Glycine betaine transport in Escherichia coli: osmotic modulation.

Exogenous glycine betaine highly stimulates the growth rate of various members of the Enterobacteriaceae, including Escherichia coli, in media with high salt concentrations (D. Le Rudulier and L. Bouillard, Appl. Environ. Microbiol. 46:152-159, 1983). In a nitrogen- and carbon-free medium, glycine betaine did not support the growth of E. coli either on low-salt or high-salt media. This molecule was taken up by the cells but was not catabolized. High levels of glycine betaine transport occurred when the cells were grown in media of elevated osmotic strength, whereas relatively low activity was found when the cells were grown in minimal medium. A variety of electrolytes, such as NaCl, KCl, NaH2PO4, K2HPO4, K2SO4, and nonelectrolytes like sucrose, raffinose, and inositol triggered the uptake of glycine betaine. Furthermore, in cells subjected to a sudden osmotic upshock, glycine betaine uptake showed a sixfold stimulation 30 min after the addition of NaCl. Part of this stimulation might be a consequence of protein synthesis. The transport of glycine betaine was energy dependent and occurred against a concentration gradient. 2,4-Dinitrophenol almost totally abolished the glycine betaine uptake. Azide and arsenate exerted only a small inhibition. In addition, N,N'-dicyclohexylcarbodiimide had a very low inhibitory effect at 1 mM. These results indicated that glycine betaine transport is driven by the electrochemical proton gradient. The kinetics of glycine betaine entry followed the Michaelis-Menten relationship, yielding a Km of 35 microM and a Vmax of 42 nmol min-1 mg of protein-1. Glycine betaine transport showed considerable structural specificity. The only potent competitor was proline betaine when added to the assay mixtures at 20-fold the glycine betaine concentration. From these results, it is proposed that E. coli possesses an active and specific glycine betaine transport system which is regulated by the osmotic strength of the growth medium.     

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.     

Evidence that Escherichia coli accumulates glycine betaine from marine sediments

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

Evidence that Escherichia coli accumulates glycine betaine from marine sediments.

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti.

Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.     

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

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.     

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.     

Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin

Abstract The addition of 1 mM glycine betaine to the growth medium of Chromatium sp. NCIMB 8379 relieved growth inhibition caused by exposure to supra-optimal Nad concentrations. Intracellular glycine betaine concentrations were dependent upon the NaCl concentration of the growth medium up to 3 M exogenous Nad. Kinetic data for the accumulation of [methyl-¹⁴C]-glycine betaine demonstrated that Chromatium sp. NCIMB 8379 possesses a constitutively expressed active transport system for glycine betaine. The transport system was saturable with respect to glycine betaine concentration and exhibited typical Michaelis-Menten type kinetics: K _m= 24 μ M, V _max= 306 nmol min⁻¹ mg protein⁻¹ at an external NaCl concentration of 1 M. The rate of glycine betaine transport decreased progressively with increasing growth medium NaCl concentration. This transport system may represent an adaptive response to growth in high osmolarity environments in this halotolerant isolate, allowing accumulation of glycine betaine from the external cell environment or recycling synthesised glycine betaine which has passively diffused from the cell.     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

Salt adaptation in <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis>: identification and characterization of a secondary glycine betaine transporter

Members of the genus Acinetobacter are well known for their metabolic versatility that allows them to adapt to different ecological niches. Here, we have addressed how the model strain Acinetobacter baylyi copes with different salinities and low water activities. A. baylyi tolerates up to 900 mM sodium salts and even higher concentrations of potassium chloride. Growth at high salinities was better in complex than in mineral medium and addition of glycine betaine stimulated growth at high salinities in mineral medium. Cells grown at high salinities took up glycine betaine from the medium. Uptake of glycine betaine was energy dependent and dependent on a salinity gradient across the membrane. Inspection of the genome sequence revealed two potential candidates for glycine betaine transport, both encoding potential secondary transporters, one of the major facilitator superfamily (MFS) class (ACIAD2280) and one of the betaine/choline/carnitine transporter (BCCT) family (ACIAD3460). The latter is essential for glycine betaine transport in A. baylyi. The broad distribution of ACIAD3460 homologues indicates the essential role of secondary transporters in the adaptation of Acinetobacter species to osmotic stress.     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-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.     

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, identified as a compatible solute in moderately halophilic bacteria. Exogenously provided ectoine was found to stimulate growth of Escherichia coli in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte or nonelectrolyte. It is accumulated in E. coli cells proportionally to the osmotic strength of the medium, and it is not metabolized. Its osmoprotective ability was as potent as that of glycine betaine. The ProP and ProU systems are both involved in ectoine uptake and accumulation in E. coli. ProP being the main system for ectoine transport. The intracellular ectoine pool is regulated by both influx and efflux systems.