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

NTV004A simple method to prepare microorganisms for AFManalysisT. Günther*, J. Raff, K. PollmannBiophysik, Helmholtz Center Dresden Rossendorf, Dresden, GermanyNowadays AFM becomes a more and more attractive method formicrobiologists to investigate Microorganisms. The technique allowsimaging over a broad magnitude scale and is not confined by the diffractionlimit. Sometimes it is interesting to measure the dimensions of an organism.The other time the question is about surface properties of a cell. Thescanning principle makes the AFM technique comparatively slow and thespecimen has to be fixed on a flat surface during the scans. It is quite simpleto dry the samples on a surface. Drying leads to a good immobilization butalso to drying artifacts like denaturation of Proteins and shrinkage of thewhole cell due to the loss of water. Therefore it is advantageous for mostbiological questions to do the imaging in liquids. Immobilization is nottrivial as result of the heterogeneous surface properties of different microorganisms. Existing preparation methods are mostly utilizing coated surfacesor lithographicaly prepared surfaces. While lithography is not an option foreveryone there is a variety of coatings available for instance poly-L-lysine orgelatine which work quite good with some microorganisms. A new methodbased on polyelectrolyte coated surfaces combined with centrifugalsedimentation shows promising results regarding the efficiency ofimmobilization. A variety of micro organisms were tested with the newmethod showing universality for many organisms. The samples wereprepared with and without fixation. Of course fixation simplifies theimaging by enhancing the stability of the samples. But even unfixedMicroorganisms can be imaged which opens the field for investigations inrespect to cell division or other dynamic processes of living cells.NTV005Development of a novel biosensor for the intracellulardetection of L-methionine and branched-chain aminoacidsN. Mustafi*, M. Bott, J. FrunzkeBiotechnology, Research Center Jülich, Jülich, GermanyMetabolite detection and quantification in single bacterial cells is one of thegreat challenges of current research in the field of White Biotechnology.Here, we report the development of a biosensor which enables theintracellular detection of L-methionine or branched-chain amino acids andtransforms this information into an optical readout, in this case theproduction of a fluorescent protein. The described biosensor will be appliedto support efforts in strain development for the production of methionine orbranched-chain amino acids and is furthermore of great value for theanalysis of production strains on a single-cell level. The sensor is based onthe Lrp-BrnFE module of Corynebacterium glutamicum, consisting of theLrp-type transcriptional regulator Lrp and its target genes brnFE encodingan export system for methionine and branched-chain amino acids. Atelevated intracellular methionine concentrations Lrp is in an active state andinduces transcription of the divergently transcribed genes brnFE. For thedevelopment of the biosensor we constructed a sensor cassette including lrp,the intergenic region of lrp and brnF, and the promoter of brnF fused to yfp,which encodes a yellow-green variant of GFP. Due to the specificitycharacteristics of Lrp, this sensor is suitable for the detection of methionineand the branched-chain amino acids L-leucine, L-valine and L-isoleucine.By in vivo measurements the relative affinity and specificity of the sensortowards its effectors was determined. Fluorescence spectroscopy andfluorescence-activated cell sorting (FACS) confirmed the general suitabilityof the system to monitor the intracellular production of methionine andbranched-chain amino acids. Thus, this sensor represents a valuable tool forefficient strain development in White biotechnology and can furthermore beapplied for the study of the population structure of industrial productionstrains.[1] Kennerknecht, N. et al (2002): Export of L-isoleucine from Corynebacterium glutamicum: a twogene-encodedmember of a new translocator family. J. Bacteriol. 184: 3947-3956.[2] Trötschel, C. et al (2005): Characterization of methionine export in Corynebacterium glutamicum.J. Bacteriol. 187: 3786-3794.NTP001Site specific mutagenesis of lysozyme immobilized onmagnetic beads as a target for specific interaction andsubsequent separation of bacteria for enrichment orisolation from complex matrices by magnetic forcesE. Diler* 1 , T. Schwartz 1 , U. Obst 1 , K. Schmitz 21 Institute of Functional Interfaces, Interface Microbiology, KarlsruheInstitute of Technology (KIT), Eggenstein-Leopoldshafen, Germany2 Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT),Karlsruhe, GermanyThe VBNC state of bacteria and low bacterial densities are big challengesfor cultivation based pathogen recovery in e.g. drinking water and foodindustry. Therefore, a new molecular biologic separation method for bacteriausing point mutated chicken c-type lysozymes immobilized on magneticbeads for bacteria separation is described. The immobilized mutatedlysozymes on magnetic beads serve as baits for the specific capture ofbacteria from complex matrices or water and can be separated by usingmagnetic racks. To avoid the bacterial cell lysis by lysozyme the protein wasmutated at amino acid position 35 leading to the exchange of the catalyticglutamate with alanin (LysE35A) and with glutamine (LysE35Q). As provedby turbidity assay with reference bacteria these changes caused theelimination of the muramidase activity from mutated lysozymes but areknown to retain their affinity for bacterial cell wall components. Themutated constructs were expressed by the yeast Pichia pastoris and secretedinto expression medium. Protein enrichment and purification was carried outby SO 3 functionalized nano-scale cationic exchanger particles. For a rapidproof of principle the proteins got biotinylated and immobilized onstreptavidin functionalized, fluorescence dye labelled magnetic beads. Theseconstructs were used for successful capture of Syto9 marked M. luteus cellsfrom cell suspension as visualised by fluorescence microscopy whichprovided a first hint for the success of the strategy.NTP002Characterization of microbial ecological systems: anindustrial applicationK.A. Stangier , B. Müller*, D. Monné Parera, Y. KumarGATC Biotech AG, Konstanz, GermanyCommon strategies for the phylogenetic characterisation of microbialecosystems are based on the „passive” DNA genome (genetic potential).GATC has developed an integrated solution to analyse such ecosystemsusing the „active” RNA. Experimental outlines will be shown to analyze acomplex industrial microbial ecosystem using a combinatory approach ofdifferent libraries and sequencing technologies. RNA Step 1: This approachdiffers from others in using the transcribed RNA („active” genome) asstarting material. Reverse transcription to cDNA is followed by anormalisation step. The normalised cDNA samples are sequenced on theRoche GS FLX. Subsequent proprietary bioinformatic analysis allows insilico separation of rRNA and mRNA. rRNA data, is used for phylogeneticanalysis. The remaining reads are assembled (de novo) and build thetranscript reference for quantification. Step 2: Total RNA starting material isdepleted of rRNA. The cDNA, derived from the remaining mRNA issequenced on the Illumina HiSeq 2000. The resulting large amount ofsequence data can be mapped to the transcripts (step 1) and quantified. Thiscombinatorial approach determines microbial diversity and abundance aswell as gene content and relative levels of gene expression. DNA For theverification of the approach, the classical standard 16S rRNA analysis usingprimers derived from conserved 16S rRNA regions is performed. Aftersequencing on the Roche GS FLX, the data are compared to the results fromthe RNA experiment. Pacific Biosciences RS With the new PacificBiosciences PacBio RS real time single molecule sequencer, reads longerthan 1,000 bp can be obtained. These read lengths enable the design ofdifferent primer sets to achieve longer and more specific 16S rRNAfragments that can be sequenced in one read. Additionally, only one read isneeded to sequence long transcripts. This will increase the accuracy of thephylogenetic studies as well as the cDNA analysis. The PacBio RS will bedeveloped for direct RNA sequencing which will lead to more preciseanalysis of ribonucleic acids. Conclusion The new approach for asimultaneous phylogenetic, qualitative, and quantitative analysis allows for aprecise look into the diversity and change in metabolic pathways ofmicrobial ecosystems.spektrum | Tagungsband 2011