Distribution of genes for synthesis of trehalose and Mannosylglycerate in Thermus spp. and direct correlation of these genes with halotolerance

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.     

Mannosylglycerate is essential for osmotic adjustment in Thermus thermophilus strains HB27 and RQ-1

We disrupted the mpgS encoding mannosyl-3-phosphoglycerate synthase (MpgS) of Thermus thermophilus strains HB27 and RQ-1, by homologous recombination, to assess the role of the compatible solute mannosylglycerate (MG) in osmoadaptation of the mutants, to examine their ability to grow in NaCl-containing medium and to identify the intracellular organic solutes. Strain HB27 accumulated only MG when grown in defined medium containing 2% NaCl; mutant HB27M9 did not grow in the same medium containing more than 1% NaCl. When trehalose or MG was added, the mutant was able to grow up to 2% of NaCl and accumulated trehalose or MG, respectively, plus amino acids. T. thermophilus RQ-1 grew in medium containing up to 5% NaCl, accumulated trehalose and lower amounts of MG. Mutant RQ-1M1 lost the ability to grow in medium containing more than 3% NaCl and accumulated trehalose and moderate levels of amino acids. Exogenous MG did not improve the ability of the organism to grow above 3% NaCl, but caused a decrease in the levels of amino acids. Our results show that MG serves as a compatible solute primarily during osmoadaptation at low levels of NaCl while trehalose is primarily involved in osmoadaptation during growth at higher NaCl levels.     

Accumulation of Mannosylglycerate and Di-myo-Inositol-Phosphate by Pyrococcus furiosus in Response to Salinity and Temperature

(sup13)C and (sup1)H nuclear magnetic resonance spectroscopy was used to identify and quantify organic solutes accumulated by the hyperthermophilic archaeon Pyrococcus furiosus in response to temperature and salinity. Di-myo-inositol-phosphate and 2-O-(beta)-mannosylglycerate were the major organic solutes accumulated in these cells. The total intracellular organic solutes increased significantly in response either to an increase in temperature or to an increase in salinity, but (beta)-mannosylglycerate accumulated mainly at high salinities, whereas the concentration of di-myo-inositol-phosphate increased dramatically at supraoptimal growth temperatures. Glutamate was present at concentrations detectable by nuclear magnetic resonance only in cells grown in low-salinity media. The intracellular levels of K(sup+) are clearly dependent on the salinity of the medium, and the concentrations of this cation are high enough to counterbalance the negative charges of (beta)-mannosylglycerate and di-myo-inositol-phosphate in the cell. The results presented here together with those previously reported for Pyrococcus woesei (S. Scholz, J. Sonnenbichler, W. Schafer, and R. Hensel, FEBS Lett. 306:239-242, 1992) strongly support a role for di-myo-inositol-phosphate in thermoprotection.     

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.     

Distribution of Genes for Synthesis of Trehalose and Mannosylglycerate in Thermus spp. and Direct Correlation of These Genes with Halotolerance

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.     

Compatible solutes in new moderately halophilic isolates

Abstract Using high performance liquid chromatography and nuclear magnetic resonance techniques, the compatible solutes of some moderately halophilic bacteria were studied. The following accepted species of moderately halophilic bacteria were included: Volcaniella eurihalina and Deleya salina among Gram-negative rods, and Salinicoccus roseus and Salinicoccus hispanicus among Gram-positive cocci. Besides these strains we have also screened other new isolates, including Marinomonas species and Gram-positive cocci and rods. The tetrahydropyrimidine carboxylic acid 'ectoine' was found to be the main compatible solute in the Gram-negative strains tested when these were grown in glucose-mineral medium. In addition, betaine was accumulated from complex media containing yeast extract. Among the Gram-positive strains investigated, the solutes proline (bacillus 30, Salinicoccus ) and hydroxyectoine (coccus 28) also played an important role, while alanine, glucose, glutamate, glutamine and trehalose occurred as minor components. We also detected two recently described compatible solutes: Nδ -acetylornithine and a homologous compound, Nε -acetyllysine. Representatives of distinct phenotypic groups of Gram-positive cocci and rods were clearly distinguished by their solute pattern.     

Stabilization of Enzymes against Thermal Stress and Freeze-Drying by Mannosylglycerate

2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications.     

Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus spp.

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, α-glutamate, di-myo-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated α-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.     

Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase

The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinus is proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo (13)C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed with D-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and D-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed in Escherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose and D-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.     

Diversity and biosynthesis of compatible solutes in hyper/thermophiles.

The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed.     

A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999     

Endogenously synthesized (-)-proto-quercitol and glycine betaine are principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new isolates of phylogenetically related thraustochytrids

We report that endogenously synthesized (-)-proto-quercitol (1D-1,3,4/2,5-cyclohexanepentol) and glycine betaine were the principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new osmotolerant isolates of thraustochytrids (strains T65, T66, and T67). The compatible solutes were identified and quantified by use of nuclear magnetic resonance spectroscopy, and their identity was confirmed by mass spectroscopy and measurement of the specific optical rotation. The cellular content of compatible solutes increased with increasing NaCl concentration of a defined medium. (-)-proto-Quercitol was the dominating solute at all NaCl concentrations tested (0.25 to 1.0 M), e.g., cells of S8 and T66 stressed with 1.0 M NaCl accumulated about 500 micromol (-)-proto-quercitol and 100 micromol glycine betaine per g dry weight. To our knowledge, (-)-proto-quercitol has previously been found only in eucalyptus. The 18S rRNA gene sequences of the four (-)-proto-quercitol-producing strains showed 99% identity, and they displayed the same fatty acid profile. The only polyunsaturated fatty acids accumulated were docosahexaenoic acid (78%) and docosapentaenoic acid (22%). A less osmotolerant isolate (strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304), did not contain (-)-proto-quercitol or glycine betaine. Thus, the level of osmotolerance and the osmolyte systems vary among thraustochytrids.     

Osmoadaptative accumulation of Nɛ-acetyl-β-lysine in green sulfur bacteria and Bacillus cereus CECT 148T

The compatible solute N(?)-acetyl-β-lysine (NeABL), thus far considered unique to methanogenic Archaea, has been found to accumulate in several strains of green sulfur bacteria (GSB) and Bacillus cereus CECT 148(T) under salt stress. A similar mixture of compatible solutes including trehalose, α-glutamate, β-glutamate and NeABL has been detected in salt-tolerant GSB strains of different phylogenetic branches. The ability of B. cereus to synthesize this compound was predicted from available genomic data, and nuclear magnetic resonance analyses of cultures grown in salt-containing media indicated that NeABL was present in the solute pools of osmotically challenged cells. The present results describe for the first time in the bacterial domain the use of this compound for osmoadaptation.     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.     

Enzyme stabilization be ectoine-type compatible solutes: protection against heating,freezing and drying

Summary The aim of this study was to elucidate the protective effect of the new compatible solutes, ectoine and hydroxyectoine, on two sensitive enzymes (lactic dehydrogenase, phosphofructokinase). The solutes tested also included (for reasons of comparison) other compatible solutes such as glycine betaine and a number of disaccharides (sucrose, trehalose, maltose). All compatible solutes under investigation displayed remarkable stabilizing capabilities. However, the degree of protection depended on both the type of solute chosen and the enzyme used as a test system. The most prominent protectants were trehalose, ectoine and hydroxyectoine, which are very often found in nature (singly or in combinationn) as part of the compatible solute cocktail of moderately halophilic eubacteria. Offprint request to: E. A. Galinski     

Diversity, biological roles and biosynthetic pathways for sugar-glycerate containing compatible solutes in bacteria and archaea

A decade ago the compatible solutes mannosylglycerate (MG) and glucosylglycerate (GG) were considered to be rare in nature. Apart from two species of thermophilic bacteria, Thermus thermophilus and Rhodothermus marinus, and a restricted group of hyperthermophilic archaea, the Thermococcales, MG had only been identified in a few red algae. Glucosylglycerate was considered to be even rarer and had only been detected as an insignificant solute in two halophilic microorganisms, a cyanobacterium, as a component of a polysaccharide and of a glycolipid in two actinobacteria. Unlike the hyper/thermophilic MG-accumulating microorganisms, branching close to the root of the Tree of Life, those harbouring GG shared a mesophilic lifestyle. Exceptionally, the thermophilic bacterium Persephonella marina was reported to accumulate GG. However, and especially owing to the identification of the key-genes for MG and GG synthesis and to the escalating numbers of genomes available, a plethora of new organisms with the resources to synthesize these solutes has been recognized. The accumulation of GG as an 'emergency' compatible solute under combined salt stress and nitrogen-deficient conditions now seems to be a disseminated survival strategy from enterobacteria to marine cyanobacteria. In contrast, the thermophilic and extremely radiation-resistant bacterium Rubrobacter xylanophilus is the only actinobacterium known to accumulate MG, and under all growth conditions tested. This review addresses the environmental factors underlying the accumulation of MG, GG and derivatives in bacteria and archaea and their roles during stress adaptation or as precursors for more elaborated macromolecules. The diversity of pathways for MG and GG synthesis as well as those for some of their derivatives is also discussed. The importance of glycerate-derived organic solutes in the microbial world is only now being recognized. Their stress-dependent accumulation and the molecular aspects of their interactions with biomolecules have already fuelled several emerging applications in biotechnology and biomedicine.     

Compatible solute biosynthesis in cyanobacteria

Compatible solutes are a functional group of small, highly soluble organic molecules that demonstrate compatibility in high amounts with cellular metabolism. The accumulation of compatible solutes is often observed during the acclimation of organisms to adverse environmental conditions, particularly to salt and drought stress. Among cyanobacteria, sucrose, trehalose, glucosylglycerol and glycine betaine are used as major compatible solutes. Interestingly, a close correlation has been discovered between the final salt tolerance limit and the primary compatible solute in these organisms. In addition to the dominant compatible solutes, many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline as secondary or tertiary solutes. In particular, the accumulation of sucrose and trehalose results in an increase in tolerance to general stresses such as desiccation and high temperatures. During recent years, the biochemical and molecular basis of compatible solute accumulation has been characterized using cyanobacterial model strains that comprise different salt tolerance groups. Based on these data, the distribution of genes involved in compatible solute synthesis among sequenced cyanobacterial genomes is reviewed, and thereby, the major compatible solutes and potential salt tolerance of these strains can be predicted. Knowledge regarding cyanobacterial salt tolerance is not only useful to characterize strain-specific adaptations to ecological niches, but it can also be used to generate cells with increased tolerance to adverse environmental conditions for biotechnological purposes.     

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.