Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis

Brevibacterium epidermis DSM 20659 is a halotolerant Gram-positive bacterium which can synthesize the osmolyte, ectoine, but prefers to take it up from its environment. The present study revealed that B. epidermis is equipped with at least one transport system for ectoine, with a maximal transport velocity of 15.7 ± 4.3 nmol/g CDW·min. The transport requires energy (ATP) and is completely inhibited by the proton uncoupler, CCCP. The ectoine uptake system is constitutively expressed at a basal level of activity and its activity is immediately 10-fold increased by hyper-osmotic stress. Initial uptake rates are not influenced by the intensity of the hyper-osmotic shock but the duration of the increased activity of the uptake system could be directly related to the osmotic strength of the assay solution. Competition assays indicate that betaine, but not proline, is also transported by the ectoine uptake system.     

Ectoine as the predominant osmolyte in the marine bacterium Marinobacter hydrocarbonoclasticus grown on eicosane at high salinities

L. FERNANDEZ-LINARES, R. FAURE, J.-C. BERTRAND AND M. GAUTHIER. 1996. Ectoine was detected by ¹³C-nuclear magnetic resonance spectroscopy in extracts of the halophilic marine bacterium Marinobacter hydrocarbonoclasticus grown at different NaCl concentrations (0.6, 1.5, 2.5 mol l^-1) in synthetic seawater on eicosane as the sole source of carbon and energy. Ectoine was synthesized as the principal osmolyte in response to stress. However, when the medium was supplemented with glycine betaine, this osmolyte was transported into the cells and replaced ectoine.     

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)     

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.     

Development of a genetic system for the moderately halophilic bacterium Halobacillus halophilus: generation and characterization of mutants defect in the production of the compatible solute proline

A procedure for markerless mutagenesis gene deletions was developed for the moderately halophilic model strain Halobacillus halophilus. Gene transfer was achieved by protoplast fusion and allelic replacement by a two-step procedure. In the first step the non-replicating plasmid integrated over the upstream or the downstream region of the target gene or operon into the chromosome to obtain single-crossover mutants. When cells were grown under non-selective conditions a second homologous recombination happened (segregation). This resulted in either the wild-type or the mutated allele. The method was used to delete the proHJA operon from H. halophilus. The mutant still produced proline and thus was not proline auxotroph but it completely lost the ability to produce proline as a compatible solute. However, growth was not impaired and the loss of the solute proline was compensated for by an increase in glutamate, glutamine and ectoine concentration. Expressions of the genes encoding the biosynthesis enzymes of theses solutes were upregulated and the activity of the key enzyme in glutamine biosynthesis, the glutamine synthetase, was increased. A model for the proline biosynthesis in the ΔproHJA mutant is discussed.     

Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.     

2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea.

A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp. and several Natronobacterium species. The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte. A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose. Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte. Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.     

Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigens

AIMS: To investigate the catabolism of ectoine and hydroxyectoine, which are the major compatible solutes synthesized by Chromohalobacter salexigens. METHODS AND RESULTS: Growth curves performed in M63 minimal medium with low (0.75 mol l(-1) NaCl), optimal (1.5 mol l(-1) NaCl) or high (2.5 mol l(-1) NaCl) salinity revealed that betaine and ectoines were used as substrate for growth at optimal and high salt. Ectoine transport was maximal at optimal salinity, and showed 3- and 1.5-fold lower values at low and high salinity respectively. The salt-sensitive ectA mutant CHR62 showed an ectoine transport rate 6.8-fold higher than that of the wild type. Incubation of C. salexigens in a mixture of glucose and ectoine resulted in a biphasic growth pattern. However, CO(2) production due to ectoine catabolism was lower, but not completely abolished, in the presence of glucose. When used as the sole carbon source, glycine betaine effectively inhibited ectoine and hydroxyectoine synthesis at any salinity. CONCLUSIONS: The catabolic pathways for ectoine and hydroxyectoine in C. salexigens operate at optimal and high (although less efficiently) salinity. Endogenous ectoine(s) may repress its own transport. Ectoine utilization was only partially repressed by glucose. Betaine, when used as carbon source, suppresses synthesis of ectoines even under high osmolarity conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is a previous step to the subsequent isolation and manipulation of the catabolic genes, so as to generate strains with enhanced production of ectoine and hydroxyectoine.     

Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.     

Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis

A production process for ectoine has been developed, using Brevibacterium epidermis DSM20659 as the producer strain. First, the optimal conditions for intracellular synthesis of ectoine were determined. The size of the intracellular ectoine pool is shown to be dependent on the external salt concentration, type of carbon source, and yeast extract concentration. Under the optimized conditions of 1 M NaCl, 50 g/L monosodium glutamate, and 2.5 g/L yeast extract, a maximum concentration of intracellular ectoine of 0.9 g/L was obtained in shake flask cultures. After optimizing the batch fermentation parameters of temperature, pH, agitation, and aeration, the yield could be further increased by applying the fed-batch fermentation principle in 1.5- to 2-L fermentors. Glutamate and yeast extract were fed to the bacterial cells such that the total glutamate concentration in the broth remained constant. A total yield of 8 g ectoine/L fermentation broth was obtained with a productivity of 2 g ectoine/L/day. After the bacterial cells were harvested from the culture broth, the ectoine was recovered from them by a two-step extraction with water and ethanol. Crystallization of the product was obtained after concentration of the extract via evaporation under reduced pressure. After this downstream process, 55% of the ectoine produced in the fermentor could be crystallized in four fractions. The first fractions were of very high purity (98%). This production process can compete with other described production processes for ectoine in productivity and simplicity. Further advantages are the relatively low amounts of NaCl needed and the absence of hydroxyectoine, often a byproduct, in the final product.     

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.     

Rapid and sensitive NMR method for osmolyte determination

We propose a rapid and sensitive method for osmolyte determination, based on one-dimensional and two-dimensional 1H NMR spectroscopy applied directly on culture of haloalkalophilic Halomonas pantelleriensis and acidothermophilic archaeon Sulfolobus solfataricus, without any extraction procedure. The osmoprotectants hydroxyectoine, ectoine, glutamate, glycine-betaine and treahalose can easily be quantified by integrating the peak areas with respect to an internal standard, and the concentrations evaluated with this method are in excellent agreement with the values previously reported. Furthermore, trace amount of osmoprotectants, often undetectable after extraction procedures, can also be evaluated.