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

nutraceutical, and sterile manufacturing environments, these technologiesunder perform due to the diversity of organisms found in theseenvironments. Whole cell proteotypic analysis with matrix-assisted laserdesorption/ionization - time of flight (MALDI-TOF) spectroscopy is anemerging rapid and inexpensive method of identifying bacteria. This studytests the accuracy, robustness, and reproducibility of this technology whenapplied to an extensive panel of known culture collection strains andfrequently seen species found in pharmaceutical, nutraceutical and sterilemanufacturing environmental monitoring programs. The MALDI-TOFmanufacturer’s recommended methods for sample processing and dataanalysis are directly compared to methods optimized in our laboratory forenvironmental monitoring. Advantages and limitations of using MALDI-TOF technology for this application are discussed. This study showed thatfewer than 70% of frequently seen environmental isolates could beaccurately identified with the MALDI Biotyper system (BrukerDaltonics). The >30% that failed to be identified were largely due tolimitations in the clinically focused Biotyper database. At Accugenix, themethods were optimized and additions made to the library such that thenumber of samples that were identified increased. While a custom databaseand method modifications are required to optimize this technology forenvironmental monitoring applications, the low cost and rapid turn aroundtime of MALDI-TOF analysis remain strong advantages for this technology.EMP034Detection and activity of halorespiring bacteria atchloroethene contaminated sitesC. Stoll*, K.R. Schmidt, A. TiehmWater Technology Center Karlsruhe, Environmental Biotechnology,Karlsruhe, GermanyChloroethenes are frequently detected in contaminated groundwater.Bacteria from several genera - e.g. Dehalobacter, Desulfomonile,Desulfitobacterium and Desulfuromonas - are able to reductivelydechlorinate / halorespire perchloroethene (PCE) and trichloroethene (TCE)to cis-1,2-dichloroethene (cDCE), whereas only bacteria from theDehalococcoides cluster are known to catalyze the complete reductivedechlorination of PCE to ethene.We investigated the distribution and growth of chloroethene-degradingmicroorganisms in groundwater samples and laboratory microcosmexperiments. Using nested PCR, we measured the occurence of thedechlorinating species in groundwater samples. The presence ofhalorespiring bacteria correlated well with hydrochemical site data, pollutantdistribution and the degree of dechlorination observed in microcosm studies.Growth of Dehalococcoides spp. in microcosms, assessed by quantitativePCR (qPCR), corresponded with the dechlorination of PCE to ethene. Aninhibition in growth was observed at lower pH, whereas Desulfitobacteriumspp. was less affected by pH changes.In conclusion, this study demonstrates that 16S-PCR detection ofhalorespiring bacteria represents a quick and easy means to estimate thedegree of reductive dechlorination of chloroethenes to occur at a given site.The authors gratefully acknowledge financial support by the GermanMinistry of Economics (BMWi, grant no KF2265705AK9).retentostat with medium supply of 50ml/h. Either a single carbon source(5mM acetate) or two carbon sources (2.5 mM acetate and 0.7 mMbenzoate) were fed and Fe(III)citrate was added as an electron-acceptor inboth cases. The lowest doubling time that was achieved during thecultivation was 229 hours, which corresponds to a growth rate of 0.003 1/h.The consumption of carbon sources, reduction of electron acceptor, cellnumbers and total protein concentration were examined. We will report onthe growth and utilization of carbon sources by G. metallireducens duringcultivation in retentostat. The analyses of expressed proteins involved indifferent substrates degradation under limited substrate conditions will bepresented. The retentostat cultivation will be used as a model to explainmicrobial physiology under natural conditions.EMP036Degradation of Amadori products in Bacillus subtilis:The physiological relevance and transcriptionalregulation of the frlBONMD operonV. Deppe* 1,2 , S. Klatte 1,2 , J. Bongaerts 1 , T. O’Connell 1 , K.-H. Maurer 1 ,F. Meinhardt 21 Henkel AG & Co. KGaA, Biotechnology, Düsseldorf, Germany2 Insitite for Molecular Miciobiology and Biotechnology, WestphalianWestphalian Wilhelms-University, Münster, GermanyAmadori products (fructosamines) constitute the first stable intermediates ofthe Maillard reaction and occur ubiquitously in nature. As rotting fruits andvegetables may contain up to 7% of the fresh mass as fructosamines,Amadori products are released into the soil and are presumed to serve assubstrates for many soil microorganisms. The Amadori product degradation(deglycation) systems of these organisms differ with respect to mechanismsas well as substrate specificities. Fructosyl amino acid oxidases of fungi andbacteria decompose extracellular Amadori products by means of oxidation,generating the respective amino acid, glucosone and H 2O 2.The fructosamine 3-kinases of mammals and homologous, related proteins,which are common to all forms of life, degrade intracellular Amadoricompounds and thus focus on cell repair functions.This study addresses to the deglycating system of the soil bacterium B.subtilis which resembles that of E. coli. A kinase (FrlD) phosphorylates thefructosamine at C6 prior to further cleavage by a deglycase (FrlB). Thephysiological importance of the encoding genes was examined, revealing thedependence of their expression for growth on fructosamines. Furthermorethe complex regulation of the corresponding transcription unit was analyzed.In addition to the known regulation by the global transcriptional regulatorCodY, the frl operon is subject to repression by the adjacent and inverselyencoded FrlR. FrlR causes the strict repression during growth on substratesother than fructosamines. The expression of frl genes increased about 33-fold with the onset of growth on Amadori products suggesting thatderepression is brought about by substrate induction. Additionally, in thefirst intergenic region of the operon a FrlR binding site was identified whichis centrally located within a 38 bp perfect palindromic sequence. There isgenetic evidence that this sequence in combination with FrlR contributes tothe remarkable decrease in the transcription downstream of the first gene ofthe frl-operon.EMP035In situ physiology of G. metallireducens under low growthrates during cultivation in retentostatS. Marozava* 1 , W. Roling 2 , R. Meckenstock 11 Institute of Groundwater Ecology, Helmholtz Center Munich, Neuherberg,Germany2 Department of Molecular Cell Physiology, Free University of Amsterdam,Amsterdam, NetherlandsThe physiology and gene regulation of microorganisms in batch laboratorycultures is well established but our knowledge of gene regulation undercarbon limited growth conditions such as in chemostats is scares. Basicallynothing is known about microbial physiology under in situ conditions insoils or sediments. The current study is devoted to examine the in situphysiology of the aromatics-degrading anaerobic deltaproteobacteriumGeobacter metallireducens under close to natural conditions. Our hypothesisis that under natural conditions (i.e. low amounts of carbon sources, lowgrowth rates, and mixed substrates) all carbon sources will be utilizedsimultaneously relatively to conditions with high substrate concentrationsand/or mixed substrates where diauxic growth prevail and one carbon sourceis preferentially consumed. Geobacter metallireducens was cultivated in aEMP037Global transcriptome analysis of soil microbialcommunitiesC. Mettel*, Y. Kim, W. LiesackDepartment of Biogeochemistry, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyGlobal transcriptome analysis, or metatranscriptomics, is defined as theanalysis of microbial gene expression patterns at the community level bymassively parallel shotgun sequencing as opposed to the total gene contentwhich is characterized by metagenomic approaches. In future,metatranscriptomics will allow us to study the functional response ofcomplex microbial communities to environmental change. This study aimedto establish a robust procedure for generating metatranscriptome data setsfrom soil microbial communities. Metatranscriptomics involves (i)extraction of total RNA, (ii) depletion of rRNA, (iii) cDNA synthesis, (iv)parallel shotgun sequencing, and (v) bioinformatic analysis. The extractionof high-quality mRNA from soil is more challenging than from most otherenvironments, mainly due to the high humic acid content. Therefore, we firstdeveloped an efficient method for extracting high-quality mRNA from soil.spektrum | Tagungsband 2011