Transient Accumulation of Glycine Betaine and Dynamics of Endogenous Osmolytes in Salt-Stressed Cultures of Sinorhizobium meliloti

The fate of exogenously supplied glycine betaine and the dynamics of endogenous osmolytes were investigated throughout the growth cycle of salt-stressed cultures of strains of Sinorhizobium meliloti which differ in their ability to use glycine betaine as a growth substrate, but not as an osmoprotectant. We present (sup13)C nuclear magnetic resonance spectral and radiotracer evidence which demonstrates that glycine betaine is only transiently accumulated as a cytoplasmic osmolyte in young cultures of wild-type strains 102F34 and RCR2011. Specifically, these strains accumulate glycine betaine as a preferred osmolyte which virtually prevents the accumulation of endogenous osmolytes during the lag and early exponential phases of growth. Then, betaine levels in stressed cells decrease abruptly during the second half of the exponential phase. At this stage, the levels of glutamate and the dipeptide N-acetylglutaminylglutamine amide increase sharply so that the two endogenous solutes supplant glycine betaine in the ageing culture, in which it becomes a minor osmolyte because it is progressively catabolized. Ultimately, glycine betaine disappears when stressed cells reach the stationary phase. At this stage, wild-type strains of S. meliloti also accumulate the disaccharide trehalose as a third major endogenous osmolyte. By contrast, glycine betaine is always the dominant osmolyte and strongly suppresses the buildup of endogenous osmolytes at all stages of the growth cycle of a mutant strain, S. meliloti GMI766, which does not catabolize this exogenous osmoprotectant under any growth conditions.     

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic Methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance ¹³C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M. portucalensis.     

Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.     

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.     

Halotolerance in Methanosarcina spp.: Role of N(sup(epsilon))-Acetyl-(beta)-Lysine, (alpha)-Glutamate, Glycine Betaine, and K(sup+) as Compatible Solutes for Osmotic Adaptation

The methanogenic Archaea, like the Bacteria and Eucarya, possess several osmoregulatory strategies that enable them to adapt to osmotic changes in their environment. The physiological responses of Methanosarcina species to different osmotic pressures were studied in extracellular osmolalities ranging from 0.3 to 2.0 osmol/kg. Regardless of the isolation source, the maximum rate of growth for species from freshwater, sewage, and marine sources occurred in extracellular osmolalities between 0.62 and 1.0 osmol/kg and decreased to minimal detectable growth as the solute concentration approached 2.0 osmol/kg. The steady-state water-accessible volume of Methanosarcina thermophila showed a disproportionate decrease of 30% between 0.3 and 0.6 osmol/kg and then a linear decrease of 22% as the solute concentration in the media increased from 0.6 to 2.0 osmol/kg. The total intracellular K(sup+) ion concentration in M. thermophila increased from 0.12 to 0.5 mol/kg as the medium osmolality was raised from 0.3 to 1.0 osmol/kg and then remained above 0.4 mol/kg as extracellular osmolality was increased to 2.0 osmol/kg. Concurrent with K(sup+) accumulation, M. thermophila synthesized and accumulated (alpha)-glutamate as the predominant intracellular osmoprotectant in media containing up to 1.0 osmol of solute per kg. At medium osmolalities greater than 1.0 osmol/kg, the (alpha)-glutamate concentration leveled off and the zwitterionic (beta)-amino acid N(sup(epsilon))-acetyl-(beta)-lysine was synthesized, accumulating to an intracellular concentration exceeding 1.1 osmol/kg at an osmolality of 2.0 osmol/kg. When glycine betaine was added to culture medium, it caused partial repression of de novo (alpha)-glutamate and N(sup(epsilon))-acetyl-(beta)-lysine synthesis and was accumulated by the cell as the predominant compatible solute. The distribution and concentration of compatible solutes in eight strains representing five Methanosarcina spp. were similar to those found in M. thermophila grown in extracellular osmolalities of 0.3 and 2.0 osmol/kg. Results of this study demonstrate that the mechanism of halotolerance in Methanosarcina spp. involves the regulation of K(sup+), (alpha)-glutamate, N(sup(epsilon))-acetyl-(beta)-lysine, and glycine betaine accumulation in response to the osmotic effects of extracellular solute.     

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.     

Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary

Seasonal changes in the leaf concentration of compatible osmolytes were investigated in three halophytic species (Lepidium crassifolium, Camphorosma annua and Limonium gmelini subsp. hungaricum) native to a salty-sodic grassland. The investigated species were shown to accumulate both carbohydrate- and amino acid-derived osmolytes. The leaf tissues of C. annua (Chenopodiaceae) preferentially stored glycine betaine and pinitol, while in L. gmelini (Plumbaginaceae) beta-alanine betaine, choline-O-sulphate, and pinitol were accumulated. In the leaves of L. crassifolium (Brassicaceae) a very high amount of proline, associated with a high level of soluble carbohydrates was found. Not only the biochemical nature of the osmolyte, but also the seasonal pattern of osmolyte accumulation showed significant species-specific fluctuations. In addition, the cellular levels of the observed osmolytes changed with the growth period and according to the environmental parameters. The highest concentrations of osmolytes were found in March, when low temperatures, hypoxic conditions and high salt concentrations were the main constraints to plant growth. The high structural diversity of osmolytes combined with their multifunctionality and the seasonal flexibility of the metabolism in plants facing multiple stresses is discussed.     

The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving Photosystem II complex

Natural osmoregulatory substances (osmolytes) allow a wide variety of organisms to adjust to environments with high salt and/or low water content. In addition to their role in osmoregulation, some osmolytes protect proteins from denaturation and deactivation by, for example, elevated temperature and chaotropic compounds. A ubiquitous protein-stabilizing osmolyte is glycine betaine (N-trimethyl glycine). Its presence has been reported in bacteria, in particular cyanobacteria, in animals and in plants from higher plants to algae. In the present review we describe the experimental evidence related to the ability of glycine betaine to enhance and stabilize the oxygen-evolving activity of the Photosystem II protein complexes of higher plants and cyanobacteria. The osmolyte protects the Photosystem II complex against dissociation of the regulatory extrinsic proteins (the 18 kD, 23 kD and 33 kD proteins of higher plants and the 9 kD protein of cyanobacteria) from the intrinsic components of the Photosystem II complex, and it also stabilizes the coordination of the Mn cluster to the protein cleft. By contrast, glycine betaine has no stabilizing effect on partial photosynthetic processes that do not involve the oxygen-evolving site of the Photosystem II complex. It is suggested that glycine betaine might act, in part, as a solute that is excluded from charged surface domains of proteins and also as a contact solute at hydrophobic surface domains.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces.

Listeria monocytogenes is a food-borne pathogen that is widely distributed in nature and is found in many kinds of fresh and processed foods. The pervasiveness of this organism is due, in part, to its ability to tolerate environments with elevated osmolarity and reduced temperatures. Previously, we showed that L. monocytogenes adapts to osmotic and chill stress by transporting the osmolyte glycine betaine from the environment and accumulating it intracellularly (R. Ko, L. T. Smith, and G. M. Smith, J. Bacteriol. 176:426-431, 1994). In the present study, the influence of various environmental conditions on the accumulation of glycine betaine and another osmolyte, carnitine, was investigated. Carnitine was shown to confer both chill and osmotic tolerance to the pathogen but was less effective than glycine betaine. The absolute amount of each osmolyte accumulated by the cell was dependent on the temperature, the osmolarity of the medium, and the phase of growth of the culture. L. monocytogenes also accumulated high levels of osmolytes when grown on a variety of processed meats at reduced temperatures. However, the contribution of carnitine to the total intracellular osmolyte concentration was much greater in samples grown on meat than in those grown in liquid media. While the amount of each osmolyte in meat was less than 1 nmol/mg (fresh weight), the overall levels of osmolytes in L. monocytogenes grown on meat were about the same as those in liquid samples, from about 200 to 1,000 nmol/mg of cell protein for each osmolyte. This finding suggests that the accumulation of osmolytes is as important in the survival of L. monocytogenes in meat as it is in liquid media.     

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.     

An osmolyte mitigates the destabilizing effect of protein crowding

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.     

COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress

The mechanism by which COX2 inhibition decreases renal cell survival is poorly understood. In the present study we examined the effect of COX2 activity on organic osmolyte accumulation in renal medulla and in cultured mouse renal medullary interstitial cells (MMICs) and its role in facilitating cell survival. Hypertonicity increased accumulation of the organic osmolytes inositol, sorbitol, and betaine in cultured mouse medullary interstitial cells. Pretreatment of MMICs with a COX2-specific inhibitor (SC58236, 10 micromol/liter) dramatically reduced osmolyte accumulation (by 79 +/- 9, 57 +/- 12, and 96 +/- 10% for inositol, sorbitol, and betaine respectively, p < 0.05). Similarly, 24 h of dehydration increased inner medullary inositol, sorbitol, and betaine concentrations in vivo by 85 +/- 10, 197 +/- 28, and 190 +/- 24 pmol/microg of protein, respectively, but this increase was also blunted (by 100 +/- 5, 66 +/- 15, and 81 +/- 9% for inositol, sorbitol, and betaine, respectively, p < 0.05) by pretreatment with an oral COX2 inhibitor. Dehydrated COX2-/- mice also exhibited an impressive defect in sorbitol accumulation (88 +/- 9% less than wild type, p < 0.05) after dehydration. COX2 inhibition (COX2 inhibitor-treated or COX2-/- MMICs) dramatically reduced the expression of organic osmolyte uptake mechanisms including betaine (BGT1) and sodium-myo-inositol transporter and aldose reductase mRNA expression under hypertonic conditions. Importantly, preincubation of COX2 inhibitor-treated MMICs with organic osmolytes restored their ability to survive hypertonic stress. In conclusion, osmolyte accumulation in the kidney inner medulla is dependent on COX2 activity, and providing exogenous osmolytes reverses COX2-induced cell death. These findings may have implications for the pathogenesis of analgesic nephropathy.     

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.     

Composition, Variation, and Dynamics of Major Osmotic Solutes in Methanohalophilus Strain FDF1

Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, beta-glutamine, and N-acetyl-beta-lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as alpha-glucosylglycerate from the C and H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. CH(3)OH-pulse/CH(3)OH-chase experiments were used to determine half-lifes for these organic solute pools in the cells. l-alpha-Glutamate showed a rapid loss of heavy isotope, indicating that l-alpha-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both beta-glutamine, which acts as an osmolyte, and alpha-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and N-acetyl-beta-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.     

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D(9)-glycine betaine, D(9)-trimethylamine-N-oxide, (13)C(2)-arsenobetaine, D(6)-DMSP, and D(4)-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r(2) values=0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

Osmolytes as modulators of conformational changes in serpins.

Protein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) alpha1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and alpha1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine N-oxide, on conformational changes in alpha1-antitrypsin. All three solutes protected native alpha1-antitrypsin against thermally induced polymerisation and inactivation in a concentration-dependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of alpha1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, alpha1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native alpha1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by alpha1-antitrypsin are discussed.     

Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another

Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.