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Posters A.Pdf INVESTIGATING THE COUPLING MECHANISM IN THE E. COLI MULTIDRUG TRANSPORTER, MdfA, BY FLUORESCENCE SPECTROSCOPY N. Fluman, D. Cohen-Karni, E. Bibi Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel In bacteria, multidrug transporters couple the energetically favored import of protons to export of chemically-dissimilar drugs (substrates) from the cell. By this function, they render bacteria resistant against multiple drugs. In this work, fluorescence spectroscopy of purified protein is used to unravel the mechanism of coupling between protons and substrates in MdfA, an E. coli multidrug transporter. Intrinsic fluorescence of MdfA revealed that binding of an MdfA substrate, tetraphenylphosphonium (TPP), induced a conformational change in this transporter. The measured affinity of MdfA-TPP was increased in basic pH, raising a possibility that TPP might bind tighter to the deprotonated state of MdfA. Similar increases in affinity of TPP also occurred (1) in the presence of the substrate chloramphenicol, or (2) when MdfA is covalently labeled by the fluorophore monobromobimane at a putative chloramphenicol interacting site. We favor a mechanism by which basic pH, chloramphenicol binding, or labeling with monobromobimane, all induce a conformational change in MdfA, which results in deprotonation of the transporter and increase in the affinity of TPP. PHENOTYPE CHARACTERIZATION OF AZOSPIRILLUM BRASILENSE Sp7 ABC TRANSPORTER (wzm) MUTANT A. Lerner1,2, S. Burdman1, Y. Okon1,2 1Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel, 2The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot, Israel Azospirillum, a free-living nitrogen fixer, belongs to the plant growth promoting rhizobacteria (PGPR), living in close association with plant roots. These bacteria are able to exert beneficial effects on plant growth and yield of many crops of agronomic importance under a variety of environmental and soil conditions. These positive effects are mainly as a result of morphological and physiological changes of the inoculated plant roots, with enhanced water and mineral uptake. Plant growth promoting substances produced by the bacteria seems to be at least partially responsible for these effects. Azospirillum cells are surrounded by a thick, dense, and tightly cell-bound layer of capsular polysaccharide (CPS) and by an outer lighter exopolysaccharide (EPS) layer bound to the cell. The EPS is involved in the attachment of Azospirillum to the plant root. Several genes involved in the Azospirillum brasilense-plant root interaction are carried on a 90 MDa plasmid called p90. p90 carries also genes involved in motility, adsorption to roots, colony morphology and genes belonging to the glycosyl- or mannosyl transferase, sugar dehydratase families and genes involved in the ABC transporter-dependent pathway (wzm and wzt). These ATP binding cassette (ABC) superfamily transporters (or traffic ATPases) are frequently involved in the translocation of complex carbohydrates across the cytoplasmic membrane. An A. brasilense wzm mutant was generated and its phenotype in comparison with the wild type strain Sp7 was evaluated. The wzm mutant was more resistance to heat, osmotic shock, osmotic pressure, desiccation and starvation but was more sensitive to elevated levels on NaCl, UV radiation and hydrogen peroxide. Differences in sensitivity to antibiotics and growth on different carbon sources were observed between the two strains. The wzm mutants also exhibited changes in cell morphology and motility. IDENTIFICATION AND CHARACTERIZATION OF THE CARBOHYDRATE ABC TRANSPORTERS IN CLOSTRIDIUM THERMOCELLUM Y. Nataf1, S. Shulami1, S. Yaron1, F. Stahl2, R. Lamed5, E.A. Bayer4, T.H. Scheper2, A.L. Sonenshien3, Y. Shoham1 1Department of Biotechnology and Food Engineering, Technion–Israel Institute of Technology, Haifa, Israel, 2Institute of Technical Chemistry, University of Hannover, Germany, 3Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA, 4Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel, 5Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel Clostridium thermocellum is an anaerobe thermophilic bacterium which efficiently degrades crystalline cellulose to soluble cellodextrins and is capable of producing ethanol. This bacterium produces a unique enzymatic complex, termed cellulosome, which integrates many glycoside hydrolases and mediates the attachment of the complex to cellulose. C. thermocellum is capable of assimilating cellodextrins which undergo phosphorylitic cleavage within the cell. This metabolic pathway allows the cell to obtain more ATP per sugar unit, since only one ATP molecule is required for the uptake of relatively large cellodextrins. Based on the recent published genome sequence of C. thermocellum, seven putative extracellular sugar binding proteins (SBP1-7) which are part of an ABC transport systems were identified. These His-tags fused proteins were purified and their ability to bind different sugars was tested using Isothermal Titration Calorimeter (ITC), Differential Scanning Calorimeter (DSC) and fluorescence quenching. Four of the binding proteins bind cellodextrins and one SPB binds laminaribiose. The ITC results indicated that all of the binding interactions were exothermic. The highest Kb values of SBP3, SBP4 and SBP5 analyses were gained with cellopentaose, suggesting that these proteins prefer to bind five sugar units. SBP7 binds only cellotriose and SBP6 interacts best with laminarbiose. Northern blot analysis indicated that the sbp3, sbp4 and sbp5 genes are part of 3.1kb, 4.0kb, and 3.3kb transcriptional units, respectively. Based on the genome sequence and the Northern blot results it appears that the ABC transporter genes are cotranscribed. E. COLI MULTIDRUG TRANSPORTER MdfA IS A MONOMER N. Sigal, O. Lewinson, S.G. Wolf, E. Bibi Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes. PROTEIN ACETYLATION/DEACETYLATION IN THE EXTREMELY HALOPHILIC ARCHAEON HALOFERAX VOLCANII N. Altman-Price, S. Barak Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel Protein acetylation and deacetylation reactions are known for sometime to be involved in many regulatory processes in eukaryotes. Recently it was shown that similar reactions have regulatory roles also in bacteria and archaea Sequence analysis of the haloarchaeon Haloferax volcanii genome enabled the identification of three putative protein acetyltransferases of the GCN5 family (Pat1, Pat2 and Elp3) and two deacetylases; (Sir2 and HdaI). Intriguingly, the gene that encodes for HdaI shares an operon with an archaeal histone homologue. We have used a gene knockout method to determine whether these putative genes are essential, and found that whereas Sir2 knockout strain can grow normally, HdaI deletion is lethal. Moreover, the specific HdaI inhibitor Trichostatin A inhibits cell growth. We also showed that Pat2 and Elp3 are “synthetic lethals”. Genetic analysis of the histone gene has shown that it is essential for growth. Site directed mutagenesis of the two unique lysine residues of the histone established a link between the histone and the acetylation/deacetylation processes in Haloferax volcanii. Changing any of the lysine residues to glutamine made the cells more sensitive to Trichostatin A while mutagenesis of the lysine in the C-terminal domain to arginine rendered the cells more resistant to that drug. IDENTIFICATION AND CHARACTERIZATION OF EXTREMOPHILE YEAST STRAINS L. Avrahami, A. Grabelsky, D. Engelberg, S. Braun Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Israel Given the global climate changes, a most urgent challenge of biotechnology research is finding extremophile strains. In the agriculture industry, for example, there is a concerning decrease in yields due to global warming. Obtaining extremophile strains is not trivial because their properties may be obtained by a combination of many genetic and epigenetic modifications. In natural extremophiles, the properties are a result of hundreds of millions of years of evolutionary selection.
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