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

200RSP025The pH-responsive transcriptional activator CadC and itslysine-dependent co-sensor LysP in E. coli: New insights intoregulatory interplay and signal transductionS. Buchner*, M. Rauschmeier*, I. Haneburger, V. Schüppel, L. Tetsch, K. JungLudwig-Maximilians-Universität München, Department Biologie I,Microbiology, Martinsried, GermanyCadC belongs to the ToxR-like transcriptional activators. This type ofregulators is characterized by a N-terminal cytoplasmic DNA-bindingeffector domain and a C-terminal periplasmic sensor domain separated bya single transmembrane helix. ToxR-like proteins combine sensoryfunction and DNA-binding activities in a single polypeptide and lack sitesfor chemical modification. CadC regulates the expression of the cadBAoperon and induces transcription under conditions of low external pH andconcomitantly available lysine. This in turn allows E. coli to adapt toacidic stress. Recently, it was demonstrated that the periplasmic domain ofCadC is responsible for pH sensing [1,2]. However, almost nothing isknown about the mechanism of signal transduction across the membrane tothe cytoplasmic effector domain of CadC. Secondary structure analysisrevealed a large unstructured cytosolic loop of unknown function betweenthe transmembrane helix and the DNA-binding domain. To investigate therole of the loop in terms of signal transduction, it was gradually truncatedor elongated. Resulting CadC-variants activated cadBA expressionindependent of external pH, implicating that the cytoplasmic loop plays animportant role in transducing the signal to the DNA-binding domain. ThepH sensor CadC is not a direct lysine sensor, but senses exogenous lysinevia an interplay with the lysine transporter LysP. Random and directedmutagenesis was performed to find LysP/CadC interaction sites and toinvestigate the interconnectivity of the two functions, regulation andtransport, of the trigger transporter LysP. This approach led to theidentification of LysP variants, which evoke altered interaction patternswith CadC and/or defects in transport activity. Further, transmembraneinteractions between LysP and CadC were analyzed in vivo using abacterial two hybrid (BACTH) system. The BACTH-study providedevidence for a constitutive interaction between sensor and triggertransporter. It is proposed that lysine-induced conformational changes ofLysP upon lysine binding and/or transport transduce the external lysinesignal to CadC by weakening the persisting protein-protein interaction.1. Eichinger A., Haneburger I., Koller C., Jung. K and Skerra A., Protein Sci. 20 (2011), p. 656-6692. Haneburger I., Eichinger A., Skerra A. and Jung K., J. Biol. Chem. 286 (2011), p. 10681-1068RSP026Copper homeostasis in Corynebacterium glutamicumX. Schuplezow*, S. Schelder, M. Bott, M. BrockerForschungszentrum Jülich GmbH, IBG-1: Biotechnologie, Jülich, GermanyCopper ions can easily alternate between an oxidised (Cu 2+ ) and a reducedstate (Cu + ) and are therefore used as cofactor of enzymes involved inelectron transport or redox reactions, such as cytochrome c oxidases ormulticopper oxidases. As high concentrations of uncomplexed copper ionscan become toxic for the cell by generating reactive oxygen species or bysulfhydryl depletion, the intracellular concentration of copper must betightly regulated to prevent toxic levels on the one hand and copperstarvation on the other hand. In prokaryotes several copper resistancesystems have been identified and characterised, but only little is knownabout the response to copper starvation conditions.Here, we have investigated the influence of elevated copper concentrationsas well as of copper starvation conditions on Corynebacteriumglutamicum, a non-pathogenic Gram-positive soil bacterium, which servesas model organism for closely related pathogenic species such asMycobacterium tuberculosis. To gain first insights into copperhomeostasis, growth and global gene expression were followed in thepresence of different copper concentrations (0 - 500 M). In this way, thecopper excess and the copper starvation stimulon were determined. Theseand subsequent experiments revealed that the recognition of and responseto elevated extracellular copper concentrations is mediated by the twocomponentsystem CopRS [1] , whereas a one-component transcriptionalregulator is responsible for adaption to increased intracellular copperconcentrations [2] . Growth of the wild type is only hardly affected undercopper starvation, whereas a deletion strain lacking the copperindependentcytochrome bd oxidase (cydAB) of the branched respiratorychain of C. glutamicum [3] exhibits a strong growth defect. The cydABmutant is strictly dependent on the copper-dependent cytochrome aa 3oxidase, which presumably is at least partially dysfunctional under copperstarvation conditions. The phenotype of the cydAB mutant and theinduction of the cydAB genes under copper starvation show thatcytochrome bd oxidase plays an important role in electron transfer frommenaquinol to oxygen under these conditions.[1] Schelder et al., 2011[2] Schelder et al., to be submitted[3] Bott and Niebisch, 2003RSP027Generating a “Gene Knock-out through Allelic Exchange” inClostridium acetobutylicumS.K. Flitsch* 1 , A. Arndt 2 , S. Noack 3 , P. Dürre 11 Universität Ulm, Institut für Mikrobiologie und Biotechnologie, Ulm, Germany2 Bundeswehrkrankenhaus Ulm, Institut für Pathologie, Ulm, Germany3 Bundesanstalt für Materialforschung, Berlin, GermanyClostridium acetobutylicum is an anaerobic, Gram-positive soil bacteriumand possesses a characteristic biphasic fermentation metabolism. Growingexponentially, the sugar substrates are metabolised to acetate and butyrate,and during the late exponential and stationary phase these acids areconverted into the solvents acetone and butanol. The aim of our work isthe construction of artificially controlled genes required for solventproduction/regulation. So, within the “COSMIC2 SysMO-project” severalgenes in C. acetobutylicum are planned to be deleted by the new “knockoutsystem”.Aldehyde ferredoxin oxidoreductase (AOR) (CA_C2018) is an oxygensensitiveenzyme that catalyses the oxidation of aldehydes to theircorresponding acids.Previous transcriptome studies for microarray shift experiments showed anupregulation of several genes of solventogenic and acidogenic metabolism,among them the CA_C2018-gene, during shift from pH 6.5 to pH 5.5. Adeletion of this aor gene in C. acetobutylicum was constructed.The “allelic exchange system” based on a homologous recombination wasused to knock-out the aor gene [1]. First, a knock-out cassette wasgenerated and then ligated into the pseudo-suicide vector pMTL-SC7515.After methylation in Escherichia coli pANS1 the vector was transformedinto C. acetobutylicum. After two independent homologousrecombinations a double-crossover C. acetobutylicum-mutant wasestablished. In further experiments, a complementation of theaorgene willbe created. Also, an overexpression mutant for this gene of C.acetobutylicum will be generated.[1] J.T. Heap, O.J. Pennington, S.T. Cartman, N.P. Minton. 2009. A modular system forClostridium shuttle plasmids. Journal of Microbiological Methods 78: 79-85.RSP028Characterization of radical SAM enzymes involved in theheme biosynthesis pathway in Methanosarcina barkeriM. Kühne*, G. LayerTechnical University of Braunschweig, Institute for Microbiology,Braunschweig, GermanyThe cyclic tetrapyrrole heme is an important cofactor for almost all livingorganisms. The heme biosynthetic pathway is partly conserved among thethree kingdoms. Thus, in eukaryotes, bacteria and archaea 5-aminolevulinic acid serves as the first common precursor for hemebiosynthesis. The first cyclic tetrapyrrole of the pathway isuroporphyrinogen III which is converted into coproporphyrinogen III ineukaryotes and most bacteria to generate heme via three further steps. Incontrast, in archaea and some bacteria an alternative heme biosynthesispathway is operative in which uroporphyrinogen III is converted intoprecorrin-2 which is then further transformed into heme [1], [2]. Recently,it was shown that in sulfate-reducing bacteria such as Desulfovibriodesulfuricans the alternative route to heme proceeds via siroheme, 12,18-didecarboxysiroheme and iron-coproporphyrin III [3]. The enzymescatalyzing the respective transformations of the intermediates representhomologs to enzymes involved in heme d 1 biosynthesis and are also foundin heme producing archaea such as the methanogen Methanosarcinabarkeri.Our studies are focused on the two heme biosynthesis enzymes NirJ1 andNirJ2 from M. barkeri which catalyze the last steps of the alternative hemebiosynthesis pathway, namely the removal of two acetate side chains from12,18-didecarboxysiroheme to form iron-coproporphyrin III (NirJ2) andthe subsequent formation of vinyl-groups at tetrapyrrole positions C3 andC8 to synthesize heme (NirJ1). Both, NirJ1 and NirJ2, belong to theradical SAM enzyme family. They contain a CxxxCxxC amino acid motifwhich is typical for members of the radical SAM superfamily and thuscontain a [4Fe-4S] cluster. Additionally, both proteins exhibit a cysteinerichC-terminus which might harbour a second iron-sulfur cluster involvedin catalysis or substrate binding. Strikingly, HemN, the enzyme thatcatalyzes the vinyl-group formation during the classical heme biosynthesispathway in bacteria, is also a radical SAM enzyme but contains only asingle [4Fe-4S] cluster. Therefore, NirJ1 and HemN catalyze an identicalreaction but use different substrates and distinct mechanisms.[1] T. Ishida, L. Yu, H. Akutsu et al. (1998) A primitive pathway of porphyrin biosynthesis and enzymologyin Desulfovibrio vulgaris. Proc Natl Acad of Sci USA 95, 4853-4858.[2] B. Buchenau, J. Kahnt, I. U. Heinemann, D. Jahn, and R. K. Thauer (2006) Heme biosynthesis inMethanosarcina barkeri via a pathway involving two methylation reactions.J Bacteriol 188, 8666-8668.[3] S. Bali, A. D. Lawrence et al.(2011) Molecular hijacking of siroheme for the synthesis of heme and d1heme. Proc Natl Acad Sci USA Early Edition.BIOspektrum | Tagungsband 2012