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.     

Lipoprotein from the osmoregulated ABC transport system OpuA of Bacillus subtilis: purification of the glycine betaine binding protein and characterization of a functional lipidless mutant.

The OpuA transport system of Bacillus subtilis functions as a high-affinity uptake system for the osmoprotectant glycine betaine. It is a member of the ABC transporter superfamily and consists of an ATPase (OpuAA), an integral membrane protein (OpuAB), and a hydrophilic polypeptide (OpuAC) that shows the signature sequence of lipoproteins (B. Kempf and E. Bremer, J. Biol. Chem. 270:16701-16713, 1995). The OpuAC protein might thus serve as an extracellular substrate binding protein anchored in the cytoplasmic membrane via a lipid modification at an amino-terminal cysteine residue. A malE-opuAC hybrid gene was constructed and used to purify a lipidless OpuAC protein. The purified protein bound radiolabeled glycine betaine avidly and exhibited a KD of 6 microM for this ligand, demonstrating that OpuAC indeed functions as the substrate binding protein for the B. subtilis OpuA system. We have selectively expressed the opuAC gene under T7 phi10 control in Escherichia coli and have demonstrated through its metabolic labeling with [3H]palmitic acid that OpuAC is a lipoprotein. A mutant expressing an OpuAC protein in which the amino-terminal cysteine residue was changed to an alanine (OpuAC-3) was constructed by oligonucleotide site-directed mutagenesis. The OpuAC-3 protein was not acylated by [3H]palmitic acid, and part of it was secreted into the periplasmic space of E. coli, where it could be released from the cells by cold osmotic shock. The opuAC-3 mutation was recombined into an otherwise wild-type opuA operon in the chromosome of B. subtilis. Unexpectedly, this mutant OpuAC system still functioned efficiently for glycine betaine acquisition in vivo under high-osmolarity growth conditions. In addition, the mutant OpuA transporter exhibited kinetic parameters similar to that of the wild-type system. Our data suggest that the lipidless OpuAC-3 protein is held in the cytoplasmic membrane of B. subtilis via its uncleaved hydrophobic signal peptide.     

Monitoring conformational changes during the catalytic cycle of OpuAA, the ATPase subunit of the ABC transporter OpuA from Bacillus subtilis

The ABC transporter (ATP-binding-cassette transporter) OpuA is one of five membrane transport systems in Bacillus subtilis that mediate osmoprotection by importing compatible solutes. Just like all bacterial and archaeal ABC transporters that catalyse the import of substrates, OpuA (where Opu is osmoprotectant uptake) is composed of an ATPase subunit (OpuAA), a transmembrane subunit (OpuAB) and an extracellular substrate-binding protein (OpuAC). In contrast with many well-known ABC-ATPases, OpuAA is composed not only of a catalytic and a helical domain but also of an accessory domain located at its C-terminus. The paradigm of such an architecture is MalK, the ABC-ATPase of the maltose importer of Escherichia coli, for which detailed structural and functional information is available. In the present study, we have applied solution FRET (F?rster resonance energy transfer) techniques using two single cysteine mutants to obtain initial structural information on the architecture of the OpuAA dimer in solution. Analysing our results in detail and comparing them with the existing MalK structures revealed that the catalytic and helical domains adopted an arrangement similar to those of MalK, whereas profound differences in the three-dimensional orientation of the accessory domain, which contains two CBS (cystathionine beta-synthetase) domains, were observed. These results shed new light on the role of this accessory domain present in a certain subset of ABC-ATPase in the fine-tuning of three-dimensional structure and biological function.     

Functional characterization of amphipathic α-helix in the osmoregulatory ABC transporter OpuA

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.     

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.     

Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background

The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.

Principal Findings

The binding of glycine betaine to purified OpuA and OpuAC (K_D = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.

Conclusions

Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.     

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined. The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator. It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport. These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.     

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.     

The compatible-solute-binding protein OpuAC from Bacillus subtilis: ligand binding, site-directed mutagenesis, and crystallographic studies

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.     

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.     

Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.     

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.     

Osmotic and chill activation of glycine betaine porter II in Listeria monocytogenes membrane vesicles

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.     

Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine

Compatible solutes play a decisive role in the defense of microorganisms against changes in temperature and increases in osmolarity in their natural habitats. In Bacillus subtilis, the substrate-binding protein (SBP)-dependent ABC-transporter OpuA serves for the uptake of the compatible solutes glycine betaine (GB) and proline betaine (PB). Here, we report the determinants of compatible solute binding by OpuAC, the SBP of the OpuA transporter, by equilibrium binding studies and X-ray crystallography. The affinity of OpuAC/GB and OpuAC/PB complexes were analyzed by intrinsic tryptophan fluorescence and the K(D) values were determined to be 17(+/-1)microM for GB and 295(+/-27)microM for PB, respectively. The structures of OpuAC in complex with GB or PB were solved at 2.0 A and 2.8 A, respectively, and show an SBP-typical class II fold. The ligand-binding pocket is formed by three tryptophan residues arranged in a prism-like geometry suitable to coordinate the positive charge of the trimethyl ammonium group of GB and the dimethyl ammonium group of PB by cation-pi interactions and by hydrogen bonds with the carboxylate moiety of the ligand. Structural differences between the OpuAC/GB and OpuAC/PB complexes occur within the ligand-binding pocket as well as across the domain-domain interface. These differences provide a structural framework to explain the drastic differences in affinity of the OpuAC/GB and OpuAC/PB complexes. A sequence comparison with putative SBP specific for compatible solutes reveals the presence of three distinct families for which the crystal structure of OpuAC might serve as a suitable template to predict the structures of these putative compatible solute-binding proteins.     

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.     

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.     

Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12

The proU locus of Escherichia coli encodes a high-affinity, binding-protein-dependent transport system (ProU) for the osmoprotectant glycine betaine. We cloned this locus into both low-copy-number lambda vectors and multicopy plasmids and demonstrated that these clones restore osmotically controlled synthesis of the periplasmic glycine betaine binding protein (GBBP) and the transport of glycine betaine in a delta (proU) strain. These clones allowed us to investigate the influence of osmolarity on ProU transport activity independent of the osmotically controlled expression of proU. ProU activity was strongly stimulated by a moderate increase in osmolarity and was partially inhibited by high osmolarity. This activity profile differs from the profile of the osmotically regulated proU expression. The proU locus is organized in an operon and the position of the structural gene (proV) for GBBP is defined using a minicell system. We determined that at least three proteins (in addition to GBBP) are encoded by the proU locus. We also investigated the permeation of glycine betaine across the outer membrane. At low substrate concentration (0.7 microM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins.     

Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli.

Glycine betaine and its precursors choline and glycine betaine aldehyde have been found to confer a high level of osmotic tolerance when added exogenously to cultures of Escherichia coli at an inhibitory osmotic strength. In this paper, the following findings are described. Choline works as an osmoprotectant only under aerobic conditions, whereas glycine betaine aldehyde and glycine betaine function both aerobically and anaerobically. No endogenous glycine betaine accumulation was detectable in osmotically stressed cells grown in the absence of the osmoprotectant itself or the precursors. A membrane-bound, O2-dependent, and electron transfer-linked dehydrogenase was found which oxidized choline to glycine betaine aldehyde and aldehyde to glycine betaine at nearly the same rate. It displayed Michaelis-Menten kinetics; the apparent Km values for choline and glycine betaine aldehyde were 1.5 and 1.6 mM, respectively. Also, a soluble, NAD-dependent dehydrogenase oxidized glycine betaine aldehyde. It displayed Michaelis-Menten kinetics; the apparent Km values for the aldehyde, NAD, and NADP were 0.13, 0.06, and 0.5 mM, respectively. The choline-glycine betaine pathway was osmotically regulated, i.e., full enzymic activities were found only in cells grown aerobically in choline-containing medium at an elevated osmotic strength. Chloramphenicol inhibited the formation of the pathway in osmotically stressed cells.     

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.