Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti.

Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.     

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.     

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Toxicity and osmoprotective activities of analogues of glycine betaine obtained by solid phase organic synthesis towards Sinorhizobium meliloti

Seven analogues of the bacterial osmoprotectant glycine betaine (GB, trimethylammonioacetate), in which the methyl groups of the Me₃N⁺ moiety are replaced by various substituents, were obtained by SPOS using Wang resin. Their biological activities (osmoprotection vs toxicity), appeared closely related to their uptake efficiency and their catabolism in the betaine-demethylating model bacterium Sinorhizobium meliloti.     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids.

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.     

Chemotaxis by Rhizobium meliloti

Summary Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

Antibacterial activity of glycine betaine analogues: involvement of osmoporters

Glycine betaine (GB) analogues were obtained using solid phase organic synthesis and assayed for their toxic activity against 15 Gram positive and Gram negative bacteria. Four benzyl derivatives of GB were selected to determine their effect on bacterial growth. Bacteriostatic and lethal effects were observed for compound 1 and compound 2, respectively. The importation of the two GB analogues into bacterial cells appeared strictly dependent on the presence of the powerful betaine membrane osmoporters; their capacity to be amassed intracellularly at molar levels from extremely dilute solutions might constitute a basis to design a new class of antimicrobial agents.     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

Polypeptide synthesis by Rhizobium bacteroids and bacteria.

When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated [35S]-methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue. The labelled polypeptides were stable for several hours in pulse-chase experiments. Changes in the concentration of H+, K+ and Mg2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides. A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed [35S]methionine. Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared. Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase. Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns. The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.     

Viability of Rhizobium bacteroids.

Bacteroids prepared from nodules of soybean and bean were tested for viability. Contrary to the prevailing view that bacteroids are nonviable, it was found that bacteroids averaged 90% viability, irrespective of Rhizobium strain, nodule age, or nodule environment.     

BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.     

Viability of Rhizobium bacteroids.

Bacteroids prepared from nodules of soybean and bean were tested for viability. Contrary to the prevailing view that bacteroids are nonviable, it was found that bacteroids averaged 90% viability, irrespective of Rhizobium strain, nodule age, or nodule environment.     

Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti.

The effect of salt stress on glycine betaine-binding activity has been investigated in periplasmic fractions released from Rhizobium meliloti 102F34 by cold osmotic shock. Binding activity was monitored by three techniques: equilibrium dialysis, filter procedure, and detection of 14C ligand-protein binding by direct non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. The three methods demonstrated the existence of a strong glycine betaine-binding activity, but only in periplasmic fractions from cells grown at high osmolarity. The non-denaturing PAGE of such periplasmic shock fluids mixed with [methyl-14C]glycine betaine showed only one radioactive band, indicating the involvement of one glycine betaine-binding protein. To determine the possible implication of this binding protein in glycine betaine uptake, transport activity was measured with cells submitted to cold osmotic shock. No significant decrease of transport activity was noticed. This lack of effect could be explained by the small quantity of periplasmic proteins released as judged by the low activity of phosphodiesterase, a periplasmic marker enzyme, observed in the shock fluid. The specificity of binding was analysed with different potential competitors: other betaines such as gamma-butyrobetaine, proline betaine, pipecolate betaine, trigonelline and homarine, or amino acids like glycine and proline, did not bind to the glycine betaine-binding protein, whereas glycine betaine aldehyde and choline were weak competitors. Optimum pH for binding was around 7.0, but approx. 90% of the glycine betaine-binding activity remained at pH 6.0 or 8.0. The calculated binding affinity (KD) was 2.5 microM. Both glycine betaine-binding activity and affinity were not significantly modified whether or not the binding assays were done at high osmolarity. A 32 kDa osmotically inducible periplasmic protein, identified by SDS-PAGE, apparently corresponds to the glycine betaine-binding protein.     

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34

Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B(12)-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment.     

Metabolism of some polyols by Rhizobium meliloti

The utilization of d-mannitol, d-arabitol, and d-sorbitol by Rhizobium meliloti was studied in extracts from mannitol-grown cells. Two different polyol dehydrogenases were induced by any of these polyols: (i) a nicotinamide adenine dinucleotide (NAD)-arabitol dehydrogenase and (ii) a NAD-sorbitol dehydrogenase, whereas polyol phosphate dehydrogenases were absent. d-Arabitol dehydrogenase was observed to act on both d-arabitol and d-mannitol, but d-sorbitol dehydrogenase acted specifically on d-sorbitol. d-Arabitol was oxidized to d-xylulose, d-mannitol and d-sorbitol were oxidized to d-fructose. An adenosine triphosphate-linked hexokinase which acts on d-fructose and absence of hexose isomerase were also detected in this organism.