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

CBP035Export the unexpected. A novel periplasmic targetingsignalA. Edwards 1 , A. Downie 1 , M. Krehenbrink* 21 John Innes Center, Norwich, United Kingdom2 Department of Biochemistry,University of Oxford, Oxford, United KingdomProteins destined for the periplasm are targeted to the Sec and TAT exportmachineries via hydrophobic N-terminal signal peptides, which are usuallycleaved after export. These signal peptides are readily recognisable, a factthat is exploited by algorithms for the prediction of the periplasmicproteome. Although the Fe/Mn superoxide dismutase (SodA) of Rhizobiumleguminosarum is exported to the periplasm, it does not carry a recognisedsignal peptide. Instead, the N-terminus of SodA is highly hydrophilic andbears no resemblance to classical signal peptides, and it remains uncleavedafter export in both R. leguminosarum and Escherichia coli. The export ofSodA is unaffected in tatC and secB mutants, but is diminished in atemperature-sensitive SecA mutant. We therefore propose that SodA export,although Sec-dependent, utilises a previously unknown targeting mechanismthat is distinct from classical periplasmic targeting. Sequence scanninganalysis revealed that a 10-amino acid sequence within SodA was sufficientto target a reporter protein to the periplasm, and mutational analysis of thissequence determined the conserved residues involved in efficientperiplasmic targeting.Our results demonstrate a novel SecB- and (classical) signal peptideindependentpathway for targeting proteins to the periplasm. The targetingmechanism may be widespread, as export of SodA to the periplasm was alsoobserved in other proteobacteria. The novel consensus motif is notrecognised by the current algorithms for predicting signal peptides, andproteins carrying it are missing from the predicted periplasmic proteomes.As proteins such as SodA play active roles in processes such aspathogenesis, these findings have wider implications for the study ofperiplasmic targeting and its role in virulence and bacterial physiology ingeneral.CBP036Metabolic changes in the murine macrophage-like tumorcell line J774A.1 after stimulation withLipopolysaccharide from E. coliP. Gierok* 1 , M. Liebeke 1,2 , M. Lalk 11 Institute of Pharmacy, Pharmaceutical Biology, Greifswald, Germany2 Biomolecular Medicine, Imperial College London, London, UnitedKingdomThe murine macrophage-like tumor cell line J774A.1 is used in numerousstudies like in vitro infections or macrophage-activation experiments. Sincethe metabolism of the macrophage plays a central role in these cellularprocesses we investigated the central metabolism by a comprehensivemetabolomic approach. Uptake and secretion of intermediates wheremonitored by extracellular metabolomics using 1 H-NMR. Investigations onthe intracellular metabolome level were performed by GC-MS and LC-MS.Since it is known that the metabolism of macrophages is affected by stimulilike pathogen-associated molecular patterns (PAMPS), we compared themetabolome data of non-stimulated cells with cells stimulated withlipopolysaccharide (LPS) from E. coli. In this study, we show that LPSaffects central metabolic pathways like glycolysis, glutaminolysis and theTCA-cycle.CBP037Structural Investigation and Mechanism of bifunctionalFructose-1,6-bisphosphate aldolase/phosphatase fromThermoproteus neutrophilusJ. Du*, W. Lü, R. Say, G. Fuchs, O. EinsleInstitute for Organic Chemistry and Biochemistry, Albert-Ludwigs-University, Freiburg, GermanyThe fructose 1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctionalenzyme with both aldolase and phosphatase activities. It is found in mostarchaeal groups and deeply branching bacterial lineages harbor thermophilicorganisms [1]. As an essential gluconeogenic enzyme, it catalysesirreversible aldol condensation of heat-labile dihydroxyaceton phosphate(DHAP) and glyceraldehyde phosphate (GAP) to FBP, and also catalysesthe hydrolysis of FBP to stabile Fructose 6-phosphate (F6P) and inorganicphosphate (Pi).In order to understand the mechanism of this bifunctional reaction, weinvestigated the structure of FBP aldolase/phosphatase in Thermoproteusneutrophilus by X-ray crystallography, activity tests, mass spectrometry andother biochemical methods. We solved the structures of this FBPaldolase/phosphatase (apo) and its complexes with DHAP, FBP and F6P atup to 1.3 Å resolution, the FBP Aldolase/Phosphatase of Sulfolobus tokodaiistructure was used as the initial search model [2]. These high resolutionstructures depict large conformational changes in distinct loops surroundingthe active center. Supported by mutational studies and mass spectrometry,these conformational changes suggest a distinct mechanism in aldolase andphosphatase reactions. These flexible loops act as a switch between aldolaseand phosphatase activities.[1] Say, R.F. and G. Fuchs (2010): Fructose 1,6-bisphosphate aldolase/phosphatase may be anancestral gluconeogenic enzyme, Nature, 464(7291): 1077-81.[2] Nishimasu, H. et al (2004):The first crystal structure of the novol class of fructose-1,6-bisphosphatase present in the thermophilic archaea , Structure, 12(6):949-59.CBP038Role of chemo- and aerotaxis in magnetotactic behaviourof Magnetospirillum gryphiswaldenseF. Popp*, D. SchülerBiocenter, Department I/Microbiology, Ludwig-Maximillian-UniversityMunich, Planegg-Martinsried, GermanyMagnetotactic bacteria (MTB) contain a chain of magnetic particles thatimparts a net magnetic moment to the cells. Passive alignment with theEarth’s magnetic field is believed to increase the efficiency of chemotacticbehaviour by reducing the complexity of a three-dimensional searchproblem in chemically stratified habitats. However, the precisecharacteristics of this behaviour as well as its interaction with chemotacticmechanisms have remained unknown.The swimming direction in various mostly uncultivated MTB has beenreported to be set by an internal magnetic field polarity that causes cells tomove in one direction with respect to the ambient magnetic field untilreaching conditions that trigger motion reversal. Although M.gryphiswaldense wild-type cells cultivated under standard conditions showno such bias, we found that magnetic swimming polarity can be selected byserial cultivation in strong magnetic fields, yielding distinct populations ofN- or S-seeking bacteria. In addition, preliminary experiments indicated thataerotaxis is the main chemotactic behaviour in M. gryphiswaldense.In order to determine molecular determinants of chemo- and aerotaxis, weperformed a genome-wide homology search in M. gryphiswaldense. Fourputative operons containing canonical chemotaxis genes cheAWYBR as wellas Methyl-accepting Chemotaxis Proteins (MCPs) and furtheruncharacterised genes were identified. Furthermore, we found that M.gryphiswaldense possesses an unusually high number of chemotaxis signaltransducers (≥ 50, compared to 5 in E. coli), a large fraction of which wasfound expressed by proteomic analysis. Amomg them, we identified threeputative aerotaxis transducers sharing homology with the E. coli proteinAer.The role that putative chemotaxis operons and aerotaxis transducers play inmagnetotaxis and magnetic swimming polarity is currently beinginvestigated by constructing single and multiple deletion mutants of allidentified chemotaxis operons and selected signal tranducers.CBP039Crystal structure of the colicin M immunity proteinC. Römer*, S. Patzer, R. Albrecht, V.B. Braun, K. ZethMax Planck Institute for Developmental Biology, Tübingen, GermanyColicins are bacterial protein toxins produced by half of E. coli naturalisolates that kill sensitive E. coli cells. Colicin M (Cma) inhibitsincorporation of murein precursors into murein. Cma producer cells areprotected by co-synthesis of an immunity protein, Cmi, that is located at theCma target site in the periplasm and anchored to the cytoplasmic membraneby an N-terminal hydrophobic sequence [1]. We resumed our previousstudies on Cma and Cmi after we had discovered that Cma activity requiresthe periplasmic FkpA prolyl cis-trans isomerase /chaperone [2].Since the hydrophobic sequence is not essential for Cmi activity [1],crystallization was performed with a soluble Cmi that lacked the N-terminus. Cmi crystals were obtained under several conditions but only onesingle crystal diffracted to a resolution of 1.95 Å. By using the recentlyspektrum | Tagungsband 2011