitrogen source, sulfate, phosphate and iron. As a basis for comparison,RNA was isolated and sequenced from cells grown under optimal(„standard”) conditions. A comparison of the relative transcript abundancesof the nutrient-limited samples with those for standard conditions revealedthat there were generally lower mRNA levels for genes involved in themajor metabolic functions, especially protein biosynthesis, photosystems,phycobiliproteins, ATP synthesis and CO 2 fixation. Nutrient limitationfurther resulted in an increase in transcripts for the nblA gene, encoding thephycobilisome degradation protein NblA, which was most prominent undernitrogen limitation. Limiting the supply of a specific nutrient generallyresulted in increased mRNA levels for genes encoding the correspondinguptake mechanisms, i. e., transporters for nitrate, ammonia, phosphate,sulfate and iron. CO 2 limitation resulted in increased transcript levels forRuBisCO and carboxysomal proteins, sbtA, coding for a bicarbonatetransporter, and the genes coding for the so-called inducible CO 2 uptakemechanism, which are related to the Type-1 NADH dehydrogenasecomplex. Transcriptional profiling further suggested that there might beadditional changes in the NADH dehydrogenase complex subunitcomposition as a result of acclimation to nutrient limitation.NTP010A novel genetically encoded FRET biosensor forquantitative detection of oxygen in living cellsJ. Potzkei* 1 , M. Kunze 2 ,S. Endres 1 , A. Heck 1 , J. Büchs 2 , K.-E. Jaeger 1 ,T. Drepper 11 Faculty of Mathematics and Natural Sciences, Institute for MolecularEnzyme Technology (IMET), Heinrich-Heine University, Jülich, Germany2 Process Engineering, RWTH Aachen, Aachen, GermanyFluorescent reporter proteins (FPs) like the green fluorescent protein (GFP)from the jellyfish Aequorea victoria enable the non-invasive quantitativereal-time analysis of complex cellular processes in vivo. However, a majordrawback of GFP and its variants is their strict limitation to aerobicbiological systems. This is primarily due to the fact that the autocatalyticsynthesis of the fluorophore depends on molecular oxygen. Therefore, werecently developed a class of fluorescent proteins which can be used underaerobic as well as anaerobic conditions (1,2,3) . These FPs carry flavinmononucleotide (FMN) as fluorophore and are thus termed FMN-bindingfluorescent proteins (FbFPs). Beside protein labeling, genetically encodedFPs can also be used as molecular biosensors allowing the online in vivomeasurement of essential parameters or metabolites. For that purpose, twodifferent FPs with overlapping emission/excitation spectra are generallyfused together via a sensory linker peptide. Thus, the presence of a certainmetabolite can be detected by a biosensor due to Förster Resonance EnergyTransfer (FRET) which only occurs after its binding to the sensor domain.Here, we present a novel FP-based biosensor that allows the detection ofmolecular oxygen for the first time. The biosensor consist of an oxygeninsensitiveFbFP domain and an O 2-sensitive YFP domain. In vitro and invivo characterization of the biosensor revealed that FRET from FbFP to YFPonly occurs in the presence but not in the absence of oxygen. Therefore, theratio of the fluorescence emission at 495nm (FbFP) relative to thefluorescence emission at 527nm (YFP) provides quantitative data of theintracellular oxygen levels during microbial growth.[1] Drepper, T. et al (2007): Reporter proteins for in vivo fluorescence without oxygen. NatBiotechnol 25: 443-445.[2] Drepper, T. et al (2010): Flavin mononucleotide-based fluorescent reporter proteins outperformgreen fluorescent protein-like proteins as quantitative in vivo real-time reporters. Appl EnvironMicrobiol 76 5990-5994.[3] Circolone, F. et al (2010): Neue Biosensoren und deren Verwendung. Patent Number: DE 10 2010037 001.NTP011To hear microbes settling down - online detection ofmicrobial biofilm formation by means of acoustic LambwavesM. Schmitt 1 , K. Schmidt 1 , M. Egert* 2 , G. Lindner 21 Institute of Sensor and Actuator Technology,Coburg University of AppliedSciences, Coburg, Germany2 Faculty of Science Coburg University of Applied Sciences, , Coburg,GermanyBiofilms are a common mode of microbial life in natural as well asindustrial and hospital environments. In the case of the latter, early detectionof biofilm formation is pivotal in preventing men and machines from lifethreateningand costly negative effects. Among the wealth of methods usedto monitor biofilm formation, biosensors appear as attractive tools due to thespeed of the detection process, suggesting a true online monitoring [1-2].However, biosensor-based biofilm monitoring still suffers from severaldrawbacks. For instance, in the case of acoustics, traditional ultrasonicsensors fail in detecting biofilm formation due to the small differences in theacoustic impedance of the biofilm in comparison to water.Here we present a new macroscopic acoustic approach, aiming at thedetection of deposits on the bottom of liquid-filled tubes and containers bymeans of Lamb waves, i.e. elastic waves propagating in thin solid mediasuch as plates or tubes [3]. Preliminary experiments with gelatine layers as abiofilm substitute proved the feasibility of this approach: Interdigitaltransducers attached to the outer wall of the liquid-filled container were usedto produce and receive the acoustic signals. Signal transmission times andsignal amplitudes of short Lamb wave pulses changed significantly with thethickness of the gelatine layer on a test surface and allowed for a reliabledetection of layers thinner than 10 μm. Subsequently, a measurement cellequipped with such transducers was passed through with a culture mediuminoculated with an overnight culture of biofilm-forming Stenotrophomonasmaltophilia cells. After 16 hours of percolation at 30°C, cell densities hadincreased to 10 8 cells / ml. Signal transmission times and amplitudesbetween the interdigital transducers had changed notably in comparison tospektrum | Tagungsband 2011