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.     

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.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.     

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.     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments

All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria. Received: 12 May 1998 / Accepted: 24 July 1998     

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.     

Microbial behaviour in salt-stressed ecosystems

Abstract: Salt stress is primarily osmotic stress, and halophilic/halotolerant microorganisms have evolved two basic mechanisms of osmoadaplation: the KCI-type and the compatible-solute type, the latter representing a very flexible mode of adaptation making use of distinct stabilizing properties of compatible solutes. A comprehensive survey, using HPLC and NMR methods, has revealed the full diversity of euhacterial compatible solutes found in nature. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. From our present knowledge of hiosynthetic pathways it appears that, apart from glycine betaine, all nitrogen-containing compatible solutes originate from two major pathways (the aspartate branch and the glutamate branch). Uptake of compatible solutes from the growth medium (environment) seems to have preference over de novo synthesis. Therefore in the natural ecosystem the solutes of primary producers (mainly glycine betaine), which are readily excreted upon dilution stress, certainly play an important role as a 'preferred' solute source for heterolrophic organisms, and as a 'vital' source for organisms unable to synthesize their own compatible solutes.     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

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.     

Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.     

The compatibility of osmotica in cyanobacteria

Abstract. The solutes accumulated by cyanobacteria in response to hyper-osmotic stress include Na⁺, K⁺, sucrose, trehalose, glucosyl-glycerol, glyeine betaine and glutamate betaine. The compatibility of several of these solutes with glutamine synthetase activity has been examined using cell-free extracts from a range of freshwater, marine and halotolerant cyanobacteria. All of the solutes tested were compatible with (i.e. non-inhibitory to) enzymic activity at physiological concentrations and the results demonstrate a rank order of compatibility which correlates with the concentrations at which the organic solutes occur in cyanobacteria, i.e. glycine betaine > polyol-derivatives > disaccharides and with the upper salinity limit for growth. The protection against inhibition by NaCl (halo-protection) afforded by these solutes to enzymic activity was also examined. Only glycine betaine was found to exert a significant halo-protective effect and this may be explained by differences in the mechanism of compatible solute function between small charged molecules and sugars/polyols.     

Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (K_m, 48 μM) to ectoine (K_m, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (K_m, 63 μM) to betaine (K_m, 333 μM) and proline (K_m, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

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.     

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.     

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Abstract Natural abundance ¹³C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.     

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (less than 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.