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

elies on sensing temporal changes in concentrations of chemoeffectorswhile bacterial cell is swimming in the gradient. Dependent on the sensoryinput, bacteria regulate their swimming duration by changing the rotationaldirection of the flagellar motor, whereby an increase in positive (attractant)stimulus when swimming up the gradient increases run duration in thisdirection.The receptor signal is transduced to the flagellar motor by a receptorassociatedcytoplasmic histidine kinase CheA and a response regulatorCheY. In addition, the chemotaxis signalling pathway contains an adaptationsystem which adjusts the activity and sensitivity of the sensory complexesby receptor methylation on four specific glutamate residues. The reactions ofreceptor methylation and demethylation are mediated by two enzymes, themethyltransferase CheR and the methylesterase CheB, respectively. Theadaptation system is necessary to ensure ligand sensing over large dynamicconcentration range and therefore to enable cells to follow attractantgradients from very low to very high concentrations.Here we investigate the mechanisms that ensure broad dynamic range of thechemotaxis system, as well as physiological factors that limit this range. Byapplying a FRET-based reporter of the intracellular pathway activity, weshow how methylation on multiple sites extends dynamic range over manyorders of magnitude. We also observe that dynamic range becomes limitedby saturation of methylation sites, with different concentration limitsobserved for different chemoeffectors. Further experiments revealed acorrelation between dynamic range of the chemotaxis system and growthinhibition of cells by high concentrations of respective chemoeffectors,suggesting that the dynamic range of the chemotaxis system has beenevolutionary tuned to physiologically beneficial ligand concentrations.CBP026Isolation of a prokaryotic cell organelle from theuniquely compartmentalized anammox bacteriaS. Neumann*, M. S.M. Jetten and L. van NiftrikDepartment of Microbiology, Institute for Water & Wetland Research,Radboud University, Nijmegen, NetherlandsThe bacteria capable of anaerobically oxidizing ammonium (anammox) havebeen discovered only quite recently [1]. Since then their significance for theglobal nitrogen cycle has become apparent due to their large contribution tothe oceanic nitrogen loss [2] and they are already applied for the removal ofammonium from municipal wastewater. Like other members of the phylumPlanctomycetes, anammox bacteria exhibit a cell compartmentalization thatis otherwise unique for prokaryotes [3]. The cells are subdivided into threecompartments. The outermost compartment is the paryphoplasm and has anunknown function, but is presumably not analogous to the periplasmic spacein Gram-negative bacteria. It is separated by an intracytoplasmic membranefrom the riboplasm, which harbors the RNA as well as DNA of the cell. Theinnermost compartment is the anammoxosome and is hypothesized to be theside of catabolism and energy generation, analogous to eukaryoticmitochondria [4-5]. Isolation of this prokaryotic cell organelle from theanammox bacterium Kuenenia stuttgartiensis was attempted by variousphysical and chemical disruption techniques and led to separation of twosubcellular fractions by Percoll density centrifugation. These wereinvestigated with immunofluorescence microscopy and transmissionelectron microscopy for their outer appearance, DNA content andhybridization with an antibody targeting the anammoxosome. Future studieswill include organelle proteomics and activity assays.[1] Strous, M., et al (2000): Missing lithotroph identified as new planctomycete. Nature. 400(6743):p. 446-449.[2] Kuypers, M.M.M. et al (2003): Anaerobic ammonium oxidation by anammox bacteria in the BlackSea. Nature. 422(6932): p. 608-611.[3] Fuerst, J.A. (2005): Intracellular compartmentation in planctomycetes. Annual Review ofMicrobiology. 59: p. 299-328.[4] Lindsay, M.R. et al (2001): Cell compartmentalisation in planctomycetes: novel types of structuralorganisation for the bacterial cell. Archives of Microbiology. 175(6): p. 413-429.[5] van Niftrik, L. et al (2008): Linking ultrastructure and function in four genera of anaerobicammonium-oxidizing bacteria: Cell plan, glycogen storage, and localization of cytochrome c proteins.Journal of Bacteriology, 190(2): p. 708-717.CBP027Interaction of Lipid II-binding lantibiotics with the wallteichoic acid precursors Lipid III and Lipid IVA. Mueller*, H. Ulm, J. Esche, H.-G. Sahl, T. SchneiderInstitute of Medical Microbiology, Immunology and Parasitology,Friedrich-Westphalian Wilhelms-University, Bonn, GermanyLantibiotics are a unique group within the antimicrobial peptidescharacterized by the presence of thioether amino acids (lanthionine,methyllanthionines). These peptides are produced by and primarily act onGram-positive bacteria and exert multiple activities at the cytoplasmicmembrane of susceptible bacteria [1]. Recently the cell wall precursor lipidII was identified as a specific target for the prototype lantibiotic nisin. Nisinbinds to lipid II, thereby inhibiting cell wall biosynthesis [2].Besides its interaction with the peptidoglycan precursors lipid I and lipid II,we show that nisin also interacts with sugar lipids involved in the synthesisof wall teichoic acid, i.e. lipid III (C55-PP-GlcNAc) and lipid IV (C55-PP-GlcNAc-ManNAc). This specific interaction with wall teichoic acidprecursors further resulted in a target-mediated pore formation, as hasrecently been shown for lipid II [3].We also show that nisin forms a complex with the various C55P-boundprecursors at a stoichiometry of 2:1 (nisin: lipid). Studies with selectedlantibiotics of the nisin sub-group, all containing the conserved lipid II -binding motif, e.g. gallidermin also showed an interaction with Lipid III andLipid IV.[1] Héchard and Sahl, (2002): Mode of action of modified and unmodified bacteriocins from Grampositivebacteria. Biochimie 84:545-557.[2] Brötz et al (1998b): Role of lipid-bound peptidoglycan precursors in the formation of pores bynisin, epidermin and other lantibiotics. Mol. Microbiol. 30:317-327.[3] Wiedemann et al (2001): Specific binding of nisin to the peptidoglycan precursor lipid II combinespore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem.276:1772-1779.CBP028In vitro and in vivo site-directed mutational analysis ofDnaA in Bacillus subtilis - aspects of its functionality inthe initiation of replicationM. Eisemann*, I. Buza-Kiss, P.L. GraumannDepartment of Microbiology, Albert-Ludwigs-University, Freiburg,GermanyThe initiator protein of chromosomal replication, DnaA, and its regulationhave intensively been studied in Escherichia coli, a model organism ofGram negative bacteria. A variety of functional capacities, such as ATPbindingand hydrolysis, oligomerization and specific DNA binding, havebeen discovered and led to a model of the underlying mechanism. Becausethe process of initiation of chromosomal replication seems to workdifferently in Gram positive bacteria, we investigated these capacities andtheir implication in initiation in Bacillus subtilis. We created several B.subtilis DnaA mutants by exchange of highly conserved amino acids thathave previously been reported for E. coli to be involved in the activitiesmentioned above. Comparative fluorescence microscopy studies of wildtypeand mutant DnaA revealed strong phenotypic effects in the frequency ofinitiation of replication, on DNA compaction, chromosomal segregation,septum formation and cell length, which are different from those phenotypesobserved in E. coli. Surface Plasmon Resonance experiments display aspecific binding affinity and binding stability to DnaA-box containing DNAfor each of the mutant DnaA forms, which correspond to the observedphenotypes in vivo. Taken together, our results suggest a novel model forhow DnaA initiates chromosomal replication in Bacillus subtilis.Funding: DFG (Deutsche Forschungsgemeinschaft)CBP029RodA influences the sites of incorporation of new cellwall material in Bacillus subtilis and colocalizes withMreB and MblC. Reimold*, M. Duong, H.J. Defeu Soufo, P.L. GraumannFaculty of Biology II/Microbiology, Albert-Ludwigs-University, Freiburg,GermanyRodA is a widely conserved bacterial protein implicated in the maintenanceof rod cell shape. We show that a functional GFP-RodA fusion largelycolocalizes with the MreB cytoskeleton at the lateral cell membrane inspektrum | Tagungsband 2011