Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-beta-synthase domains are required for its osmoregulatory function

The plant pathogen Pseudomonas syringae may cope with osmotic stress on plants, in part, by importing osmoprotective compounds. In this study, we found that P. syringae pv. tomato strain DC3000 was distinct from most bacterial species in deriving greater osmoprotection from exogenous choline than from glycine betaine. This superior osmoprotection was correlated with a higher capacity for uptake of choline than for uptake of glycine betaine. Of four putative osmoregulatory ABC transporters in DC3000, one, designated OpuC, functioned as the primary or sole transporter for glycine betaine and as one of multiple transporters for choline under high osmolarity. Surprisingly, the homolog of the well-characterized ProU transporter from Escherichia coli and Salmonella enterica serovar Typhimurium did not function in osmoprotection. The P. syringae pv. tomato OpuC transporter was more closely related to the Bacillus subtilis and Listeria monocytogenes OpuC transporters than to known osmoprotectant transporters in gram-negative bacteria based on sequence similarity and genetic arrangement. The P. syringae pv. tomato OpuC transporter had a high affinity for glycine betaine, a low affinity for choline, and a broad substrate specificity that included acetylcholine, carnitine, and proline betaine. Tandem cystathionine-beta-synthase (CBS) domains in the ATP-binding component of OpuC were required for transporter function. The presence of these CBS domains was correlated with osmoregulatory function among the putative transporters examined in DC3000 and was found to be predictive of functional osmoregulatory transporters in other pseudomonads. These results provide the first functional evaluation of an osmoprotectant transporter in a Pseudomonas species and demonstrate the usefulness of the CBS domains as predictors of osmoregulatory activity.     

Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.     

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.     

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.     

Halotolerant cyanobacterium Aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT(A. halophytica)) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetT(A. halophytica) is acidic, 4.58. BetT(A. halophytica) specifically catalyzed the transport of betaine. Choline, gamma-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT(A. halophytica). Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT(A. halophytica) is a Na(+)-betaine symporter. Betaine uptake activities of BetT(A. halophytica) were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT(A. halophytica) showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT(A. halophytica) were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na(+)-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT(A. halophytica) is the first identified transporter for compatible solutes in cyanobacteria.     

Uncoupling of choline-O-sulphate utilization from osmoprotection in Pseudomonas putida

The genomic context of the recognized bet genes for choline-O-sulphate (COS) utilization in Pseudomonas putida KT2440 is such that betC (choline sulphatase) lies adjacent to an ATP-binding cassette transporter and a LysR type regulator, but well away from betBA, encoding enzymes for transformation of choline into glycine betaine. The consequences of such genetic layout of the functions for COS metabolism have been examined with a suite of genetic and biochemical approaches. An early clue of the utilities of the betencoded products was exposed by the phenotypes of a betC deletion. This mutant still accumulated intact COS but failed to use this compound as carbon or nitrogen source. Furthermore, betC expression was downregulated at high salt concentrations, showing that the principal role of this gene lied in COS metabolism, not in osmoprotection. In contrast, the betBA genes were required for choline transformation into the highly effective compatible solute glycine betaine (and the concomitant endurance to high salt) and also for its utilization as carbon or nitrogen source. Thus, unlike in the cases of Bacillus subtilis and Sinorhizobium meliloti, betC is unrelated to osmoprotection in Pseudomonas putida while the betBA genes are required for both betaine synthesis and tolerance to high osmotic pressure.     

Cellular Choline and Glycine Betaine Pools Impact Osmoprotection and Phospholipase C Production in Pseudomonas aeruginosa

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.     

Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.     

The BCCT family of carriers: from physiology to crystal structure

Increases in the environmental osmolarity are key determinants for the growth of microorganisms. To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from scarce environmental sources as effective osmoprotectants. A number of these systems belong to the BCCT family (betaine-choline-carnitine-transporter), sodium- or proton-coupled transporters (e.g. BetP and BetT respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, an L-carnitine/γ-butyrobetaine antiporter that is not involved in osmotic stress responses. The glycine betaine transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in an occluded conformation in complex with its substrate glycine betaine and two crystal structures of CaiT in an inward-facing open conformation in complex with L-carnitine and γ-butyrobetaine were reported recently. These structures and the wealth of biochemical data on the activity control of BetP in response to osmotic stress enable a correlation between the sensing of osmotic stress by a transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.     

Salt adaptation in <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis>: identification and characterization of a secondary glycine betaine transporter

Members of the genus Acinetobacter are well known for their metabolic versatility that allows them to adapt to different ecological niches. Here, we have addressed how the model strain Acinetobacter baylyi copes with different salinities and low water activities. A. baylyi tolerates up to 900 mM sodium salts and even higher concentrations of potassium chloride. Growth at high salinities was better in complex than in mineral medium and addition of glycine betaine stimulated growth at high salinities in mineral medium. Cells grown at high salinities took up glycine betaine from the medium. Uptake of glycine betaine was energy dependent and dependent on a salinity gradient across the membrane. Inspection of the genome sequence revealed two potential candidates for glycine betaine transport, both encoding potential secondary transporters, one of the major facilitator superfamily (MFS) class (ACIAD2280) and one of the betaine/choline/carnitine transporter (BCCT) family (ACIAD3460). The latter is essential for glycine betaine transport in A. baylyi. The broad distribution of ACIAD3460 homologues indicates the essential role of secondary transporters in the adaptation of Acinetobacter species to osmotic stress.     

Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Identification of osmo-dependent and osmo-independent betaine-choline-carnitine transporters in Acinetobacter baumannii: role in osmostress protection and metabolic adaptation

Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.     

Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.     

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity ( K _m, 2.6 μM) and, although it also binds betaine ( K _m, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX ( K _m, 24 μM) and the betaine-specific SBP BetX ( K _m, 0.6 μM). Unlike most ABC transporter loci, caiX , betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.     

Isolation and Characterization of ButA,a Secondary Glycine Betaine Transport System Operating in <Emphasis Type="Italic">Tetragenococcus halophila</Emphasis>

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.Received: 19 November 2002/Accepted: 19 December 2002     

Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline.

Exogenously provided glycine betaine functions as an efficient osmoprotectant for Bacillus subtilis in high-osmolarity environments. This gram-positive soil organism is not able to increase the intracellular level of glycine betaine through de novo synthesis in defined medium (A. M. Whatmore, J. A. Chudek, and R. H. Reed, J. Gen. Microbiol. 136:2527-2535, 1990). We found, however, that B. subtilis can synthesize glycine betaine when its biosynthetic precursor, choline, is present in the growth medium. Uptake studies with radiolabelled [methyl-14C]choline demonstrated that choline transport is osmotically controlled and is mediated by a high-affinity uptake system. Choline transport of cells grown in low- and high-osmolarity media showed Michaelis-Menten kinetics with Km values of 3 and 5 microM and maximum rates of transport (Vmax) of 10 and 36 nmol min-1 mg of protein-1, respectively. The choline transporter exhibited considerable substrate specificity, and the results of competition experiments suggest that the fully methylated quaternary ammonium group is a key feature for substrate recognition. Thin-layer chromatography revealed that the radioactivity from exogenously provided [methyl-14C]choline accumulated intracellularly as [methyl-14C]glycine betaine, demonstrating that B. subtilis possesses enzymes for the oxidative conversion of choline into glycine betaine. Exogenously provided choline significantly increased the growth rate of B. subtilis in high-osmolarity media and permitted its proliferation under conditions that are otherwise strongly inhibitory for its growth. Choline and glycine betaine were not used as sole sources of carbon or nitrogen, consistent with their functional role in the process of adaptation of B. subtilis to high-osmolarity stress.     

The Sinorhizobium meliloti ABC transporter Cho is highly specific for choline and expressed in bacteroids from Medicago sativa nodules

In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth. Using a PCR strategy, we identified three chromosomal genes (choXWV) which encode components of an ABC transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas the best homology scores were obtained with components of betaine ProU-like systems, Cho is not involved in betaine transport. Site-directed mutagenesis of choX strongly reduced (60 to 75%) the choline uptake activity, and purification of ChoX, together with analysis of the ligand-binding specificity, showed that ChoX binds choline with a high affinity (KD, 2.7 microM) and acetylcholine with a low affinity (KD, 145 microM) but binds none of the betaines. Uptake competition experiments also revealed that ectoine, various betaines, and choline derivatives were not effective competitors for Cho-mediated choline transport. Thus, Cho is a highly specific high-affinity choline transporter. Choline transport activity and ChoX expression were induced by choline but not by salt stress. Western blotting experiments with antibodies raised against ChoX demonstrated the presence of ChoX in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa roots. The choX mutation did not have an effect on growth under standard conditions, and neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that the remaining choline uptake system(s) still present in the mutant strain can compensate for the lack of Cho transporter.     

Betaine is a highly effective organic osmolyte but does not appear to be transported by established organic osmolyte transporters in mouse embryos

Betaine protects early preimplantation mouse embryos against increased osmolarity in vitro, functioning as an organic osmolyte. Betaine is effective at very low external concentrations, with half-maximal protection of 1-cell embryo development to blastocysts at approximately 50 microM, making it one of the best osmoprotectants for mouse preimplantation embryos. We performed studies designed to determine whether known high-affinity organic osmolyte transporters could account for the ability of betaine to act as an organic osmolyte in preimplantation embryos. We found no evidence in 1-cell embryos of transport by a betaine/GABA transporter (BGT1), the osmoregulated betaine transporter found in a number of cell types, as betaine and GABA did not inhibit each other's transport. Instead, all saturable GABA transport in embryos was apparently via the beta-amino acid transporter. We also found that the glycine transporter, GLY, which mediates osmoprotective transport of glycine in early preimplantation embryos, does not appear to transport betaine. Finally, increased osmolarity did not induce any detectable System A amino acid transporter activity, which is osmotically-inducible in other cells and can transport betaine. There does appear, however, to be a saturable betaine transporter in 1-cell mouse embryos, as considerable 14C-betaine transport was measured which was substantially inhibited by excess unlabeled betaine. Our data imply that betaine functions as an organic osmolyte in embryos due to its saturable transport via a mechanism distinct from known osmolyte transporters. We propose that an unidentified high-affinity betaine transporter may be expressed in early embryos and mediate transport of betaine as an organic osmolyte.     

OusB, a broad-specificity ABC-type transporter from Erwinia chrysanthemi, mediates uptake of glycine betaine and choline with a high affinity

The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi.     

Small-molecule inhibition of choline catabolism in Pseudomonas aeruginosa and other aerobic choline-catabolizing bacteria

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.