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

OTP033The acyltransferases KirCI and KirCII involved inSupramolecular Templating of kirromycin biosynthesisE.M. Musiol*, T. Härtner, A. Kulik, W. Wohlleben, T. WeberInstitute of Microbiology and Infection Medicine (IMIT), Biotechnology,Eberhard-Karls-University, Tübingen, GermanyStreptomyces collinus Tü 365 is the producer of kirromycin, which is apolyketide antibiotic. Kirromycin binds to the elongation factor Tu andinhibits the bacterial protein biosynthesis. The skeleton of this antibiotic issynthesized by a large complex of type I polyketide synthases and nonribosomalpeptide synthetases (PKS I/NRPS complex), encoded by thegenes kirAI-kirAVI and kirB.KirAI-KirAV, except the NRPS, KirAIII, belong to PKSs type I with „trans-AT”-architecture. These megaenzymes possess no acyltransferase domainsintegrated in the PKS modules. In contrast KirAVI is of the classical „cis-AT”-type PKS, of which the ATs are part of the PKS protein.Two separate genes, kirCI and kirCII with similarity to acyltransferaseswere identified in the kirromycin gene cluster. To characterize the role ofkirCI and kirCII in kirromycin biosynthesis, mutants in these genes wereconstructed and analyzed for kirromycin production. The inactivation ofkirCI (ΔkirCI) resulted in a significant reduction of kirromycin production.In ΔkirCII kirromycin synthesis was completely abolished. Both mutantswere successfully complemented with the wild type genes. Thecomplemented strains produced the antibiotic at levels comparable with thewild type. This data indicate that both genes are involved in kirromycinbiosynthesis and the gene kirCII is essential for the production of thisantibiotic.For kirromycin assembly, a selective loading of ACPs with the buildingblocks malonyl-CoA and ethylmalonyl-CoA is required. This function ispresumably carried out by KirCI and KirCII, respectively. To confirm thishypothesis and to determine the specificity of KirCI and KirCII an in vitroACP loading assay was developed.Therefore KirCI, KirCII and two selected ACPs were expressed in E. coliand purified. The proteins were used in the in vitro assay and the loading ofmalonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA to the ACPs wasmonitored by autoradiography and HPLC-ESI-MS. The experiments showedthat KirCI loads specifically malonyl-CoA onto ACP4 and the secondenzyme, KirCII is the first, biochemically characterized „trans-AT” withhigh specificity for ethylmalonyl-CoA and transfers this substrate to ACP5.Thus, the specific recognition mechanism of the ACP of module 4 and 5 bythe ATs, KirCI and KirCII, respectively is at least in part determined by theACP. To our knowledge, such interaction mechanism, where free-standingproteins that provide building blocks, dock site-specific to the „recipient”-protein to achieve structural diversity in polyketides was not characterizeduntil now.[1] Wolf, H. and H. Zähner (1972): Arch. Mikrobiol., 83, 147-15[2] Weber, T. et al (2008): Chem. Biol.,15, 175 - 188.OTP034Analysis of the chlamydial amidase AmiAA. Klöckner*, A. Gaballah, H.-G. Sahl, B. HenrichfreiseInstitute of Medical Microbiology, Immunology and Parasitology,Pharmaceutical Microbiology, University of Bonn, Bonn, GermanyFor intracellular bacteria there is no need to maintain osmotic stabilizationby means of a cell wall and peptidoglycan has not been reliably detected inthe obligate intracellular genus Chlamydia so far. Nevertheless, chlamydiaeare susceptible to antibiotics that target cell wall biosynthesis, a paradoxknown as the chlamydial anomaly. A genome-wide search withinchlamydiae has identified a nearly complete pathway for peptidoglycanbiosynthesis.Recently, we demonstrated in vitro activity of the chlamydial enzymesMraY and MurG that catalize the last two steps of the biosynthesis of themembrane bound cell wall block lipid II. We discussed the hypothesis thatmaintaining lipid II biosynthesis in cell wall lacking bacteria reflects anessential role of this precursor in procaryotic cell division. [Henrichfreise,Schiefer et al. 2009. Mol Microbiol. 73: 913-23.].Here, we investigate the fate of lipid II in Chlamydia. To check whether thepeptide chain of lipid II is released from its carrier (C55-P) by amidaseaction, as previously suggested [Ghuysen and Goffin. 1999. Antimicrob.Agents Chemother. 43:2339-2344.], amidase AmiA from Chlamydiapneumoniae was overproduced in Escherichia coli and purified. ChlamydialAmiA exhibited in vitro activity in dye release assays using RemazolBrilliant Blue R-dyed peptidoglycan as substrate.Our findings strengthen the hypothesis that in chlamydiae lipid II issynthesized and further processed, including recycling of the carrier C55-P.A deeper insight into the peptidoglycan biosynthesis machinery and thechlamydial anomaly on molecular level will provide a basis for the design ofnovel anti-infective strategies against Chlamydia.OTP035Completing the Inventory: Systematic Deletion Analysisof Secondary Zinc Uptake Systems in Cupriavidusmetallidurans CH34 to Understand Multiple MetalHandlingA. Kirsten* 1 , M. Herzberg 1 , J. Seravalli 2 , G. Grass 2 , D.H. Nies 11 Division of Molecular Microbiology, Martin-Luther-University Halle-Wittenberg, Halle, Germany2 University of Nebraska, Lincoln, USACupriavidus metallidurans is adapted to high concentrations of transitionmetal cations. The bacterium harbors a variety of metal efflux systems,which are the basis of its metal resistance. It is able to maintain cellularmetal homeostasis even at high concentrations of many heavy metals inparallel. C. metallidurans contains four CorA paralogs of the metalinorganic transport (MIT) protein family of magnesium transport systems,ZupT of the ZRT/IRT protein family ZIP of zinc/iron transporters and PitA,which imports metal phosphate complexes. Multiple deletion muntants wereconstructed to characterize the contribution of each system to transitionmetal import. All of these transporters were regulated by zinc availability.While expression of zupT was induced up-regulated under conditions of zincstarvation, that of the other genes was down-regulated at high zincconcentrations. Only corA 1 expression was influenced by the magnesiumconcentration. This identified ZupT as the main zinc uptake system underconditions of low zinc availability, PitA as cation-phosphate uptake system,CorA 1 as main secondary magnesium uptake system, CorA 2 and CorA 3 asback-up systems for metal cation importOTP036Structure and function of PilQ, a unique secretin of theDNA transporter from the thermophilic bacteriumThermus thermophilus HB27J. Burkhardt 1 , B. Averhoff* 1 , J. Vonck 21 Institute für Molecular Bio Science, Molecular Microbiology andBioenergetics, Goethe-Univesity, Frankfurt, Germany2 Department of Structural Biology, Max Planck Institute of Biophysics,Frankfurt, GermanyThermus thermophilus HB27 is known for its extremely high competencefor natural transformation and its ability to take up DNA from members ofthe archaea, bacteria and eukarya. A genome-wide genetic screen followedby mutant studies led to the identification of 16 distinct proteins [1]. One ofthe competence proteins, the secretin-like protein PilQ, was found to beessential for DNA binding and uptake in HB27 [2]. Here we report theisolation, structural and functional analyses of a unique PilQ from T.thermophilus. Native PAGE, gel filtration chromatography andelectrophoretic mobility shift analyses indicated that PilQ forms amacromolecular homopolymeric complex that binds dsDNA. Electronmicroscopy showed that the PilQ complex is 15 nm wide and 34 nm longand consists of an extraordinary stable „cone” and „cup” structure and fivering structures with a large central channel. Moreover, the electronmicroscopic images together with secondary structure analyses combinedwith structural data of T2SS and T3SS secretins suggest that the individualrings are formed by conserved domains of alternating α-helices and β-sheets.The unprecedented length of the PilQ complex correlated well with thedistance between inner and outer membrane of T. thermophilus. Indeed,PilQ was found immunologically in both membranes indicating that the PilQcomplex spans the entire cell periphery of T. thermophilus. This is consistentwith the hypothesis that PilQ accommodates a PilA4 comprisingpseudopilus mediating DNA transport across OM and periplasmic space in asingle step process [3].[1] Averhoff, B. (2009): FEMS Microbiol. Rev. 33:611-626.[2] Schwarzenlander, C. et al (2009): Environ. Microbiol. 11:801-808.[3] Burkhardt, J. et al (2010): J. Biol. Chem., submitted.spektrum | Tagungsband 2011