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

130forS. Typhimurium. Uncovering the function of the ORF 10 gene productand the identification of potential interaction partners would provideessential information to understand the relevance of the whole island in theemerging monophasic variant of Salmonella Typhimurium.U. Dobrindt, B. Hochhuth, U. Hentschel, J. Hacker, Nature Reviews2(2004), p. 414.S. Trüpschuch, J.A. Laverde Gomez, I. Ediberidze, A. Flieger, W. Rabsch, Int J MedMicrobiol300(2010), p. 279.MPP2-FGImportant codon positions and unusual anomalies in microbial16S RNA sequencesS. LawrenceUniversity of Cambridge and Sci-Tech(South), Earth Sciences andBiochemistry Research, Cambridge, United KingdomIn most microbial RNA seqeunces there are particular regions of thesequence that show a priority for important translations especially whenthe organism is producing specific substances for its own survivalmechanisms and for incorporation and use in both intercellular andintracellular activities. These codon sequences are needed for theproduction of polysaccharides which are used both inside and outside thecell wall so are both exopolysaccharides and intrapolysaccharides.However these particular codon sequences are not always as regular asexpected and have some unusual anomalies especially with the advent ofAAA and AAAA repetitions. These may not seem unusual at first but theirimportance becomes apparent with the gradual production of the intra andextracellular products. The two species that will be considered that providesuch unusual sequences are firstly xanthomonas, a plant pathogen andsecondly clostridia, a human pathogen. The important codon sequencesand anomalies for these species will be considered.MPP3-FGComplete fiber structure of the trimeric autotransporter adhesinSadAM.D. Hartmann, A.N. Lupas, B. Hernandez Alvarez*Max Planck Institute for Developmental Biology, Department of ProteinEvolution, Tuebingen, GermanyTrimeric autotranporter adhesins (TAAs) represent a group of nonfimbrial,non-pilus adhesins that are widespread in -, -, and g-proteobacteria. They include a number of prominent pathogenicity factorsincluding Yersinia YadA, Neisseria NadA and Bartonella BadA that areinvolved in pathogen adhesion as well as in the defence against hostresponses. TAAs are targeted by the type Vc secretion pathway throughthe outer membrane into the extracellular space. Their architecture followsa general head-stalk-anchor assembly from the N- to the C-terminus.TAAs are highly modular multidomain proteins with a variable number ofhead and stalk domains that are linked by several types of connectordomains. The highly conserved C-terminal membrane anchor harbours theautotransporter function and defines the protein family [1].In order to explore the domain diversity of trimeric autotransporteradhesins, we set out to produce a dictionary approach (daTAA, available athttp://toolkit.tuebingen.mpg.de/dataa) which allows the detailed andautomated annotation of TAAs from sequence data [2]. daTAA providesinformation on the sequence, structure and function of so far 25 differentdomain types as well as the rules by which these are combined to form theobserved long fibers on the cell surface.As complete TAA fibers are not amenable for X-ray crystallography, weturned to solve the structures of single domains in order to assemble theminto the full fiber in silico in a later step. The Salmonella adhesin SadAserved as a perfect model as it is a highly complex adhesin composed ofdifferent types of domains. Closest SadA homologues are found in almostall enterobacteria, such as UpaG, an adhesin involved in the infectionprocess of uropathogenic E. coli. Exploiting the observation that almost alldomain types of TAAs begin and end in coiled-coil segments, weproduced a pASK IBA - based expression vector system that fuses theextremely stable trimeric pII variant of the GCN4 leucine zipper in registerto the N- and C-terminal ends of the domain constructs [3, 4]. We solvedthe structure of all exemplars of domain types of SadA by molecularreplacement and assembled them together with homology models ofisolated domains into a complete structural model of the full SadA fiber.Our work successfully approved the applicability of the dictionaryapproach to understand the structural organization and to perform theannotation of this complex class of proteins.1. D. Linke, T. Riess, I.B. Autenrieth, A. Lupas and V.A. Kempf. Trends Microbiol.,14(2006), p. 264.2. P. Szczesny and A. Lupas.Bioinformatics.24(2008), p. 1251.3. B. Hernandez Alvarez, M.D. Hartmann, R. Albrecht, A.N. Lupas, K. Zeth and D. Linke. Protein Eng DesSel.21(2008), p. 11.4. M.D. Hartmann, O. Ridderbusch, K. Zeth, R. Albrecht, O. Testa, D.N. Woolfson, G. Sauer, S.Dunin-Horkawicz, A.N. Lupas and B. Hernandez Alvarez. Proc Natl Acad Sci U S A. 106 (2009), p.16950.OTV001The first structure of a LanI protein, SpaI: The proteinconferring autoimmunity against the lantibiotic subtilin inBacillus subtilis reveals a novel foldN.A. Christ* 1,2 , S. Bochmann 1 , D. Gottstein 2,3 , E. Duchardt-Ferner 1,2 ,U. Hellmich 1,2 , S. Düsterhus 1 , P. Kötter 1 , P. Güntert 2,3 , K.-D. Entian 1,4 ,J. Wöhnert 1,2,41 Goethe University Frankfurt, Institute for Molecular Bioscience, Frankfurt amMain, Germany2 Goethe University Frankfurt, Center of Biomolecular Magnetic Resonance,Frankfurt am Main, Germany3 Goethe University Frankfurt, Institute of Biophysical Chemistry, Frankfurt amMain, Germany4 Goethe University Frankfurt, Cluster of Excellence “MacromolecularComplexes", Frankfurt am Main, GermanyThe careless use of many antibiotics in the past lead to emergingresistances even against ‘last resort’ drugs such as vancomycin. Thus,there is an urgent need for structurally novel antimicrobial agents.Lantibiotics are small ribosomally synthesized peptide antibiotics withposttranslational modified amino acids resulting in the characteristiclanthionine and methyllanthionine bridges.Bacillus subtilis ATCC 6633 produces the lantibiotic subtilin whichdamages the cell wall of gram-positive bacteria. SpaI is a 16.8 kDalipoprotein which is part of the self-protection system of B. subtilis againstsubtilin. It is attached to the outside of the cytoplasmic membrane via acovalent diacylglycerol anchor. SpaI together with the ABC-transporterSpaFEG protects the membrane from subtilin insertion.We solved the structure of a 15 kDa biologically active fragment of SpaIby NMR which is the first structure of any LanI (lanthionine immunity)protein from lantibiotic producing strains. A search in the DALI databaseindicated a novel fold for SpaI. Our data show that SpaI has as mainly -strand structure with seven -strands and two -helices 1 . NMRinvestigations of a full length construct of SpaI lacking the diacylglycerolanchor suggest that the 30 N-terminal amino acids are unfolded in theabsence of a membrane. However, this N-terminal stretch showsinteractions with liposomes in NMR titration experiments. When mutatingthis stretch in vivo the SpaI mediated immunity of B. subtilis againstsubtilin is not affected and lipobox mutants of SpaI are still found in themembrane fraction.Our results are the first step on the way to understand subtilinautoimmunity of B. subtilis on a structural level at atomic resolution.1 Christ N.A., Duchardt-Ferner E., Düsterhus S., Kötter P., Entian K.D. and Wöhnert J.,Biomol.NMR Assign. in press.OTV002Analysis of SpaI-mediated lantibiotic immunity in Bacillus subtilisS. Bochmann* 1 , N. Christ 1,2 , P. Kötter 1 , S. Düsterhus 1 , J. Wöhnert 1,2,3 , K.-D. Entian 1,31 Goethe University Frankfurt, Institute of Molecular Biosciences, Frankfurt amMain, Germany2 Goethe University Frankfurt, Center of Biomolecular Magnetic Resonance,Frankfurt am Main, Germany3 Goethe University Frankfurt, Cluster of Excellence “MacromolecularComplexes, Frankfurt am Main, GermanyLantibiotics are lanthionine-containing peptides [1] that exhibitantimicrobial as well as pheromone-like autoinducing activity [2]. Bacillussubtilis ATCC 6633 produces the cationic pore-forming lantibioticsubtilin, which acts on Gram-positive microorganisms by interfering withthe lipid II cycle essential for peptidoglycan biosynthesis [3]. Selfprotection of the producer cells is mediated by the lipoprotein SpaI and theSpaFEG ABC-transporter [4]. SpaI as typical lipoprotein is anchored to theouter membrane via a diacylglycerol moiety.Different SpaI mutations were generated to elucidate the mechanism ofSpaI-mediated immunity. In contrast to other membrane boundlipoproteins, replacement of the cysteine within the lipobox-motif “LSAC”by alanine did not release the protein from the membrane. This resultindicates that the membrane interaction of the mature protein occurs alsoin the absence of lipid-modification. Based on structural elucidation, twodomains (domain 1 and domain 2) were identified, which are indispensablefor SpaI function. Surprisingly, if amino acid residues of domain 1 werenewly aligned, the mutated SpaI D1mix protein was still functional. Thecurrent data suggest that the overall charge of domain 1 is decisive for itsfunction, and not its primary sequence. Domain 2 is also indispensable forSpaI function and needs to be entirely conserved.Our current data suggest that the N-terminal domain of SpaI is importantfor membrane association in addition to the diacylglycerol anchor.1. Schnell, N., et al.,Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with foursulphide-rings. Nature, 1988. 333(6170): p. 276-8.2. Stein, T., et al.,Dual control of subtilin biosynthesis and immunity in Bacillus subtilis. Mol Microbiol,2002. 44(2): p. 403-16.3. Brotz, H., et al.,Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epiderminand other lantibiotics. Mol Microbiol, 1998. 30(2): p. 317-27.4. Klein, C. and K.D. Entian,Genes involved in self-protection against the lantibiotic subtilin produced byBacillus subtilis ATCC 6633. Appl Environ Microbiol, 1994. 60(8): p. 2793-801.BIOspektrum | Tagungsband 2012