Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.     

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.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria

The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria.

The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells.     

Mitochondrial mediation of environmental osmolytes discrimination during osmoadaptation in the extremely halotolerant black yeast Hortaea werneckii

We have investigated the mitochondrial responses to hyperosmotic environments of ionic (4.5 M NaCl) and non-ionic (3.0 M sorbitol) osmolytes in the most halo/osmo-tolerant black yeast, Hortaea werneckii. Adaptation to both types of osmolytes resulted in differential expression of mitochondria-related genes. Live-cell imaging has revealed a condensation of mitochondria in hyperosmotic media that depends on osmolyte type. In the hypersaline medium, this was accompanied by increased ATP synthesis and oxidative damage protection, whereas adaptation to the non-ionic osmolyte resulted in a decrease in ATP synthesis and lipid peroxidation level in mitochondria. A proteomic study of the mitochondria revealed preferential accumulation of energy metabolism enzymes in the hypersaline medium, and accumulation of protein chaperones in the non-ionic osmolyte. The HwBmh1/14-3-3 protein, localized to mitochondria in hypersaline conditions, and not at optimal salinity, suggesting its role in differential perception of ionic and non-ionic osmolytes in H. werneckii.     

Novel insights into the role of potassium for osmoregulation in Halomonas elongata

The role of K(+) in osmoregulation of the halophilic bacterium Halomonas elongata was investigated. At lower salinities (0.51 M NaCl), K(+) was the predominant cytoplasmic solute (1.25 micro mol mg protein(-1)). At higher salinities (1.03 M NaCl) ectoine became the main cytoplasmic solute (1.57 micro mol mg protein(-1)), while the K(+) content remained unchanged. In response to osmotic upshock, cells of H. elongata simultaneously accumulated ectoine and K(+) glutamate. The ectoine and K(+) glutamate levels in osmotically stressed cells exceeded the level of cells adapted to high salinities. The increase in K(+) glutamate was long lasting (>120 min) and not transient, as described for non-halophiles. Regulation of the synthesis of ectoine and glutamate was proven to occur mainly at the level of enzyme activity. Limitation of K(+) inhibited the growth of salt-adapted H. elongata cells, especially at high salinities, and caused a decrease of the intracellular organic solute content, inhibition of respiration, and an abolition of the cell's ability to respond to osmotic stress. The saturation constant K(S) for K(+) was estimated to be 105 micro M at a salinity of 0.51 M NaCl, indicating that an uptake system of medium affinity is responsible for K(+) accumulation in H. elongata.     

Lysine-2,3-aminomutase and beta-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of Nepsilon-acetyl-beta-lysine and growth at high salinity

The compatible solute N(epsilon)-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N(epsilon)-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N(epsilon)-acetyl-beta-lysine formation from alpha-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei G?1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei G?1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Delta(abl) mutants of M. maripaludis no longer produced N(epsilon)-acetyl-beta-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N(epsilon)-acetyl-beta-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.     

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.     

Osmolytes and membrane lipids in the adaptation of micromycete Emericellopsis alkalina to ambient pH and sodium chloride

The accumulation of low molecular weight cytoprotective compounds (osmolytes) and changes in the membrane lipids composition are of key importance for the adaptation to stress impacts. However, the reason behind the wide variety of osmolytes present in the cell remains unclear. We suggest that specific functions of osmolytes can be revealed by studying the adaptation mechanisms of the mycelial fungus Emericellopsis alkalina (Hypocreales, Ascomycota) that is resistant to both alkaline pH values and high sodium chloride concentrations. It has been established that the fungus uses different osmolytes to adapt to ambient pH and NaCl concentration. Arabitol was predominant osmolyte in alkaline conditions, while mannitol prevailed in acidic conditions. On the salt-free medium mannitol was the main osmolyte; under optimal conditions (pH 10.2; 0.4 M NaCl) arabitol and mannitol were both predominant. Higher NaCl concentrations (1.0–1.5 M) resulted in the accumulation of low molecular weight polyol - erythritol, which amounted up to 12–14%, w/w. On the contrary, changes in the composition of membrane lipids were limited under pH and NaCl impacts; only higher NaCl concentrations led to the increase in the degree of unsaturation of membrane lipids. Results obtained indicated the key role of the osmolytes in the adaptation to the ambient pH and osmotic impacts.     

Changes in major intracellular osmolytes in L-929 cells following rapid and slow application of hyperosmotic media

Cultured L-929 cells respond to media-made hyperosmotic (600 mOsmol/kg H2O) by addition of NaCl, sorbitol or proline by adjusting successively their intracellular level in different osmolytes: Na+, K+, amino acids and sorbitol. In the NaCl medium, Na+ and K+ are first to increase. Their concentration is then down-regulated while they are replaced by less disrupting osmolytes: amino acids and sorbitol. The amino-acid level is also adjusted with respect to the increase in sorbitol which starts only after 24 h, depending on the induction of aldose reductase. A similar evolution in the amount of these osmolytes is observed, with different time scales and amplitudes, depending on whether the osmotic shocks are applied abruptly or slowly, in a more physiological way. The interplay between the osmolytes is also different depending on their availability in the external medium. Such complex evolutions indicate that a cascade of interacting signals must be considered to account for the overall regulation process. It can hardly be fitted into a model implicating a single primary signalling event (early increase in ions or decrease in cell volume) as usually postulated. Also, the volume up-regulation is not significantly different in the different conditions, showing that it is not primarily dependent on the adjustment of the intracellular osmolarity which is reached immediately upon cell shrinkage and is maintained all over, independently of the availability and changes in nature of the osmolytes.     

Responses of photosynthetic apparatus of the halotolerant microalga <Emphasis Type="Italic">Dunalliella maritima</Emphasis> to hyperosmotic salt shock

Chlorophyll fluorescence induction curves were used as a means to assess the functional condition of the photosynthetic apparatus in cells of the halotolerant green microalga Dunaliella maritima (Massjuk) (division Chlorophyta) exposed to hyperosmotic salt shock of various intensities. The shock was caused by the transfer of algal cells grown in the medium with 0.5 M NaCl to the media with elevated NaCl concentrations (1.0, 1.5, and 2.0 M). Parameters of chlorophyll fluorescence (F ₀, F _m, F ₀′, F _t′) were measured by means of a specialized pulse-amplitude-modulation fluorometer PAM 2100. In addition, the rate of photosynthetic oxygen evolution as well as the intracellular Na⁺ and glycerol content (the main osmolyte in this microalga) were determined. The hyperosmotic salt shock was found to elevate the intracellular Na+ content and reduce the functional activity of PSII in D. maritima. The suppression of PSII activity was evident from the decrease in the maximal quantum yield of photochemical energy conversion in PSII, the decreased rate of linear electron transport, the increased reduction of the primary acceptor Q_A, and the suppression of photosynthetic O₂ evolution. The functional activity of PSII recovered gradually along with restoration of osmotic and ionic balance in algal cells. It is proposed that PSI ensures energy supply during cell responses of D. maritima to hyperosmotic salt shock.     

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.     

Turgor regulation in a novel Halomonas species

The mechanisms by which a novel eubacterium, identified as belonging to the genus Halomonas, adapted to increases in the extracellular osmotic potential were investigated. It was shown that the ability of the bacterium to grow after hyperosmotic shock was dependent on the presence of potassium ions. Growth of the bacterium in 2 M NaCl medium could be limited by low concentrations of K⁺ and this enabled the affinity for K⁺ to be determined (K _s=21.5 M). Rubidium salts could be substituted for those of potassium, but the lowest concentration of Rb⁺ that allowed growth in 2 M NaCl medium was 50-fold greater than the minimum concentration of K⁺. ¹³C-NMR spectroscopy and HPLC analysis were used to demonstrate the accumulation of organic solutes in the cytoplasm after exposure to high salinities. The major osmolyte was ectoine, but glutamate and ectoine hydroxide were also present. Addition of exogenous glycine betaine to 3.25 M NaCl medium resulted in the accumulation of high intracellular concentrations of glycine betaine in the bacterium. This reduced the level of ectoine accumulation but did not fully inhibit the synthesis of this compound in the cytoplasm.Abbreviation Specific growth rate (generations/h)     

The osmophobic effect: natural selection of a thermodynamic force in protein folding

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments and these osmolytes protect intracellular macromolecules against the denaturing environmental stress. In natural selection of organic osmolytes as protein stabilizers, it appears that the osmolyte property selected for is the unfavorable interaction between the osmolyte and the peptide backbone, a solvophobic thermodynamic force that we call the osmophobic effect. Because the peptide backbone is highly exposed to osmolyte in the denatured state, the osmophobic effect preferentially raises the free energy of the denatured state, shifting the equilibrium in favor of the native state. By focusing the solvophobic force on the denatured state, the native state is left free to function relatively unfettered by the presence of osmolyte. The osmophobic effect is a newly uncovered thermodynamic force in nature that complements the well-recognized hydrophobic interactions, hydrogen bonding, electrostatic and dispersion forces that drive protein folding. In organisms whose survival depends on the intracellular presence of osmolytes that can counteract denaturing stresses, the osmophobic effect is as fundamental to protein folding as these well-recognized forces.     

Living with urea stress

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments. These osmolytes protect intracellular macromolecules against denaturing environmental stress. In contrast to the usually benign effects of most organic osmolytes, the waste product urea is a well-known perturbant of macromolecules. Although urea is a perturbing solute which inhibits enzyme activity and stability, it is employed by some species as a major osmolyte. The answer to this paradox was believed to be the discovery of protective osmolytes (methylamines). We review the current state of knowledge on the various ways of counteracting the harmful effects of urea in nature and the mechanisms for this. This review ends with the mechanistic idea that cellular salt (KCl/NaCl) plays a crucial role in counteracting the effects of urea, either by inducing required chaperones or methylamines, or by thermodynamic interactions with urea-destabilised proteins. We also propose future opportunities and challenges in the field.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.