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.     

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.     

The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K_d value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.     

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.     

Stress responses ofBacillus subtilis to high osmolarity environments: Uptake and synthesis of osmoprotectants

A decrease in the water content of the soil imposes a considerable stress on the voil-living bacteriumBacillus subtilis: water exits from the cells, resulting in decreased turgor and cessation of growth. Under these adverse circumstances,B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated viade novo synthesis, butB. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute ofB. subtilis is the potent osmoprotectant glycine betaine. This trimethylammonium compound can be taken up by the cell through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment,B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence ofB. subtilis against high osmolarity stress.     

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.     

Ectoine functions as an osmoprotectant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC

We report here that the cyclic amino acid ectoine functions as an osmoprotectant for the soil bacterium Bacillus subtilis. Growth experiments with a set of B. subtilis strains that carry defined mutations in the glycine betaine transport systems OpuA, OpuC and OpuD and the choline transport system OpuB revealed that ectoine was specifically accumulated via the ABC-transport system OpuC. Competition experiments employing unlabeled ectoine and radiolabeled glycine betaine showed that the OpuC transport system has a low affinity for ectoine with a K_i value of approximately 1.5 mM. Ectoine was identified by ¹H NMR spectroscopy in the solute pool of cells grown in the presence of ectoine. Ectoine could not be used by B. subtilis as sole carbon or nitrogen source. Our data thus characterise ectoine as a metabolically inert stress compound for B. subtilis and establish a crucial role for the ABC-transport system OpuC for the acquisition of the osmoprotectant ectoine from the environment.     

Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4 degrees C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4 degrees C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.     

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.     

High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

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.     

Biochemical and structural analysis of the Bacillus subtilis ABC transporter OpuA and its isolated subunits

Adaptation of microorganisms to changing osmotic conditions is a prerequisite for survival and cellular vitality for most microorganisms. In the Gram-positive soil bacterium Bacillus subtilis, five transport systems catalyze the uptake of compatible solutes across the plasma membrane that allow the growth of B. subtilis over a wide range of osmotic conditions. Focus of this review is the osmoprotectant uptake A (OpuA) transporter, a member of the family of substrate-binding protein (SBP)-dependent ATP-binding cassette (ABC) transporters that mediates the uptake of the compatible solutes glycine betaine and proline betaine. OpuA is composed of three subunits: a nucleotide-binding domain (OpuAA) located in the cytosol, a transmembrane domain (OpuAB), and a SBP (OpuAC), which binds glycine betaine and proline betaine with high specificity and targets it to OpuAB for ATP-dependent translocation across the plasma membrane. After a brief introduction in the field of bacterial osmoadaptation, we will summarize our recent findings about the biochemical and structural analysis of the components of the OpuA systems. Our studies covered both the isolated subunits of the OpuA transporter and initial investigations of the whole transporter in vitro.     

Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.     

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.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

Role of the Glycine Betaine and Carnitine Transporters in Adaptation of Listeria monocytogenes to Chill Stress in Defined Medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.     

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.     

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.