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

203First they were discovered in plants, but later were also described in fungi,cyanobacteria and other prokaryotes. In plants, phytochromes control awide variety of developmental processes, while their function inprokaryotes is widely unknown. Most bacterial phytochromes contain ahistdine-kinase domain suggesting that signal transduction occurs via atwo-component regulatory system.Pseudomonas aeruginosais one of thefirst heterotrophic bacteria in which a phytochrome has been identified.With the two genes bphO and bphP P. aeruginosa possesses the twonecessary components to assemble a red-light photoreceptor system:bphOcodes for the heme oxygenase to generate the chromophore biliverdin IXand bphP,encoding the apo-phytochrome. So far, no correspondingphytochrome response regulator has been identified yet.bphO and bphP form a bicistronic operon whose expression is controlledby the alternative sigma factor RpoS. New analyses provide an additionalregulation of bphP by the quorum sensing-regulator LasR.This exceptionalregulation is currently addressed to study in more detail. To investigate thefunction of bphO and bphP chromosomal knock-out mutants wereconstructed and analysed. However, no significant phenotypical differencebetween the mutants and wild type were observed. A combination ofexpression profile experiments and proteome analyses revealed a link to abphP-mediated stress response. The most downregulated genes are used ina genetic screen to identify the corresponding response regulator of BphPto gain further insight into the function of the phytochrome inP.aeruginosa and the components of its regulon. In addition someproteome phosphorylation studies will be presented.RSP041A heme-based redox sensor in the methanogenic archaeonMethanosarcina acetivoransB. Molitor* 1 , M. Staßen 2 , M. Rother 2 , N. Frankenberg-Dinkel 11 Ruhr University Bochum, Physiology of Microorganisms, Bochum, Germany2 TU Dresden, Institute for Microbiology, Dresden, GermanyBased on a bioinformatics study, the protein MA4561 fromMethanosarcina acetivorans was originally predicted to be aphytochrome-like protein [1]. Phytochromes are red light sensingphotoreceptors with a bound linear tetrapyrrole chromophore at aconserved cysteine residue in either a PAS or a GAF domain. MA4561consists of two alternating PAS and GAF domains fused to a C-terminalkinase domain.While we were able to show that recombinantly produced and purifiedprotein does not bind any linear tetrapyrrole chromophores, UV-visspectroscopy revealed the presence of a heme tetrapyrrole cofactor. Incontrast to many other known heme-containing proteins, the heme wasfound to be covalently bound via one vinyl side chain to cysteine 656 inthe second GAF domain. This GAF domain by itself is sufficient forcovalent attachment. The heme cofactor is redox active and is able tocoordinate carbon monoxide in its reduced state. Interestingly, the redoxstate of the heme cofactor has a strong influence on autophosphorylationactivity. While reduced and CO-bound protein does not autophosphorylate,the oxidized protein gives a strong autophosphorylation signal. Twodimensionalthin-layer chromatography identified serine and tyrosineresidues as phosphorylation sites.Based on its genomic localization, MA4561 is most likely a sensor kinaseof a two-component system. The transcriptional regulator MA4560 (MsrG)encoded downstream of MA4561is directly involved in transcriptionalactivation ofmtsH, which encodes a methyltransferase/corrinoid fusionprotein involved in methylsulfide metabolism [2, 3]. On the basis of ourresults a model in which MA4561 acts as a heme-based redox sensor ispresented.[1] Karniol, B. et al.,Biochem J(2005)392(1), 103-116[2] Bose, A.et al.,Mol Microbiol (2009)74(1), 227-238[3] Oelgeschläger, E., and Rother, M., Mol Microbiol (2009) 72(5), 1260-1272RSP042Functional Analysis of Additional Circadian Clock Proteins inSynechocystissp. PCC 6803H.-T. Deinzer*, J. Holtzendorff, A. Wilde, A.K. BäckerUni Giessen, Mikrobiology, Giessen, GermanyCircadian rhythms, oscillations with approximately 24 h periods drivingmany physiological activities, are found in most eukaryotes. Amongprokaryotes, exclusively cyanobacteria are known to harbour an internalclock. Work on the model strain for the circadian clock, Synechococcuselongatus PCC 7942 has shown that the interaction of only 3 proteins,KaiA, KaiB and KaiC encoded by the kaiABC gene cluster is essential forthe generation of circadian rhythms of gene expression. The timing processitself is based on rhythmic changes in the autophosphatase-, autokinaseandATPase- activity of the hexameric KaiC protein.A few cyanobacteria show variations among their circadian clock genecomposition, such as the loss of kaiA in the case of Prochlorococcus. Incontrast, the genome of Synechocystissp. PCC 6803 holds an additionalkaiC2B2 operon and two orphan kaiB3 and kaiC3 genes in addition to thekaiABC gene cluster. We are currently investigating the function of theseadditional kai genes.Analysis of Synechocystis kai mutants indicates that kaiC2 is an essentialgene. Knockdown mutants of the kaiC2B2 operon display a bleachedphenotype. Biochemical characterization of purified KaiC2 proteinsuggests that it possesses kinase activity and might interact withcomponents of the phycobilisome as well as with the transcriptionmachinery. Further biochemical characterization will yield insights intoKai protein complex formation, as well as ATPase activity andphosphorylation cycles of the three different KaiC proteins fromSynechocystis.RSP043Model of the synthesis of trisporic acid in Mucorales showingbistabilityS. Werner 1 , A. Schroeter 1 , C. Schimek 2 , J. Wöstemeyer 2 , S. Schuster* 11 University of Jena, Dept. of Bioinformatics, Jena, Germany2 University of Jena, Institute of General Microbiology and MicrobialGenetics, Jena, GermanyAn important substance in the signaling between individuals of Mucor-likefungi is trisporic acid (TA). This compound, as well as some of itsprecursors, serves as a pheromone in mating between (+)- and (-)-matingtypes. Moreover, intermediates of the TA pathway are exchanged betweenthe two mating partners. Here, we present mathematical modelsof the synthesis pathways of TA in the two mating types of an idealizedMucor-fungus, based on differential equations. These models include thepositive feedback of TA on its own synthesis. We compare three submodelsin view of bistability, robustness and the reversibility oftransitions. Our modelling study showed that, in a system whereintermediatesare exchanged, a reversible transition between the two stable steady statesoccurs, while an exchange of the end product leads to an irreversibletransition. The reversible transition is physiologically favoured, becausethe high-production state of TA must come to an end eventually.Moreover, the exchange of intermediates and TA is compared with the 3-way handshake widely used by computers linked in a network.RSP044Cysteine formation with Corynebacterium glutamicum andintracellular sensing of O-acetyl-serineK. Hoffmann*, M. Bott, L. EggelingFZ Jülich GmbH, Institute of Bio- and Geosciences, IBG I: Biotechnology,Jülich, GermanyWe succeeded to engineer Corynebacterium glutamicum into a superior L-serine producing microorganism. L-Serine is a precurser of L-cysteine andboth amino acids are required for pharmaceutical purposes. Consequently,it is of interest to study the two step conversion of L-serine to L-cysteinemediated by serine acetyltransferase (SAT, cysE) and O-acetylserinesulfhydrylase (OASS, cysK). The L-cysteine synthesis involves theintermediate O-acetyl-serine (OAS) which is demonstrated to interact invitro with the transcriptional regulator CysR. We fused the CysR targetcysI to EYFP to construct pSenOAS. Presence of pSenOAS resulted inincreased fluorescence of cultures with elevated OAS levels, illustratingthat in vivo OAS interacts with CysR to cause increased cysI transcription.The system established allows the detection of cells with elevated OASlevels at the single cell-level and the differentiation and sorting of singlecells according to their cytosolic OAS concentration via FACS(Fluorescence Activated Cell Sorting).The L-serine producer accumulated already 1 mM L-cysteine. Uponoverexpression of cysE 5.8 mM L-cysteine accumulated, and upon thecombined expression of cysE plus cysK 7.3 mM L-cysteine was found. Thework illustrates that C. glutamicum is a promising candidate for theoverproduction of L-cysteine, and that FACS selection is a tool for furtherstrain development.RSP045Identification of the target promoters of Qdr1 and Qdr2, twotranscriptional regulators of 2-methylquinoline degradationby Arthrobacter nitroguajacolicus Rü61aH. Niewerth*, S. FetznerWestfälische Wilhelms-Universität Münster, Institut für MolekulareMikrobiologie und Biotechnologie, Münster, GermanyArthrobacter nitroguajacolicus Rü61a is a Gram-positive soil bacteriumwhich is able to utilize 2-methylquinoline as source of carbon and energy.The genes that are required for the conversion of 2-methylquinoline toanthranilate are clustered in two divergently oriented “upper pathway”operons (pAL1.003-006 and pAL1.007-011). A third operon (pAL1.019-023) codes for enzymes involved in anthranilate degradation via coenzymeBIOspektrum | Tagungsband 2012