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

mechanism, thus these enzymes function as AHL-lactonases. Finally ourdata suggest that the autoinducer hydrolases are of importance forrhizosphere colonization.OTP041The overlapping gene pair htgA/yaaW in Escherichia coliO157:H7 EDL933L. Fellner* 1 , K. Neuhaus 2 , R. Landstorfer 2 , S. Simon 1 , D. Oelke 1 , S. Scherer 21 Department of Data Analysis and Visualization, University of Konstanz,Konstanz, Germany2Department of Microbiology at the Central Institute for Food andNutrition Research (ZIEL), Technical University Munich, Freising,GermanyOverlapping genes are defined as a pair of genes whose coding regions arepartially or completely overlapping. Since DNA consists of twocomplementary strands and triplets code for the amino acids, six openreading frames would be possible in theory. Overlapping genes were firstobserved in bacteriophages, but in bacteria they are assumed to be rare. Afew examples have been reported for Pseudomonas fluorescens andStreptomyces coelicolor, but most bacterial overlapping genes are not verywell characterized [2, 3]. In this work we examine the controversialoverlapping gene pair htgA/yaaW in Escherichia coli O157:H7 EDL933.htgA has been described as heat inducible in 1993 [1]. Since then, theexperimental evidence is scarce and htgA is now classified as (obsolete)synonym to yaaW in the databases. This is astonishing insofar as htgA andyaaW are at the same locus, but in opposite orientation. Several records ofmicroarray experiments in the databases suggest differential, strand specificregulation of the htgA/yaaW-region.First, we analyzed the transcription of htgA and yaaW at six different growthconditions. Next, we cloned the suspected promoter regions of both genes infront of a gfp reporter. Third, one of the overlapping open reading frameswas destroyed by introducing a mutation causing a stop codon, which issilent on the other frame. Both mutants were tested under several growthconditions for a phenotype. Preliminary results suggest that both, htgA andyaaW, are weakly transcribed. The latter varies with different growthconditions. The existence of HtgA is still under investigation.[1] Missiakas, D. et al (1993): The Escherichia coli heat shock gene htpY: mutational analysis,cloning, sequencing, and transcriptional regulation. J Bacteriol 175: 2613-2624.[2] Silby, M.W. and S.B. Levy (2008): Overlapping protein-encoding genes in Pseudomonasfluorescens Pf0-1. PLoS Genet 4: e1000094.[3] Tunca, S. et al (2009): Two overlapping antiparallel genes encoding the iron regulator DmdR1 andthe Adm proteins control siderophore and antibiotic biosynthesis in Streptomyces coelicolor A3(2).Febs J 276: 4814-4827.OTP042Comparison of two prokaryotic expression systems forthe gamma-cyclodextrin glucanotransferase fromBacillus sp. G-825-6S. Washeim*, C. Foellner, W. ZimmermannDepartment of Microbiology and Bioprocess Technology, University ofLeipzig, Leipzig, GermanyCyclodextrin glucanotransferases (CGTases) convert starch to a mixture ofcyclic and linear oligosaccharides. The cyclic oligosaccharides, known ascyclodextrins have the ability to form inclusion complexes with manyorganic molecules altering their stability, solubilty, or bioavailabilty.Cyclodextrins are therefore of interest for applications in pharmaceutical,food and cosmetic industries. Gamma-CGTases are microbialglucanotranferases mainly producing gamma-cyclodextrin consisting of 8glucose units. The gamma-CGTase gene (cgtS) of the alkalophilic Bacillussp. G-825-6 was codon-optimized for usage with E. coli and B. subtilisallowing an expression in both hosts. E. coli BL21 (DE3) [pET-20b(+)::cgtS] and B. subtilis DB 430 [pHT08::cgtS] were used and theefficiency of the systems was compared. The transformation efficiency wasanalyzed by PCR resulting in 4 positive E. coli and 7 positive B. subtilisclones. Both vector systems were IPTG-inducible and contained a His-tag.E. coli and B. subtilis were grown in batch cultures at 37 °C. The inductionwas performed at an OD 600 of 1,0 for 14 h at room temperature. Crudeprotein extract obtained by sonification of the harvested cells was used fordetermination of starch-hydrolyzing activity by a colorimetric assay. UsingE. coli as expression system, clones containing starch-hydrolyzing activitycould be obtained while no activity was detected with the B. subtilis clones.The His-tag fusion protein obtained with the E. coli expression system waspurified and employed for the synthesis of cyclodextrins. As main endproducts, beta-cyclodextrin composed of 7 glucose units and gammacyclodextrinin a ratio 1:3 were detected by HPLC with pulsedamperometric detection.OTP043A Glycogen Synthase Defect Mutant of Clostridiumacetobutylicum ATCC 824K. Zimmermann*, R. Uhlig, R.-J. FischerDepartment of Microbiology, University of Rostock, Rostock, GermanyThe transition phase of the growth of the Gram-positive, spore-forminganaerobe Clostridium acetobutylicum is characterized by severalmorphological changes. At the beginning swollen and cigar shaped cells,clostridial stages, are formed. In the cells a polymeric carbohydrate, socalled granulose is accumulated in the form of granules. Thismacromolecule is defined as an amylopectin-like structure with slightlybranched (2 % of 1.6-linkages) glucose molecules. Granulose is expected tobe a energy- and carbon storage, putatively serving as a prerequisite forsporulation.In C. acetobutylicum, the synthesis of granulose is believed to be encodedby the gene products of the glg-Operon: GlgC (cac2237, Glucose-1-phosphate-adenyltransferase), GlgD (cac2238, ADP-Glucosepyrophosphorylase),GlgA (cac2239, Glycogen [Granulose] synthase) andCac2240 (a protein of unknown function). As only one glycogen synthase isannotated in the genome of C. acetobutylicum, glgA was expected to play acrucial role in the biosynthesis of granulose.Here we report on the construction of a specific glgA defect mutant using theClosTron ® technology [1]. Individual mutant strains were selected, whichare unable to accumulate granulose. Molecular analysis (Southern Blot andPCR investigations) proved the correct insertion of the „knock out” genecassette.Furthermore, results of the phenotypic characterisation are presented. Thisdata include iodine staining of colonies and cells in comparison to thewildtype, growth analysis (optical density, pH, product spectrum) and firstcomparative sporulation assays.[1] Heap (2007): J Microbiol Methods 70:452-464.OTP044Changes in abundance and activity of the biocenosisduring provoked process failureM. Liebrich*, M. Kasina, A. Kleyböcker, D. Seyfarth, H. WürdemannInternational Center for Geothermal Research, Helmholtz Center, Potsdam,GermanyOne solution to reduce the effects of climate change due to increasing CO 2emissions is the anaerobic fermentation of organic material. The knowledgeabout the interaction of the complex biological processes in biogas plants islimited up to date, e.g. the behaviour of the biocenosis under stressconditions.Due to the lack of process understanding biogas plants often run below theirmaximum loading rate. In order to maximise the space-time-yield of abiogas plant changes of the microbial community during shock loading,over-acidification and deacidification were monitored in several labexperiments.During these experiments the formation of different aggregates wasobserved, that had an influence on the process stability. The size ofaggregates was depended on the amount of additives used to stabilize theprocess during over-acidification.The biological samples were examined using different molecular biologymethods, to observe changes in the abundance and activity of themicroorganisms involved: Genetic fingerprinting (Denaturing gradient gel electrophoresis,DGGE) for the characterisation of the dominant species of thebiocenosis qPCR (quantitative real-time-polymerase chain reaction) toquantify the metabolic activity of the groups of microorganismsinvolved FISH (fluorescence in situ hybridization) to quantify the differentgroups of microorganismsspektrum | Tagungsband 2011