Biochimie (1991) 73, 1187-1193 © Soci6t~ fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

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Characterization of an osmoregulated periplasmic glycm. betaine-binding protein in AzospiriUum brasilense sp7 N Riou, MC Poggi, D Le Rudulier* Laboratoire de Biologie Vdgdtale et Microbiologie, lIRA CNRS 1114, Facultd des Sciences et des Techniques, Universitd de Nice-Sophia Antipolis, Parc Valrose, 06034 Nice Cedex, France (Received 24 December 1990; accepted 15 March 1991)

Summary --- 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 [methyl14C] glycine betalne 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 isoelectfic focusing. This protein bound glycine betaine with high affinity (Kv of 3 gM), but had no affinity for either other betaines (proline betaine, y-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.

transport / glycine betaine / binding protein / AzospiriUum brasUense

Introduction The property to adapt to changes in the osmotic strength of the environment is a crucial trait for organisms. To withstand fluctuations in the osmolarity of the environment, bacteria accumulate low-molecularweight solutes such as potassium, glutamate, proline, glycine betaine or other betaines [1-3]. Among the compatible solutes, glycine betaine is one of the most important osmoprotective solutes. In Salmonella typhimurium and Escherichia coli, the uptake of glycine betaine is mediated by two transport systems: Pro P and Pro U [4-6]. The activities of the two transport systems are enhanced by exposure of the cells to osmotic stress. In the Pro P system, the osmotic control is primarily the result of a post-transcriptional modification of the transport protein, transcription of prop was only increased three-fold by growth at high osmolarity [4, 7]. On the contrary, the expression of *Correspondence and reprints Abbreviations: SDS-PAGE: sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis; GBBP: glyeine betainebinding protein.

the proU locus is mainly regulated at the transcriptional level, increasing over 100-fold with enhanced osmotic pressure [5, 6]. The proU operon of enteric bacteria belongs to a family of related transport systems which utilize a periplasmic binding protein [8-10]. Analysis of the nucleotide sequence [11, 12] of the proU operon of E coli indicates that this locus contains three genes, proV, proW, and proX. The proV gene encodes a hydrophilic protein with homology to the energy-coupling component of binding-protein-dependent transport systems. The proW gene encodes a hydrophobic polypeptide thought to be located in the cytoplasmic membrane. The proX gene encodes the periplasmic glycine betaine-binding protein (GBBP). In Azospirillum brasilense glycine betaine plays also an important role in osmoregulation [13, 14]. This compound is accumulated in cells grown at high osmolarity and is not catabolized. As in E coli and S typhimurium, glycine betaine transport in A brasilense sp7 is strongly stimulated by elevated osmolarity, and significantly reduced in cells subjected to an osmotic cold shock, suggesting a transport mechanism involving a periplasmic GBBP [14].

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In this paper, we report the existence of a periplasmic G B B P in A brasilense sp7, as well as its identification and specificity properties. Using an immunoprecipitation technique and Western blotting associated with immunodetection, we also show that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with G B B P produced by A brasilense grown at high osmolarity.

Materials and methods Chemicals [Methyl-t4C] choline (2.15 MBq lamol-I) was purchased from Amersham Corp (Amersham, UK). [Methyl.14C]glycine betaine was prepared enzymatically from [methyl-14C].choline using choline oxidase from Alcaligenes sp (Sigma Chemical Co), and purified using paper electrophoresis [15]. Radioactive proline betaine was synthetized from L-[U-14C] proline (9.62 MBq lamol-l, CEA, Gif-sur-Yvette, France). [14Ch,butyrobetaine (0.59 MBq lamol-l) was kindly provided by J Deshusses (University of Geneva, Switzerland). Glycine betaine, glycine betaine aldehyde, choline, trigonelline were purchased from Sigma Chemical Corp. Homarine, proline betaine, 7-butyrobetaine and pipecolate betaine were obtained as mentioned earlier [15]. All other chemicals were of reagent grade and available commercially. Bacterial strains and growth condition Azospirillum brasilense, strain sp7 (ATCC 2914) was maintained in nutrient broth rich medium [16]. Cultures were grown aerobically at 30°C in K minimal medium [ 17], except that the carbon source used was sodium lactate (5 g !-I) instead of malate. The nitrogen source was 20 mM NH4CI. E coli strain MC 4100 was obtained from E Bremer (University of Konstanz, Germany), and grown aerobically at 37°C in LB medium or minimal medium 63 [15]. The osmolarity of minimal medium was increased by the addition of NaCI. The osmotic pressure of the different media was measured by freezing-point depression with an H Robling micro-osmometer (Bioblock Scientific, Illkirch, France). Bacterial growth was monitored spectrophotometrically at 420 nm, and the protein concentration of bacterial cultures was quantified by the Lowry method [ 18]. Preparation of periplasmic fractions and binding assays Periplasmic fractions were isolated from cells grown in lowsalt-medium (K medium) or high-salt-medium (K medium with 0.3 M NaCI) by cold osmotic shock [14, 19]. Binding assays were done by the filter binding procedure [20]. Samples of periplasmic shock fluid (200 lag protein) were incubated at 20°C for 30 min with the appropriate amount of [methyl-14C] glycine betaine (2.5 kBq), in final volume of 50 lal. Proteins were precipitated by adding 2 ml of ice cold-saturated ammonium sulphate solution, and collected by filtration onto 0.45 gm nitrocellulose filters (Millipore Corp). The radioactivity of the filters was determined by scintillation counting. This procedure was used for three purposes. First, to determine the glycine betaine-binding affinity using various amounts of substrate (0.1 to 50 laM). Second, to analyse the binding activity at various pH (from 5.5 to 8.5). Third, to measure the effect of the osmolarity of the binding assay by adding an increased amount of NaCI.

Protein gel electrophoresis and autoradiography Binding activity could be detected by direct polyacrylamide gel electrophoresis of the complex ligand-protein in nondenaturing conditions [21 ]. Aliquots of the concentrated shock fluids (100 lag) from low-and-high-salt-grown cells, were resuspended in Laemmli sample buffer [22] without SDS and [~mercaptoethanol (pH 6.8) and mixed with radioactive [14C] glycine betaine, at a final concentration of 10 laM. The samples were left at room temperature for 30 rain, and were subjected to non-denaturing PAGE (10% gel) in a discontinuous system. The running buffer in which SDS was omitted contained 0.025 M Tris-HCl and 0.192 M glycine (pH 8.2). In order to shorten the time of electrophoresis, the Bio-Rad mini Protean II slab cell system (Bio-Rad Labs, Richmond, CA) was used. The analyses were performed with a constant voltage setting of 200 V, usually for approximately 45 min. The gels were then quickly dried on Whatman 3 MM paper, and autoradiographed using Kodak films (Kodak X-Omat) during approximately one week. Parallel protein migrations (30 Izg per well) were performed for staining with Coomassie blue. Staining can also be clone after autoradiography by rapidly scraping the paper from the gel with distilled water. In order to identify the GBBP, a preparative non-denaturing gel was performed. Each well was loaded with the same amount of periplasmic proteins (100 ~tg), and after the run, one lane was stained to identify the band that binds glycine betaine. The appropriate area of the unstained gel was cut out, and the gel slice was electro-eluted using a model 422 electro-eluter system (Bio-Rad Labs). The eluted proteins were submitted to SDS-PAGE (7.5 to 17.5% linear gradient). Molecular weight standards were from Bio-Rad Labs.

lsoelectric focusing Polyacrylamide gel isoelectric focusing was performed using a Bio-Rad mini protean slab cell, with a mixture of Bio-Rad ampholytes (pH range from 5.0 to 7.0). The mixture of standard proteins (Sigma) contained [~-lactoglobulin A from bovine milk (p/, 5.1), carbonic anhydrase II from bovine erythrocytes (pl, 5.9), carbonic anhydrase I from human erythrocytes (p/, 6.6), and myoglobine from horse heart (p/, 6.8). The proteins were separated during 3.5 h under 750 V after progressive prefocusing of the gel (55 min).

lmmunoprecipitation Immunoadsorbed antt-GBBP antibody from E coil were kindly provided by M Villarejo (University of California, Davis, USA). Immunoprecipitation and SDS-PAGE were performed as previously described [23] with periplasmic proteins (3 ~g) from low- and high-salt-grown cells of A brasilense sp7, and from 0.5 M NaCl-grown cells of E coli. The proteins were labeled with Nal25I (37 MBq), in the presence of Iodo-Gen iodination reagent (100 l.tg) prepared as described previously •[24], and purchased from Pierce Europe BV (dud Beijerland, The Netherlands). Immunoprecipitated soluble proteins were separated by one-dimensional PAGE (12.5%), in the presence of 3% (w/v) SDS [22]. Similar experiments were performed with non-immune rabbit serum as control. Results were revealed by autoradiography.

Western blotting Periplasmic proteins (3 lag) from low- and high-salt-grown cells of A brasilense and from 0.5 M NaCl-grown cells of

Glycine betaine-binding protein in A brasilense E coli were solubilized in Laemmli buffer [22] without 13mercaptoethanol. They were submitted to a gradient (7.517.5% acrylamide) SDS-PAGE, and transferred onto nylon membrane (Hybond C, Amersham) [25] by using a Bio-Rad mini trans-blot electrophoretie transfer cell as recommended by the manufacturer. The blot was treated with the immunoadsorbed anti-GBBP antibody from E coli. Immunoreactive bands were visuall.zed by using the biotinylated horseradish peroxidase system [26].

Results Binding activity

We have previously demonstrated that the glycine betaine-binding activity is at least six-fold higher in cells grown at high osmolarity (0.3 M NaCI) than in cells grown at low osmolarity [14]. Thus, the glycine betaine-binding affinity, assayed with the filter binding procedure [10, 20], was determined using only periplasmic fractions isolated from cells grown at high osmolarity (fig 1). The maximal capacity of binding was 725 pmol/mg of periplasmic protein with a free ligand concentration of 50 l.tM; this value is approximately equivalent to that obtained with periplasmic fraction prepared from S typhimurium LT2

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cells grown in the presence of 0.3 M NaCI [10]. Assuming that one molecule of protein binds one molecule of glycine betaine, the calculated Kt, was found to be 3 l.tM. Non-specific binding determined with bovine serum albumin was negligible. The pH dependence of the binding activity was also assayed by measuring the binding of [methyl-laC] glycine betaine with periplasmic fractions isolated fi'om cells grown at high osmolarity (fig 2). Optimum pH for binding was between 7.0 and 7.5 with almost 80 and 30% reduction of binding at pH 5.5 and 8.5, respectively. Since the activity of glycine betaine transport was dependent on the osmolarity, a possible role for binding proteins in this modulation was investigated by raising the osmolarity of the binding assay. With periplasmic shock fluids from cells grown at low or high osmolarity, increasing the ionic strength of the assay buffer with 0.3 M NaCI had no significant effect on the glycine betaine-binding activity. Interestingly, the binding capacity was not inhibited by high ionic

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Glycine betaine ~MJ Fig 1. Glycine betaine-binding activity in periplasmic shock fluid of A brasilense sp7. Periplasmic fractions were isolated from cells grown at high osmolarity (K medium + 0.3 M NaC1). Binding activity was assayed using the filter binding procedure: 10-I.tl aliquots of periplasmic shock fluid (200 [.tg protein) were equilibrated 30 min at 20°C in 10 mM Tris-HCl, pH 7.5 with the final glycine betaine concentrations indicated on the abscissa, in a final volume of 50 gl; the samples were treated with cold-saturated ammonium sulphate solution as described in Materials and methods. Each point is the mean of duplicates carried out on two independent periplasmic shock fluids.

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pH Fig 2. Effect of pH on glycine betaine-binding activity periplasmic shock fluid of A brasilense sp7. Binding [methyl:4C] glycine betaine was assayed as in figure with 10 lttM glycine betaine. Each point is the mean duplicates carried out on two independent preparations periplasmic fractions.

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active band corresponds to the most intense band observed after Coomassie staining. On the contrmT, shock fluid from cells grown at low osmolarity did not show any labeled band. It is also clear that the amount of proteins found at the same position as the radioactive band was much lower in cells obtained in the absence of NaCI than in cells maintained in the presence of salt. Using non-denaturing PAGE with larger plates (20 x 20 cm) and much longer migration (14 h at 70 V) still allowed the detection of the GBBP (data not shown). When periplasmic shock fluid from E coil MC4100 cells grown at 0.5 M NaCI was incubated with [methyl-~4C] glycine betaine at pH 6.8 and subjected to non-denaturing PAGE as previously, the GBBP characterized in this bacterium [8, 9] showed a much less negative entire charge than the GBBP from A brasilense (data not shown). The specificity of the binding was assayed by incubation of periplasmic proteins from 0.3 M NaCIgrown cells with various unlabeled potential competitors (1 mM final) prior to the addition of [methyl-~4C] glycine betaine (10 I.tM). Fractions were submitted to non-denaturing PAGE and then to autoradiography (fig 4). When unlabeled glycine betaine was added, the labeling totally disappeared, demonstrating the specificity of the binding phenomenon (fig 4, track 2). Mixing periplasmic proteins with other betaines (proline betaine, T-butyrobetaine, pipecolate betaine, trigonelline, homarine), or structurally related compounds (glycine, betainal, choline, proline) had no significant effect on the intensity of the radioactive band, showing that the specificity of the binding phenomenon is very high. Confirmation of this narrow specificity was obtained by using available ~4C betaine as ligand (ie choline, ¥-butyrobetaine, proline betaine) instead of t4C glycine betaine. Absolutely no binding could be detected with any of these radioactive betaines.

strength. These data show that the modulation of glycine betaine transport activity is not simply a consequence of modified substrate binding by periplasmic proteins. Indeed, similar conclusions have been formulated in the case of E coli [8], and S typhimurium [ 10].

Non-denaturing polyacrylamide gel electrophoresis Crude shock fluids from A brasilense cells grown at high or low osmolarity were incubated with [methyl~4C] glycine betaine as described in Materials and methods, and subjected to non-denaturing PAGE and autoradiography (fig 3). Coomassie blue staining (fig 3A) of the two periplasmic fractions showed a relative abundance of highly negative charged proteins in low-salt-grown cells compared to highsalt-grown ceils. On the autoradiogram (fig 3B), only one radioactive band was found when the cells were grown at high osmolarity showing the presence of the label bound to one protein. Interestingly, this radio-

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Fig 3. Non-denaturing PAGE 10% gel (A), and autoradiography (B) of periplasmic proteins from A brasilense sp7. Proteins were released by cold osmotic shock from cells grown at low osmolarity (-) and high osmolarity 0.3 M NaC! (+). A. 30 ~tg amount of protein were stained with Coomassie blue. B. 100 /.tg amount of proteins was incubated with 10 ~tM [methyi-~4C] glycine betaine, subjected to non-denaturing PAGE, and then autoradiographed.

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Fig 4. Autoradiography of periplasmic proteins from A brasilense sp7, subjected to 10% non-denaturing PAGE. Proteins were from cells grown in K medium with 0.3 M NaCl. A 100 l~g amount of proteins was incubated with 10 gM [methyl-14C] glycine betaine in the absence (track 1) or the presence (tracks 2 to 11) of 1 mM unlabeled betaines or analogues: glycine betaine, glycine, glycine betaine aldehyde, choline, proline, proline betaine, y-butyrobetaine, pipecolate betaine, trigonelline, homarine, respectively from track 2 to track 11.

Glycine betaine-bindingprotein in A brasilense Characterization of the GBBP:SDS-PAGE and isoelectric focusing Crude periplasmic shock fluids from cells grown at low or high osmolarity were separated by one-dimensional SDS-PAGE using a 7.5 to 17.5% linear gradient of aerylamide as resolving gel. Proteins electro-eluted from the band which binds [methyl-14C] glycine betaine on a non-denaturing gel were also submitted to the same migration (fig 5). Following this simple process, the purification of the glycine betaine-binding protein was quite good, as judged by Coomassie blue staining. A single major band appeared with a molecular mass of 32 kDa, a value in very close agreement with results obtained in E coil [8, 9] and S typhimurium [10, 27, 28], It is clear that this protein was found in a large amount in periplasmic shock fluid extracted from cells grown at high osmolarity, whereas it was very weakly represented in cells grown at low osmolarity. The differences in the patterns were not investigated further, but it is obvious that other periplasmic proteins were more abundant under salt stress. Interestingly, when glycine betaine

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Fig 5. SDS-PAGE (7.5 to 17.5% linear gradient) of periplasmic proteins from A brasilense sp7, and electroeluted GBBP. Proteins (30 l.tg) were from cells grown at low osmolarity (track 2), or high osmolarity, 0.3 M NaCI (track 3). The GBBP (1 l.tg, track 5) was electro-eluted from the band which binds [methyl-14C] glycine betaine on a preparative non-denaturing gel. The molecular mass markers (tracks 1 and 4) were from top of the gel: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (1a.4 kDa). The gel was stained with Coomassie blue.

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(1 raM) was added to the growth medium with 0.3 M NaCI, the amount of the 32 kDa protein was greatly reduced (data not shown), as previously observed in E coli [8]. lsoelectric locusing analysis in a pH gradient of 5.0 to 7.0 showed that the GBBP focused as a single band with a pl between 5.9 and 6.6. A standard protein with a p! within this range would be needed to get more accurate determination. Immunoprecipitation assays and Western blotting After solubilization and immunoprecipitation, periplasmic proteins from A brasilense cells grown at low or high osmolarity were analyzed by SDS-PAGE and autoradiographed. Periplasmic proteins from E coli cells grown at high osmolarity (0.5 M NaCI) were used as a positive control, lmmunoadsorbed antiGBBP antibody from E coli revealed intensively the 32-kDa protein from E coli as expected (fig 6, track 1), and also the GBBP from A brasilense obtained at high osmolarity (track 2). No labeling was seen with proteins from A brasilense cells grown at low osmolarity (track 3). Western blotting of GBBP from A brasilense with anti-GBBP from E coli also showed that cross-reaction occurred only with proteins from cells grown at high osmolarity (fig 7), demonstrating that GBBP was absent or very weakly represented in this extract. Discussion The results described in this study are a continuation of our recent studies which demonstrate the existence of a high-affinity osmoregulated glycine betaine uptake system in A brasilense, and also suggest that uptake is periplasmic-protein-dependent [14]. In the present work, we have detected a GBBP by direct non-denaturing polyacrylamide gel electrophoresis, using a method already utilized for characterization of ),-butyrobetaine binding-protein in Agrobacterium, leucine-binding protein, and ribose-binding protein in E coli [29]. In A brasilense sp7 we have shown that an osmotically inducible 32-kDa periplasmic protein is a GBBP with a KD of 3 l.tM for substrate binding. For the following reasons, it is very likely that this protein is involved in the transport process of glycine betaine. Firstly, the dissociation constant is in very good agreement with the Kt of the transport system which has been previously measured as 10 l.tM [ 14]. Secondly, the binding protein is not found in cells grown at low osmolarity in which very low transport activity occurs. Thirdly, we have previously shown significant reduction of transport after osmotic cold shock [14], with concomitant release of the 32-kDa protein. In addition, this protein has a very high specific binding

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Fig 6. lmmunoprecipitation of GBBP from E coli (track !), and A hrasUense sp7 (tracks 2 and 3). Periplasmic proteins were labeled with l"5I, and immunoprecipitation was conducted a:~ described in Materials and methods. After SDS-PAGE (12.5% gel), labeled immunoprecipitated proteins were revealed by autoradiography. E coli MC 4100 was grown at high osmolarity, 0.5 M NaCI (track 1); A brasilense sp7 was obtained in the presence of 0.3 M NaCl (track 2), or in the absence of salt (track 3). Molecular mass markers were as in figure 5.

activity, and no detectable affinity for other betaines such as proline betaine, 7-butyrobetaine, pipecolate betaine, trigonelline, homarine, or related compounds like choline, glycine betaine aldehyde, glycine and proline. It is noteworthy that the GBBP from A brasilense has many similarities with the GBBP already well characterized in E coli [8, 9]. In addition to the same molecular weight, very close KD, identical osmoregulated synthesis, and narrow specificity, it should also be mentioned that the glycine betainebinding activity of both periplasmic proteins is not altered whether or not the binding assay is done at high osmolarity. Furthermore, biosynthesis of both proteins is strongly reduced when growth medium of high-osmolarity is supplemented with 1 mM glycine betaine [6, 8]. Moreover, immunoadsorbed antibody

Fig 7. Immunoblotting of GBBP from A brasilense sp7 and E coli MC 4100. Periplasmic proteins were subjected to a gradient (7.5-17.5% acrylamide) SDS-PAGE (3 ~g per track), transferred onto nylon membrane, and Western blotted with immunoadsorbed anti-GBBP antibody from E coli. Immunoreactive bands were visualized by using the biotinylated horseradish peroxidase system. A brasilense cells were grown at low or high osmolarity (tracks 2 and 3, respectively). E coli cells were grown at high osmolarity (track 4). Molecular markers are indicated as kDa (track 1): pyruvate kinase (58.0), fumarase (48.5), lactic dehydrogenase (36.5), and triosephosphate isomerase (26.6).

directed against the purified GBBP from E coli crossreacts with the 32-kDa protein synthesized by A brasilense cells grown at high osmolarity. Finally, despite a quite broad phylogenetic distance between the Enterobacteriaceae and Azospirillum strains [30], it is quite likely that the function and the structure of the GBBP are well conserved, even if entire charges are different. In order to get more informations on this uptake system, we are currently using the p r o U locus and smaller DNA segments of E coli as a probe, and looking for hybridization with total digested DNA of

Glycine betaine-binding protein in A brasilense

A brasilense sp7. In fact, since A brasilense is a nitrogen-fixing bacterium, it has already demonstrated strong homology between several nif genes of A brasilense and Klebsiella pneumoniae nif genes [31, 32]. Similarly, osmoregulatory genes and particularly the proU locus might have been conserved during molecular evolution. Besides the GBBP from E coli and S typhimurium, the existence, purification, and properties of 7-butyrobetaine-binding protein have been reported in Agrobacterium sp [29, 33]. This 52-kDa protein involved in 7-butyrobetaine transport did not recognize glycine betaine [29]. Several additional differences between this 7-butyrobetaine-binding protein from Agrobacterium and the OBBP from A brasilense should be noticed. For example, the 7-butyrobetaine-binding protein is inducible by the substrate whereas the level of the GBBP is strongly reduced upon glycine betaine supplementation into the growth medium. In addition, on the contrary to glycine betaine uptake, the rate of 7-butyrobetaine transport is not enhanced by increasing the osmolarity of the medium [33]. This is in agreement with the fact that 7-butyrobetaine does not show any osmoprotective activity in Agrobacterium which uses this molecule as the sole source of both carbon and nitrogen [33]. These data demonstrated that among soil isolated bacteria, different betaines can function as energy source or osmoprotectant. Interestingly, R meliloti, another soil bacterium has the ability to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant [2, 34]. An in-depth study of betaines uptake and metabolism in various organisms should contribute to elucidation of the complex mechanism by which multiple nutritional and osmotic control systems interact.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Acknowledgments

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We are grateful C Elmerich and E Bremer for providing A brasilense and E coli strains, respectively. We are indebted to M Villarejo and J Deshusses for generous gift of immuno-

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adsorbed anti-OBBP antibody and radioactive 7-butyrobetaine, respectively. Thanks are due to JL Cousin and G Ponzio (INSERM U 210, Nice) for assistance in labeling of periplasmic proteins and immunoprecipitation assays. We appreciate the assistance of H Le Bds. The research was supported by the Centre National de la Recherche Scientifique.

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References

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1 Epstein W (1986) FEMS Microbiol Rev 39, 73-78 2 Le Rudulier D, Strom AR, Dandekar AM, Smith LT, Valentine RC (1984) Science 224, 1064-1068 3 Csonka LN (1989) Microbiol Rev 53, 121-147

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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...
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