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

199RSP020Regulation of anaerobic aromatic hydrocarbons degradation inAromatoleum aromaticum under anaerobic growth conditionA. Ashraf*, J. Heider, T. KraushaarPhilipps Universität Marburg, Biologie/Mikrobiologie, Marburg, GermanyThe denitrifying Betaproteobacterium Aromatoleum aromaticum utilizes awide range of aromatic compounds under anoxic condition, among themthe hydrocarbons ethylbenzene or toluene. The genes coding for theenzymes of anaerobic toluene metabolism are induced coordinately in thepresence of toluene, whereas those coding for the enzymes of anaerobicethylbenzene metabolism are induced sequentially in the presence ofethylbenzene and the intermediate acetophenone, respectively. Threeoperons coding for two-component regulatory systems were identified inthe genome sequence of A.aromaticum as possible candidates for affectingthe induction of all toluene-catabolic genes (tdiSR) and the induction ofethylbenzene-catabolic genes by ethylbenzene (ediSR) and theintermediate acetophenone (adiRS). We show here that the (adiRS) operonis indeed involved in the acetophenone-dependent regulation of geneexpression. The function of these gene products was investigated bygenetic and biochemical studies: adiSR deletion mutant of A. aromaticumwas unable to grow on either ethylbenzene or acetophenone and wascomplemented by adding the adiRS genes. Moreover, the predictedacetophenone-sensing histidine kinase (AdiS) was overproduced in E. coliand its biochemical properties, i.e. ligand binding, are in line with itsproposed function.[1]-Heider, J., and G. Fuchs.1997. Anaerobic metabolism of aromatic compounds. Eur JBiochem243:577-96[2]-R. Rabus, M. Kube, A. Beck,,F. Widdel and R. Reinhardt. Genes involved in the anaerobicdegradation of ethylbenzene in a denitrifying bacterium, strain EbN1. Arch Microbiol (2002)178:506-516RSP021The W-/Se-containing class II benzoyl-CoA reductase complexin obligately anaerobic bacteriaC. Löffler* 1 , J. Seifert 2 , H.-J. Stärk 3 , M. Boll 11 University , Biochemistry, Leipzig, Germany2 Helmholtz Centre for Environmental Research, Proteomic, Leipzig, Germany3 Helmholtz Centre for Environmental Research, Analytic, Leipzig, GermanyBenzoyl-Coenzyme A (CoA) is a central intermediate in the anaerobicdegradation of aromatic compounds and serves as substrate for benzoyl-CoA reductases (BCRs). There are two completely different classes ofBCRs which both yield the nonaromatic product cyclohexa-1,5-diene-1-carbonyl-CoA [1,2]. Class I BCRs of facultative anaerobes, referred to asBcrABCD, are ATP-dependent, [4Fe-4S] clusters containing enzymes. Incontrast, strictly anaerobic bacteria are proposed to employ a W-/Zn-/FeS-/Flavin-/Se-containing, ATP-independent BamBCDEFGHI complex. Theactive site containing components BamBC were purified and characterizedfrom the aromatic compound degrading model organism Geobactermetallireducens [1]. The remaining BamDEFGHI subunits are consideredto be involved in the ATP-independent electron activation reaction. Weprovide evidence that class II BCRs are composed of the predicted highmolecular BamBCDEFGHI complex. Initial data indicate that the electrontransfer to the aromatic ring is driven by an electron bifurcation process.(1) Kung et al. (2009), PNAS 106 : 17687-92(2) Löffler et al. (2011) Environ Microbiol 13(3) : 696-709RSP022Metabolome and transcriptome analysis of P. aeruginosa in achronic lung infection modelA. Pelnikevich* 1 , L. Whielmann 1 , D. Schomburg 2 , B. Tümmler 11 Medizinische Hochschule Hannover, Hannover, Germany2 Technical University Braunschweig, Braunschweig, GermanyPseudomonas aeruginosais an ubiquitous environmental soil bacteriumand an opportunistic pathogen of humans, animals and plants. It causeschronic infections in patients with cystic fibrosis (CF), chronic obstructivepulmonary disease and bronchiectasis.We studied the control of virulence factor production depending onmetabolic pathways and the transcriptomic state of the organism tounderstand the activation of specific virulence programs of P. aeruginosa.We analysed the metabolome and transcriptome of P. aeruginosa invarious media and growth phases.P. aeruginosa PA14 is an acute infection clinical isolate obtained from aburnwound of a patient. It displays pathogenicity in a variety of geneticallytractable model hosts and mice.P. aeruginosa RN7 is a clone of PA14 strain. It is a CF-isolate, which wasisolated short after the infection of a patient. RN7 causes chronic disease inexperiments with mice.P. aeruginosa TBCF10839 is a highly virulent strain, which belongs to amajor clone in the P. aeruginosa population. It is a pilin-deficient strainthat produces large amounts of alginate and shows high resistance againstphagocytosis. Being a strong producer of virulence effector proteins, itcauses substantial airway pathology in mice after intratracheal instillation.In an integrative approach of both data sets will be combined to reveal aholistic picture of the adaptive pathway regulation of P. aeruginosa in alung infection and identificate key determinants for the chroniccolonization of the human lung.RSP023The importance of the GAF domain for K + -sensing in thesensor kinase KdpD in Escherichia coliH. Schramke* 1 , G. Gabriel 1 , C. Vilhena 2 , R. Heermann 1 , K. Jung 11 Ludwig-Maximilians-Universität, Department 1, Mikrobiologie,Martinsried/München, Germany2 Universidade de Lisboa, Faculdade de Farmácia, Lisbon, PortugalPotassium is the most abundant cation in bacteria and important fordifferent cellular functions. The high affinity K + transporter KdpFABC ofE. coli assures the uptake of K + when it is limited in the environment. Theproduction of KdpFABC is regulated by the two-component systemKdpD/KdpE, which comprises the membrane-integrated histidine kinaseKdpD and the soluble response regulator KdpE. KdpD specificallyphosphorylates and dephosphorylates KdpE and therefore regulates theactivation and termination of kdpFABC transcription, respectively [1]. K +has an inhibitory effect on the kinase activity of KdpD in vitro, but a K + -binding site is yet unknown. The kinase activity is also inhibited by Rb + ,but not by Cs + . New bioinformatic methods revealed that KdpD contains aGAF domain in the C-terminal cytoplasmic region. GAF domains areprominent ligand binding sites and were first identified in cGMP-specificcyclic nucleotide phosphodiesterase, adenylyl cyclase and the transcriptionfactor FhlA. The replacement of the GAF domain of KdpD with the GAFdomain of a conserved protein 3e0Y of Geobacter sulfurreducens led to aKdpD variant, which caused kdpFABC transcription independent of theextracellular K + concentration. Hence this KdpD variant was unable tosense K + . By using site-directed and random mutagenesis three aminoacids were identified - two inside and one outside of the GAF domain -which might form a K + -binding site.[1] Heermann and Jung, FEMS Microbiol Lett. 2010 Mar; 304(2):97-106.RSP024The histidine kinase SgmT is a c-di-GMP receptor andregulates synthesis of an extracellular matrix proteaseT. Petters* 1 , X. Zhang 1 , J. Nesper 2 , A. Treuner-Lange 1 , N. Gomez Santos 1 ,M. Hoppert 3 , U. Jenal 2 , L. Søgaard-Andersen 11 MPI for terrestrial Microbiology, Ecophysiology, Marburg, Germany2 Biozentrum, Basel, Switzerland3 Georg-August-Universität, Göttingen, GermanyMyxococcus xanthus cells are covered by an extracellular matrix composedof exopolysaccharides and proteins, which is indispensable for type pilidependentmotility and fruiting body formation in response to starvation.The orphan DNA binding response regulator DigR plays a role in theregulation of extracellular matrix composition. Using a two-tiered strategy,we genetically and biochemically identify the orphan hybrid histidinekinase SgmT, which contains an N-terminal GAF domain and a C-terminalGGDEF domain, as the partner kinase of DigR. By EMSA and DNase Ifootprinting experiments, we identify the DigR binding site in thepromoter of the fibA gene, which encodes a metalloprotease and is themost abundant protein in the extracellular matrix. Whole-genomeexpression profiling experiments in combination with the identified DigRbinding site allowed the identification of candidate members of the DigRregulon and suggest that SgmT/DigR regulate the expression of genescoding for secreted proteins of unknown function, FibA as well asenzymes involved in secondary metabolite synthesis. Our data demonstratethat the N-terminal GAF domain is the primary sensor domain in SgmTand that the C-terminal GGDEF domain binds the second messenger bis-(3’-5’)-dimeric cyclic-GMP (c-di-GMP) in vitro and functions as a c-di-GMP receptor in vivo to spatially sequester SgmT upon c-di-GMP binding.We suggest that SgmT activity is regulated by two sensor domains, theGAF domain and the GGDEF domain, and that binding of ligand to theGAF domain results in SgmT activation and binding of c-di-GMP to theGGDEF domain results in spatial sequestration of SgmT insulating theSgmT/DigR from cross-talk from other signalling systems.BIOspektrum | Tagungsband 2012