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

More recently, we have applied these methods to cytosolic fraction of 100Sribosomes, a dimerized form of 70S ribosomes associated with starvation inE. coli [3]. It was possible to purify in silico a particular ribosomalarrangement of the two 70S ribosomes that form a dimer. In contrast to thelateral contact of the 30S subunits observed in dense polysomes, theresolved 100S arrangement show that the contact of small subunits is frontaland implies a possible participation of the S9, S10 and S2 proteins as well as16S rRNA. Moreover, this 100S ribosomal arrangement has been detected intomograms of intact E. coli cells specifically in stationary phase, whichreinforce the physiological role of 100S ribosomes as a storage form ofribosomes important for cell survival.[1] Ortiz, J. O. et al. (2006): Struct Biol. 156:334.[2] Brandt, F. et al (2009): Cell 136:261.[3] Ortiz, J.O. et al. (2010): J. Cell Biol. 190:613.ISV27No abstract submitted!ISV28Protein and RNA Dynamics in Living CellsZ. Luthy-SchultenDepartment of Chemistry, University of Illinois, Urbana, USASignaling pathways in RNA:protein complexes involved in translation areidentified by community network analysis derived from moleculardynamics simulations. These complexes include the amino-acyl-tRNAsynthetases, the elongation factor EF-Tu, and the ribosome. A dynamiccontact map defines the edges connecting nodes (amino acids andnucleotides) in the physical network whose overall topology is presented asa network of communities, local substructures that are highlyintraconnected, but loosely interconnected. While nodes within a singlecommunity can communicate through many alternate pathways, thecommunication between monomers in different communities has to takeplace through a smaller number of critical edges or interactions which areevolutionarily conserved. The time dependent variation of these networksduring tRNA migration is consistent with kinetic data and reactionmechanisms suggested at each step of translation.In bacterial cells, translation involves thousands of these RNA:proteincomplexes which occupy a large portion of the cell volume and make amajor contribution to the extrinsic noise of gene expression. Using data fromproteomics, cryo-electron tomography, and in vivo single moleculefluorescence experiments, we study the inducible lac genetic switch in amodeled E. coli cell. Compared to models in which the spatial heterogeneityis ignored, the in vivo model for fast-growing cells predicts an overalllowering of cellular noise, due to the influence of molecular crowding onrepressor binding rates. The smaller slow-growing cells have a largerinternal inducer concentration which lead to a significant decrease in thelifetime of the repressor-operator complex, an increase in the mean numberof transcriptional bursts, and mRNA localization. The long time simulationsof biochemical pathways under in vivo cellular conditions, were calculatedwith a lattice-based, reaction-diffusion model that runs on graphicsprocessing units.[1] Chen, K. et al (2010): Biophys. J. 99, 3930-3940.[2] Trabuco, L. et al (2010): J. Mol. Biol. 402, 741-760.[3] Alexander, R. et al (2010): FEBS Lett. 584, 376-386.[4] Sethi, A. et al (2009): PNAS 106, 6620-6625.[5] Roberts, E. et al (2009): Proc. 8th IEEE Intl. Meeting on High Performance Comp. Biol.ISV29Biosynthesis and remodeling of bacterial membranelipidsO. Geiger*, C. Sohlenkamp, I.M. López-LaraCenter for Genomic Sciences, National Autonomous University of Mexico,Cuernavaca, MexicoThe model bacterium Escherichia coli contains the phospholipidsphosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE) asmajor membrane lipids and biosyntheses and functionalities of individualmembrane lipids have mainly been studied in this organism. However, inother bacteria, additional and alternative membrane lipids are found and inmany cases neither their biosyntheses nor their functionalities areunderstood. Some Gram-negative bacteria have phosphatidylcholine (PC) orsphingolipids in their standard repertoire, whereas many Gram-positiveshave glycosylated diacylglycerols and lysyl-phosphatidylglycerol in theirmembranes. Notably, phosphatidylinositol is an essential lipid forMycobacterium tuberculosis. Steroid and hopanoid lipids only occur insome bacteria.Bacterial membrane lipid composition should not be considered as aninvariable constant, but rather as the result of a steady-state, characteristicfor a given physiological condition. Under certain stress conditions, specificnew membrane lipids can be formed in order to minimize the stress exerted.For example, challenge of proteobacteria with acid causes modifications ofpre-existing membrane lipids, resulting in the formation of lysylphosphatidylglycerolor hydroxylations of ornithine-containing lipids. Underphosphorus-limiting conditions of growth, some bacteria form membranelipids lacking phosphorus such as ornithine-containing lipids, or thediacylglycerol (DAG)-based glycolipids, sulfolipids, and betaine lipids.In Sinorhizobium meliloti, a Gram-negative soil bacteria able to establishnitrogen-fixing root nodules with their respective legume host plants, thezwitterionic phospholipids PE and PC of its membrane are degraded uponphosphorus limitation by a specific phospholipase C to the respectivephosphoalcohol and DAG [1]. DAG in turn is the lipid anchor from whichbiosyntheses are initiated during the formation of phosphorus-free, DAGbasedmembrane lipids. Inorganic phosphate (Pi) can be liberated from thephosphoalcohol. Obviously, in S. meliloti under phosphate-limitingconditions, membrane phospholipids provide a pool for metabolizable Pi,which in turn can be used for the synthesis of other essential phosphoruscontainingbiomolecules.[1] Zavaleta-Pastor et al (2010): Proc. Natl. Acad. Sci. USA 107:302-307.ISV30Regulation of membrane homeostasis in PseudomonasaeruginosaY.-M. ZhangBiochemistry and Molecular Biology, Medical University of South Carolina,Charleston, USAMembrane lipid biogenesis is a vital facet of bacterial physiology that istightly regulated at both biochemical and genetic level. Bacterial survivaldepends on membrane lipid homeostasis and on the ability to adjust lipidcomposition to acclimatize the bacterial cell to optimize growth in diverseenvironments. The most energetically expensive membrane lipidcomponents to produce are the fatty acids, which determine the viscosity ofthe membrane and, in turn, influence many crucial membrane-associatedfunctions. Thus, bacteria have evolved sophisticated mechanisms to finelycontrol the expression of the genes responsible for the metabolism of fattyacids. These regulatory mechanisms adjust the level and activity ofbiosynthetic enzymes to match the demand for new membrane. The versatilehuman pathogen Pseudomonas aeruginosa contains both saturated fattyacids (SFAs) and monounsaturated fatty acids (UFAs) in the membrane. InP. aeruginosa, the predominant UFA synthesis is carried out by the FabA-FabB pathway of the type II fatty acid synthase. The two key componentsfor UFA production FabA and FabB are co-transcribed in a fabAB operon.Two oxygen-dependent desaturases, DesA and DesB, supplement the FabA-FabB pathway for UFA synthesis in P. aeruginosa, which is the firstbacterium identified that has more than one pathway for UFA synthesis.These three complementary pathways for UFA formation allow theubiquitous P. aeruginosa to survive in various environments. The FabA-FabB pathway is active under all growth conditions and produces themajority of the UFAs. Because DesA introduces double bonds into existingfatty acyl chain of phospholipids, it allows the bacterium to quickly modifythe membrane properties to adapt to abrupt changes in growth conditions.DesB allows P. aeruginosa to modify the composition of exogenous fattyacids being transported into the cell. The FabA-FabB and DesB pathwaysfor UFA synthesis are coordinately regulated by a TetR-familytranscriptional factor DesT, which senses the composition of cellular acyl-CoA pool to fine tune the expression of the pathway enzymes. Saturatedacyl-CoAs stabilize a conformation that cannot bind DNA, whileunsaturated acyl-CoAs stabilize a conformation that binds DNA. Recentlywe found that the content of cis-vaccenate in the membrane plays a key rolein the pathogenicity of P. aeruginosa. Reduced level of cis-vaccenate leadsto decreased fluidity of the membrane and defects in the secretion of variousextracellular virulence factors, biofilm formation, and motility. Therefore,membrane homeostasis is essential for both survival and virulence of P.aeruginosa, and may provide new strategies for the development of anti-Pseudomonas treatments.spektrum | Tagungsband 2011