Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Growth, pectate lyase production and solute accumulation by Erwinia chrysanthemi under osmotic stress: effect of osmoprotectants

Glycine betaine strongly stimulated the growth rate of five strains of Erwinia chrysanthemi when grown in a synthetic medium at 0·986, 0·983 and 0·980 a _w (NaCl) whereas in four strains, little effect was observed compared with the control. Proline, dimethyl glycine, carnitine and pipecolic acid also actedas osmoprotectants. Glutamate and trehalose, commonly accumulated by enteric bacteria in response to osmotic stress, failed to act as osmoprotectants when supplied exogenously. Glycine betaine and pipecolic acid partially overcame the inhibition of pectate lyase release by NaCl in strain ECC. ¹³C NMR spectroscopy of two osmotically-stressed strains showed that glycine betaine was accumulated intracellularly from synthetic media containing the exogenous osmoprotectant. However, both strains also synthesized and accumulated trehalose in addition to glycine betaine in response to osmotic stress in complex media containing glycine betaine.     

Calcium Requirement for Fast Axonal Transport in Frog Motoneurons

Abstract: Calcium is required to sustain fast axonal transport in sensory neurons of frog and cat. We studied the Ca²⁺ dependence of fast axonal transport in the motoneurons of the lower spinal cord from frog. The accumulation of acetylcholinesterase at a crush on the ventral roots was used to follow axonal transport. Two types of experiments were performed: modification of the medium bathing the ventral roots, alone, and modification of the medium bathing the spinal cord and ventral roots. Incubation (17-18 h) of the ventral roots in Ca²⁺-free medium markedly inhibited acetylcholinesterase transport, a finding that demonstrates a Ca²⁺ requirement for fast axonal transport in motoneurons; when 4 m M MgCl₂ was added to the Ca²⁺-free medium, transport was also greatly reduced. During incubation of the ventral roots in normal medium supplemented with 0.18 m M CoCl₂ transport proceeded normally; but when the Co²⁺ concentration was raised to 1.8 m M , transport was diminished as drastically as in the Ca²⁺-free medium. Incubation of the spinal cord and ventral roots in medium containing 0.18 m M CoCl₂ did not reduce the accumulation of acetylcholinesterase at the crush. Similarly, accumulation of acetylcholinesterase at a crush on the dorsal root was not significantly reduced by exposure of the dorsal root ganglion and root to 0.18 m M Co²⁺. Exposure of sensory cell bodies to 0.18 m M Co₂₊ thus produces differential effects on transport of acetylcholinesterase and on transport of newly synthesized radiolabeled protein.     

Adaptation of Escherichia coli to high osmolarity environments: Osmoregulation of the high-affinity glycine betaine transport system ProU

Abstract: A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Fscherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor. Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor. The physiological reaction of the cell to the changing environmental condition is a highly coordinated process. Loss of turgor triggers a rapid influx of K⁺ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate. The increased intracellular concentration of K⁺-glutamate allows the adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, K⁺-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K⁺ ions with compounds that are less d eleterious for the cell's physiology. These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine. One of the most important compatible solutes for bacteria is glycine betaine. This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline. In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes.     

A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: Nε-acetyl-β-lysine can be substituted by glutamate and alanine

N^ε-acetyl-β-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase ( ablA ) and the β-lysine acetyltransferase ( ablB ) abolished the production of N^ε-acetyl-β-lysine in Methanosarcina mazei Gö1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Gö1 N^ε-acetyl-β-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to α-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 ± 0.05 μmol (mg protein)⁻¹ representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Gö1.     

Osmoadaptation of Thiocapsa roseopersicina OP-1 in batch and continuous culture: Accumulation of K+ and sucrose in response to osmotic stress

Abstract Increasing growth medium NaCl concentration inhibited the growth of Thiocapsa roseopersicina OP-1 due to both an increase in the lag phase of the growth cycle and a reduction in specific growth rate. Addition of 0.05% w/v acetate to the growth medium stimulated growth at all NaCl concentrations, but this stimulation was greatest at supra-optimal NaCl concentrations. Optimal growth under all conditions tested in both batch and continuous culture was recorded at a salt concentration of 0.3 M NaCl. The intracellular concentrations of both K⁺ and sucrose increased linearly with increasing growth medium NaCl concentration indicating as osmoregulatory role for these solutes. Time courses of osmoadaptation in batch culture demonstrated a biphasic response to osmotic stress. The initial phase consisted of a rapid accumulation (within 30 min) of K⁺ from the growth medium. This was followed by a slower synthesis of sucrose which partially replaced intracellular K⁺ during the second phase of osmoadaptation.     

Influence of exogenously supplied potassium and sodium salts on the abscisic acid-induced proline accumulation in barley leaf segments

A stimulation of the abscisic acid (ABA)-induced increase in proline was observed in leaf segments of barley ( Hordeum vulgare L. cv. Georgie) if K⁺ or Na⁺ were supplied in the external medium as salts of monovalent anions such as NO₃, Br, Cr and I, but not when sulphate or phosphate were used. To a lesser extent, the effect was evident also with RbCl, but it did not occur when chlorides of Li⁺. Cs⁺, NH₄⁺, Mg:⁺ and Ca²⁺ were used. Both KC1 and NaCl in the concentration range 2–100 m M influence the ABA-dependent proline accumulation to the same extent; the increase induced was about 100% at 10 m M , and reached a maximum between 60 and 100 m M. The effect is not due to the osmotic activity of the salts and does not seem to depend on changes in K⁺ and Na⁺ levels within the leaf tissue, but it is somehow linked to their external concentration. The existence of a specific interaction between ABA and K⁺ or Na⁺, possibly at the cell membrane level, is proposed.     

A comparative study of the effects of NaCl salinity on respiration, photosynthesis, and leaf extension growth in Beta vulgaris L. (sugar beet)

Abstract. Three parameters influencing the capacity for carbon accumulation, i.e. photosynthesis, respiration, and leaf extension growth, were studied in Beta vulgaris L. (sugar beet) cultured in nutrient solution containing 0.5 to 500 mol m⁻³ NaCl. Leaf extension growth was the parameter most sensitive to salinity: the initial rate of leaf extension and final leaf length each declined linearly with increase in external NaCl concentration. Photosynthetic O₂ evolution of thin leaf slices did not decline until salinity levels reached 350 to 500 mol m⁻³ NaCl, while respiratory O₂ consumption was not affected by salinity throughout the range. The results suggest that the influence of salinity on the capacity for carbon accumulation in B. vulgaris occurs primarily through reduction in the area of photosynthetic surface.     

Effects of copper on active and passive Rb+ influx in roots of winter wheat

The effects of copper (CuCl₂) on active and passive Rb⁺(⁸⁶Rb⁺) influx in roots of winter wheat grown in water culture for 1 week were studied. External copper concentrations in the range of 10–500 μ M in the uptake nutrient solution reduced active Rb⁺ influx by 20–70%, while passive influx was unaffected (ca 10% of the Rb⁺ influx in the Cu-free solution). At external Rb⁺ concentrations of up to 1 m M , Cu exposure (50 μ M decreased V_max to less than half and increased K_m to twice the value of the control. Short Cu exposure reduced the K⁺ concentration in roots of low K⁺ status. Pretreatment for 5 min in 50 μ M CuCl₂ prior to uptake experiments reduced Rb⁺ influx by 26%. After 60 min pretreatment with Cu, the corresponding reduction was 63%. Cu in the cultivation solution impeded growth, especially of the roots. The Cu concentration in the roots increased linearly with external Cu concentration (0–100 μ M ) while Cu concentration in the shoots was relatively unchanged. The K⁺ concentration in both roots and shoots decreased significantly with increased Cu in the cultivation solutions. Possible effects of Cu on membranes and ion transport mechanisms are discussed.     

Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.     

13C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Bacterium Ba1 and Vibro costicola

The synthesis and uptake of intracellular organic osmolytes (compatible solutes) were studied with the aid of natural abundance 13C NMR spectroscopy in two unrelated, moderately halophilic eubacteria: Ba1 and Vibrio costicola. In minimal media containing 1 M NaCl, both microorganisms synthesized the cyclic amino acid, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (trivial name, ectoine) as the predominant compatible solute, provided that no glycine betaine was present in the growth medium. When, however, the minimal medium was supplemented with glycine betaine or the latter was a component of a complex medium, it was transported into the cells and the accumulating glycine betaine replaced the ectoine. In Ba1, grown in a defined medium containing glucose as the single carbon source, ectoine could only be detected if the NaCl concentration in the medium was higher than 0.6 M; the ectoine content increased with the external salt concentration. At NaCl concentrations below 0.6 M, alpha,alpha-trehalose was the major organic osmolyte. The concentration of ectoine reached its peak during the exponential phase and declined subsequently. In contrast, the accumulation of glycine betaine continued during the stationary phase. The results presented here indicate that, at least in the two microorganisms studied, ectoine plays an important role in haloadaptation.     

K+ status and ABA affect both exudation rate and hydraulic conductivity in sunflower roots

Twenty‐day‐old sunflower plants ( Helianthus annuus L. cv. Sun‐Gro 380) grown in nutrient solutions with different KCl levels were used to study the effects of K⁺ status of the root and of abcisic acid (ABA) on the exudation rate (J_v), the hydraulic conductivity of the root (L_p), the fluxes of exuded K⁺ and Na⁺ (J_K and J_Na), and the gradient of osmotic pressure between the xylem and the external medium. J_v and L_p increased in direct proportion to the K⁺ starvation of the root. Also addition of ABA (4 µ M ) at the onset of exudation in the external medium made J_v and L_p rise, and this effect also increased with the degree of K⁺ starvation. Similarly, K⁺ starvation and ABA promoted both the flux of exuded Na⁺ and the accumulation of Na⁺ in the root. We suggest that ABA acts as a regulating signal for the radial transport of water across the root, and that potassium may be an effector of this mechanism.     

Potassium requirement of pyruvate kinase extracted from leaves of halophytes

Pyruvate kinase enzymes were partially purified from leaves of halophytes, Atriplex gmelini C. A. Mey., Chenopodium acuminatum Wild, and Spergularia salina J. et C. Presl., grown hydroponically in the presence of 250 m M NaCl in a greenhouse, to determine their K_m values for potassium. The values were all ca 10⁻³ M , as also reported for the glycophyte enzymes. However, the K_m values were reduced by 60 to 70% by the addition of betaine to a concentration of 1 M .     

Potassium-Stimulated Release of Radiolabelled Taurine and Glycine from the Isolated Rat Retina

Abstract: The release of preloaded [³H]glycine and [³H]taurine in response to a depolarising stimulus (12.5-50 m M KCl) has been studied in the superfused rat retina. High external potassium concentration immediately increased the spontaneous efflux of [3H]glycine, the effect of 50 m M K⁺ apparently being abolished by omitting calcium from the superfusing medium. In contrast, although high potassium concentrations increased the spontaneous emux of [³H]taurine from the superfused rat retina, this release was not evident until the depolarising stimulus was removed from the superfusing medium. The magnitude of this "late" release of [³H]taurine was dependent on external K⁺ concentrations, and appeared immediately after cessation of the stimulus irrespective of whether it was applied for 4, 8, or 12 min. Potassium (50 m M )-induced release of taurine appeared partially calcium-dependent, being significantly reduced (p < 0.01) but not abolished by replacing calcium with 1 mM EDTA in the superfusate. High-affinity uptake systems for both [³H]glycine and [³H]taurine were demonstrated in the rat retina in vitro ( K _m values, 1.67 μ M and 2.97 μ M ; V_max values, 19.3 and 23.1 nmol/g wet weight tissue/h, respectively). The results are discussed with respect to the possible neuro-transmitter roles of both amino acids in the rat retina.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila

Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T. halophila.     

Mechanisms of Sodium Transport at the Blood-Brain Barrier Studied with In Situ Perfusion of Rat Brain

Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V _max of 50.1 nmol/g/min and a K _m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na⁺/H⁺ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na⁺-K⁺-2Cl⁻/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co ₂ of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na⁺/H⁺ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl⁻/HCO₃⁻ exchange, lead to net NaCl secretion and obligate water transport into the brain.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Ectoine as the predominant osmolyte in the marine bacterium Marinobacter hydrocarbonoclasticus grown on eicosane at high salinities

L. FERNANDEZ-LINARES, R. FAURE, J.-C. BERTRAND AND M. GAUTHIER. 1996. Ectoine was detected by ¹³C-nuclear magnetic resonance spectroscopy in extracts of the halophilic marine bacterium Marinobacter hydrocarbonoclasticus grown at different NaCl concentrations (0.6, 1.5, 2.5 mol l^-1) in synthetic seawater on eicosane as the sole source of carbon and energy. Ectoine was synthesized as the principal osmolyte in response to stress. However, when the medium was supplemented with glycine betaine, this osmolyte was transported into the cells and replaced ectoine.