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

65CEV016Elucidation of the N-glycosylation pathway in thethermoacidophilic archaeon Sulfolobus acidocaldariusB. Meyer* 1 , B. Zolghadr 2 , E. Peyfoon 3 , M. Pabst 2 , M. Panico 3 ,H.R. Morris 3 , P. Messner 2 , C. Schäffer 2 , A. Dell 3 , S.-V. Albers 11 Max-Planck-Institut für terrestrische Mikrobiologie, Molecular Biology ofArchaea, Marburg, Germany2 Universität für Bodenkultur Wien, Department of NanoBiotechnology, Vienna,Austria3 Imperial College London, Division of Molecular Biosciences, London,United KingdomGlycosylation is the most dominant form of post translation proteinmodification. It is proposed that more than 2/3 of the eukaryotic proteinsare modified by the attachment of sugar molecules. Due to the commonoccurrence of glycosylation in eukaryotic proteins, it was long believedthat glycosylation is a restricted to this domain of life, however, when in1976 Mescher and Strominger purified the S-Layer protein fromHalobacterium salinarium, which contained glycans covalently linked toasparagine residues, questions evoked how N-glycosylation occurs inBacteria and Archaea.So far the N-glycosylation process in crenarchaeotais still uncovered. Here, we will report the first results elucidating the N-glycosylation pathway in the thermoacidophilic archaeon Sulfolobusacidocaldarius. Deletion studies of selected genes coding forglycosyltransferases mediating the transfer of activated sugar precursors toa lipid carrier and the key enzyme of the glycosylation theoligosaccharyltransferase, showed the essential properties of the N-glycosylation process in Sulfolobus. Furthermore S. acidocaldariusexhibited a unique composition and branched structure of the N-linkedoligosaccharide, which is linked by a chitobiose core to the S-Layerprotein, known to be present in the N-glycans of Eukarya and so far notfound in other Archaea.CEP001Interaction between histidine kinase and ABC-transporter:new regulatory pathway in antimicrobial peptide resistancemodules of Bacillus subtilisS. Dintner*, S. GebhardLMU Mikrobiologie, Department I, Martinsried, GermanyThe genome of Bacillus subtilis contains three loci (bceRSAB, psdRSAB,yxdJKLM), which are very similar in gene organization and in sequence,are involved in resistance to various peptide antibiotics. The encodedmodules are comprised of a two-component regulatory system (TCS) andan ATP-binding-cassette (ABC) transporter. Both the permease and sensorkinase components show unusual domain architecture: the permeasescontain ten transmembrane helices with a large extracellular loop betweenhelices 7 and 8, while the sensor kinases lack any obvious input domain.Strikingly, in the Bce and Psd modules the ABC-transporter and TCS havean absolute and mutual requirement for each other in both sensing of andresistance to their respective antimicrobial compounds, suggesting a novelmode of signal transduction in which the transporter constitutes the actualsensor. Database searches revealed the wide-spread occurrence of suchmodules among Firmicutes bacteria, and parallel phylogenetic analysisshowed that transporters and TCSs have co-evolved. Based on thesefindings, we hypothesize the formation of a sensory complex between bothcomponents, likely involving direct protein-protein interactions betweenthe transport permease and histidine kinase. This is supported by initialresults from bacterial two-hybrid assays. To further validate ourhypothesis, both the transporter (BceAB) and the histidine kinase (BceS)were expressed heterologously in E. coli cytoplasmic membranes andcould be purified to high yields. Physical interaction between both proteincomponents will be tested by subsequent in vitro interaction and copurificationstudies, combined with in vivo cross-linking experiments.Taken together, our results show that Bce-type ABC-transporters andTCSs have co-evolved to form self-sufficient detoxification modulesagainst antimicrobial peptides, and suggest a novel signaling mechanisminvolving formation of a sensory complex between transport permease andsensor kinase.CEP002Mapping functional domains of colicin M, a protein toxin fromE. coliS. Helbig* 1 , S. Patzer 1 , K. Zeth 1 , C. Schiene-Fischer 2 , V. Braun 11 Max Planck Institute for Developmental Biology, Department of ProteinEvolution, Tübingen, Germany2 Max Planck Research Unit of Enzymology of Protein Folding, Halle, GermanyColicin M (Cma), a protein toxin from E. coli, is a novel phosphataseconcerning sequence, structure and substrate specificity. It is is importedinto the periplasm of sensitive cells via a receptor-dependent energycoupledprocess. E. coli and closely related strains are killey by inhibitionof murein biosynthesis; Cma cleaves the phosphate ester bond between thelipid carrier and the murein precursor. This mode of action is unique forCma. With 271 amino acid residues, it is the smallest of all knowncolicins. Its fold is unique among colicins and even among all knownproteins. The protein forms a compact structure, which makes it difficult todelineate the functional domains which are well-separated in most othercolicins [1].To study these functional domains of Cma, mutants in the variouspredicted domains were isolated and characterized with special emphasison the activity domain. The active site is located in a surface-exposedregion. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. Thisresidue is exposed at the Cma surface and is surrounded by Asp225,Tyr228, Asp229, His235 and Arg236; replacement of each residue withalanine inactivated Cma. We propose that Asp226 directly participates inphosphate ester hydrolysis and that the surrounding residues contribute tothe active site. All these residues are strongly conserved in Cma-likeproteins of other species.Moreover, we found that the hydrophobic helix 1, that extends from thecompact Cma structure, binds the toxin to the FhuA receptor in the outermembrane and is thereby involved in its uptake [3].Killing of cells by Cma strictly depends on the periplasmic peptidyl prolylcis/trans isomerase/chaperone FkpA [4]. Because of its compact structurethe colicin must unfold during translocation across the outer membraneund refold in the periplasm to be toxic. This is supported by FkpA thatpresumably assists in refolding by cis/trans isomerisation of one or a fewprolyl bonds.To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15proline residues individually with alanine and found four mutants withreduced activities. P107A displayes 10%, P129A, P176A and P260A show1% activity. Three of them were not imported, the remaining P176Amutant is structural identical to wild-type Cma which makes it unlikelythat the mutation changes the phosphatase active site that is located farfrom this proline residue. In an in vitro peptide assay FkpA isomerized theCma prolyl bond Phe175-Pro176 at a high rate. These results suppose thatthis bond is most likely targeted by FkpA in the activation of Cma in theperiplasm [4].[1] Zeth et al. (2008) Crystal structure of colicin M, a novel phosphatase specifically imported byEscherichia coli. J Biol Chem. 283(37):25324-31[2] Hullmann et al. (2008) Periplasmic chaperone FkpA is essential for imported colicin M toxicity.Mol Microbiol 69 (4):926-37[3] Helbig and Braun (2011) Mapping functional domains of colicin M. J Bacteriol. 193(4):815-21[4] Helbig et al. (2011) Activation of colicin M by the FkpA prolyl cis-trans isomerase/chaperone.J Biol Chem. 286(8):6280-90CEP003Oligomeric structure of the energy transducing ExbB-ExbD-TonB complexA. Pramanik*, V. BraunMax Planck Institute for Developmental BIology, Protein Evolution, Tübingen,GermanyIn Escherichia coli and other Gram-negative bacteria energy coupled outermembrane transporters allow the entry of scarce substrates, toxic proteins,and bacterial viruses (phages) into the cells. The required energy is derivedfrom the proton-motive force, which is transduced by the ExbB-ExbD-TonB protein complex from the cytoplasmic membrane. Little is knownabout the structure and stoichiometry of this complex, which is required toelucidate the mechanisms of energy harvesting at the cytoplasmicmembrane and concomitant energy transfer to the outer membranetransporters. We found that C-terminally His6 tagged ExbB and and StrepTagged ExbD are as functional as wild type. We solubilized an ExbBoligomer and an ExbB-ExbD subcomplex from the cytoplasmic membranewith the help of the detergents decyl and undecyl maltoside. We havepurified tagged ExbB oligomer and ExbB-ExbD complex by affinitychromatograph followed by size exclusion chromatography. We havecharacterized the protein complex in solution by Blue Native PAGE, sizeexclusion chromatography and small angle X-ray scattering (SAXS). Allthe methods indicated that there are 4-6 ExbB monomers in the complex.To understand the definite stoichiometry of the complexes we used laserinducedliquid bead ion desorption mass spectrometry (LILBID-MS). Atmoderate desorption laser energies we determined the oligomeric structureof ExbB to be mainly hexameric (ExbB 6), with minor amounts of trimers(ExbB 3), dimers (ExbB 2), and monomers (ExbB 1). Under the sameconditions ExbB-ExbD formed a complex consisting of ExbB 6ExbD 1, witha minor amount of ExbB 5ExbD 1. At higher desorption laser intensities,ExbB 1 and ExbD 1 and traces of ExbB 3ExbD 1, ExbB 2ExbD 1, ExbB 1ExbD 1,ExbB 3, and ExbB 2 were observed. Since the ExbB 6 complex and theExbB 6ExbD 1 complex remained stable during solubilization andsubsequent chromatographic purification on nickel-nitrilotriacetateagarose, Strep-Tactin, and Superdex 200, and during native blue gelelectrophoresis, we conclud that ExbB 6 and ExbB 6ExbD 1 aresubcomplexes on which the final complex including TonB is assembled.1. Pramanik, A., et al., Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichiacoli as revealed by LILBID mass spectrometry. Biochemistry, 2011.50(41): p. 8950-6.2. Pramanik, A., et al., ExbB protein in the cytoplasmic membrane of Escherichia coli forms astable oligomer. Biochemistry, 2010.49(40): p. 8721-8.BIOspektrum | Tagungsband 2012