Characterization of an osmoregulated periplasmic glycine betaine-binding protein in Azospirillum brasilense sp7.
Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock. Non-denaturing PAGE in the presence of [methyl-14C] glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells. The binding activity was absent in periplasmic fractions from cells grown at low osmolarity. SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000. The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing. This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline). Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity. Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.
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Laboratoire de Biologie Végétale et Microbiologie, URA CNRS 1114, Faculté des Sciences et des Techniques, Université de Nice-Sophia Antipolis, France.
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Le Rudulier
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D000596:Amino Acids / Q000032:analysis; D015806:Azospirillum brasilense / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007525:Isoelectric Focusing; D026901:Membrane Transport Proteins; D009994:Osmolar Concentration; D033902:Periplasmic Binding Proteins; D011233:Precipitin Tests
D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C053437:PROX protein, E coli; D033902:Periplasmic Binding Proteins; D001622:Betaine