Glycine betaine and potassium ion are the major compatible solutes in the extremely halophilic methanogen Methanohalophilus strain Z7302.

Methanohalophilus strain Z7302 was previously isolated from a hypersaline environment and grows over a range of NaCl concentrations from 1.7 to 4.4 M. We examined the relationships between cell growth rate, cell volume, and intracellular solute concentrations with increasing salinity. This extremely halophilic methanogen synthesized three zwitterionic compounds, beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine, and also accumulated potassium ion as compatible solutes to balance the external and internal osmotic pressures. Potassium and glycine betaine were the predominant compatible solutes when Methanohalophilus strain Z7302 was grown at high external NaCl concentrations and approached intracellular levels of 3 and 4 M, respectively.     

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.     

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.     

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.     

Free amino acid dynamics in marine methanogens. beta-Amino acids as compatible solutes.

Methanogenic archaebacteria respond to osmotic stress by accumulating a series of organic molecules which function as compatible solutes. In two strains of marine methanogenic archaebacteria, Methanogenium cariaci and Methanococcus thermolithotrophicus, four key organic solutes are observed: L-alpha-glutamate, beta-glutamate, N epsilon-acetyl-beta-lysine, and betaine. The first three of these are synthesized de novo; betaine is transported into the Mg. cariaci cells from the medium. Mesophilic Mg. cariaci will preferentially transport betaine from the extracellular medium if it is present to counterbalance the external NaCl. In its absence it synthesizes N epsilon-acetyl-beta-lysine as the dominant osmolyte. This zwitterionic compound occurs at levels in Mg. cariaci which are considerably greater (based on mumol/mg of protein) than in Mc. thermolithotrophicus grown in media of the same ionic strength. Intracellular potassium ion concentrations, determined by 39K NMR spectroscopy and atomic absorption, differ significantly in the two cells. In Mc. thermolithotrophicus, intracellular K+ is balanced by the total concentration of anionic amino acid species, glutamate, and beta-glutamate. Turnover of the organic solutes has been monitored using 13C-pulse/12C-chase, and 15N-pulse/14N-chase experiments. Both beta-amino acids exhibit slower turnover rates when compared to L-alpha-glutamate or aspartate, consistent with their roles as compatible solutes. Biosynthetic information for the beta-amino acids is also provided by 13C-labeling experiments. beta-Glutamate shows a lag in 13C uptake from 13CO2, indicative of its biosynthesis from a precursor (probably a macromolecule) not in equilibrium with the soluble L-alpha-glutamate pool. Confirmation of a novel route for beta-glutamate synthesis and the production of the beta-lysine moiety from the diaminopimelate pathway is deduced from [13C2]acetate labeling patterns.     

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.     

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

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.     

Metabolism of chloride in halophilic prokaryotes

While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions. Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin. Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance. However, some species show a specific requirement for chloride. In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent. In this organism the expression of a large number of proteins is chloride regulated. Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride. A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber. It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms.     

相容性溶质四氢嘧啶及其羟基化衍生物的研究进展

四氢嘧啶类化合物是嗜盐以及耐盐菌胞内合成的一类能够抵御外界高盐胁迫的相容性溶质,概述了四氢嘧啶及其衍生物的理化特征以及在嗜盐微生物中抵御外界高渗透压的作用机理,主要阐述了四氢嘧啶类相容性溶质的生物合成途径、膜运输机理、分泌释放机制、高密度发酵生产等方面在细胞、分子水平上的最新研究进展以及前景展望。并且综述了四氢嘧啶类在精细化工、生物医药及生物制造等行业的应用研究以及发展前景,探讨了未来的研究方向。     

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.     

Organic osmolytes in methanogenic archaebacteria

Methanogenic archaebacteria have developed unique ways of dealing with osmotic stress. While several of them have transport systems capable of internalizing betaine, an osmolyte in many eubacteria, in general they have developed de novo synthesis of a novel series of beta-amino acids as compatible solutes. 13C-NMR spectroscopy has been the key tool in elucidating both the identity of these organic osmolytes and in investigating their dynamics.     

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.     

Transport of Compatible Solutes in Extremophiles

Salt-tolerant as well as moderately halophilic and halophilic organisms have to maintain their turgor. One strategy is to accumulate small organic compounds, compatible solutes, by de novo synthesis or uptake. From a bioenergetic point of view, uptake is preferred over biosynthesis. The transport systems catalyzing uptake of compatible solutes are of primary or secondary nature and coupled to ATP hydrolysis or ion (H+, Na+) symport. Expression of the transporter genes as well as the activity of the transporters is regulated by salinity/osmolarity and one of the key questions is how salinity or osmolarity is sensed and the signal transmitted as far as to gene expression and transporter activation. Recent studies shed light on the nature and the activation mechanisms of solute transporters in extremophiles, and this review summarizes current knowledge on the structure, function and osmo- or salt-regulation of transporters for compatible solutes in extremophiles.     

Glycine betaine is the main organic osmotic solute in a stratified microbial community in a hypersaline evaporitic gypsum crust

We have examined the organic osmotic solutes content within the stratified microbial communities in an evaporitic gypsum crust found in an evaporation pond (~194 g/l total dissolved salts) of the salterns of the Israel Salt Company, Eilat. We extracted intracellular solutes from the upper three pigmented layers of the crust: a yellow-orange layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, and a layer colored red-purple by purple sulfur bacteria; dense communities of heterotrophic bacteria were present in all layers. The solutes were analyzed by Raman spectroscopy, ¹H and ¹³C nuclear magnetic resonance, and HPLC. All layers contained glycine betaine as the only detectable osmotic solute; ectoine and other solutes known to be produced by many halophilic and halotolerant prokaryotes were not found. In this first attempt to assess the osmotic solute content within complex natural communities of halophilic microorganisms, the predominant role of glycine betaine as an osmolyte was established. Most heterotrophic bacteria cannot produce glycine betaine but preferentially use it when it is supplied. Presence of glycine betaine produced by the photoautotrophic members of the community, therefore, may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost.     

Biosynthesis and fate of compatible solutes in extremely halophilic phototrophic eubacteria

Abstract Most halophilic and halotolerant eubacteria are able to accumulate compatible solutes from their environment during salt stress. They are usually able to synthesize sugars and amino acids. These compatible solutes, however, are of less importance in extremely halophilic eubacteria, where usually glycinebetaine or ectoine is required. Extremely halophilic phototrophic sulfur bacteria of the genus Ectothiorhodospira are able to synthesize the three compatible solutes glycinebetaine, trehalose and ectoine. While glycinebetaine is the major compatible solute under all conditions the percentage of trehalose and ectoine varies depending on the availability of nitrogen sources. Using acetate plus bicarbonate as simultaneous substrates the three compatible solutes were analysed by ¹³C-NMR spectroscopy. The label found indicated the following biosynthesis pathways: glycine, derived from glyoxylate out of the Kornberg cycle, undergoes a three-fold methylation with S-adenosylmethionine as methyl donor. The latter is derived from the tetrahydrofolate pathway. Several enzymes of this pathway have been found and are under investigation. The labelling of trehalose indicates that the Calvin cycle is blocked in the presence of acetate. The two glucose moieties of trehalose are linked by trehalose-6-phosphate synthase. The enzyme was characterized. Ectoine is synthesized from aspartate via aspartophosphate, aspartate semialdehyde and α, β-diaminobutyrate. Dilution stress leads to rapid excretion of betaine and ectoine, followed by immediate uptake to balance overshoot excretion. Trehalose is not excreted under dilution stress but is degraded by trehalase and subsequently metabolized.     

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.     

Lysis of Halobacteria in Bacto-Peptone by Bile Acids

All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.     

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.     

Adaptation of anaerobic biomass to saline conditions: Role of compatible solutes and extracellular polysaccharides

This study investigated the role of compatible solutes, extracellular polysaccharides (EPS), and nutrients on anaerobic biomass when stressed with salinity. When 1 mM of osmoregulants glycine betaine, α-glutamate and β-glutamate were added separately to serum bottles containing biomass not adapted to sodium, and fed with glucose and 35 g NaCl/L, all the compatible solutes were found to alleviate sodium inhibition, although glycine betaine was found to be the most effective. The effect of glycine betaine on different anaerobic bacterial groups under salinity stress was monitored using VFAs, and showed that methanogens were more protected than propionate utilisers. Moreover, the addition of 1 mM of glycine betaine to anaerobic biomass not adapted to salinity resulted in significantly higher methane production rates compared with anaerobic biomass that was exposed for 4 weeks to 35 g NaCl/L. Interestingly, under saline batch conditions when the medium was replaced totally the culture produced less methane than when only new substrate was added due to compatible solutes cycling between the media and the cell. The elimination of macronutrients from the medium was found to have a more pronounced negative effect on biomass under saline compared with nonsaline conditions, and because of the synthesis of N-compatible solutes sufficient nutrients should always be present. On the other hand, the absence from the medium of micronutrients did not further reduce biomass activity under salinity. Finally, a higher production of EPS was obtained from biomass exposed to higher salt concentrations, and its composition was found to change under different saline conditions and time. As a result, biomass under saline conditions had a slightly higher mean flock size compared with the biomass that was not subjected to salt.