Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.     

Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance

Intracellular ion concentration and respiratory activity in the marine cyanobacterium Spirulina subsalsa was analyzed during cell transition from saline to hypersaline medium. During salt upshock, an early phase of Na⁺ and Cl^- influx was observed, followed by an adaptation phase where both Na⁺ and Cl^- were excluded from the cell. Respiration in intact cells was enhanced during salt upshock. S. subsalsa spheroplasts exhibited a high rate of O₂ uptake, which was further enhanced in cells grown in hypersaline medium, upon addition of NaCl to the assay mixture. This effect was found to be specific to sodium ions. Plasma membrane fractions from cells grown in hypersaline medium exhibited a high rate of cytochrome oxidase activity, which was further stimulated by NaCl, and was sensitive to DCCD. Immunoblot analysis of Spirulina plasma membrane polypeptides with anti-cytochrome oxidase serum demonstrated high content of 53.4 kDa polypeptide of cytochrome oxidase, which was enriched in membranes obtained from hypersaline Spirulina cells. The enhanced respiration, and more specifically the enrichment of cytochrome oxidase activity in salt-adapted cells in situ, as well as its stimulation by NaCl in vitro and inhibition by DCCD, suggest that cytochrome oxidase is involved in the extrusion of sodium ions from cells of the salt-tolerant Spirulina subsalsa.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide m-chlorophenyl hydrazone - TMPD N, N, N, N, tetramethyl p-phenylenediamine dichloride     

Physiological Aspects of Adaptation of the Marine Microalga Tetraselmis (Platymonas) viridis to Various Medium Salinity

We studied the capability of the marine microalga Tetraselmis (Platymonas) viridis to adapt to low and high medium salinity. The normal NaCl concentration for growth of this alga is 0.5 M. It was shown that T. viridis cells could actively grow and maintain osmoregulation and cytoplasmic ion homeostasis in the wide range of external salt concentrations, from 0.01 to 1.2 M NaCl. Using the plasma membrane vesicles isolated from T. viridis cells grown at various NaCl concentrations (0.01, 0.05, 0.5, 0.9, and 1.2 M), we studied the formation of the phosphorylated intermediate of Na⁺-ATPase, the enzyme responsible for Na⁺ export from the cells with a mol wt of ca. 100 kD. Na⁺-ATPase was shown to function in the plasma membrane even in the cells growing at an extremely low NaCl concentration (0.01 M). When alga was grown in high-salt media, the synthesis of several proteins with molecular weights close to 100 kD was induced. The data obtained argue for the hypothesis, which was put forward earlier, that a novel Na⁺-ATPase isoform is induced by T. viridis growing at high NaCl concentrations.     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

Osmoregulation in the Halotolerant Alga Asteromonas gracilis

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.     

Effect of 24-epibrassinolide on biomass, growth and free proline concentration in Spirulina platensis (Cyanophyta) under NaCl stress

In this study, biomass, growth and free proline concentration were investigated in Spirulina platensis treated with different concentrations of NaCl (50, 100, 150, and 200 mM) and 24-epibrassinolide (24-epiBL) hormone (0.5, 1.0, and 3.0 μM) over 5 days. As a result of analysing the cultures under salinity stress, it was determined that biomass and growth rate decreased significantly, while proline concentration increased considerably under salinity stress. The increase in the concentration of proline suggests a role in response to NaCl stress. Among the cultures treated with different concentrations of 24-epiBL, maximum growth was determined at the cultures at 1.0 μM 24-epiBL. Algal growth was also greater at the 0.5 and 3.0 μM concentrations of 24-epiBL with respect to control cultures. With respect to control, 24-epiBL affected growth rate and biomass positively, but proline concentration did not change. Among the cultures supplied with different combinations of NaCl and 24-epiBL, growth rate increased in 150/0.5 and 150/3.0 mM/μM concentrations, but was maximal for the culture containing 150/1.0 mM/μM combination. The increase in algal growth suggests a role for 24-epiBL in partially alleviated to NaCl stress. These results suggest that 24-epiBL may have a protective role for S. platensis reducing the inhibitor effects of salinity stress.     

Effects of silicon on enzyme activity and sodium,potassium and calcium concentration in barley under salt stress

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown in a hydroponics system containing 120 mol m^-3 NaCl only and 120 mol m^-3 NaCl with 1.0 mol m^-3 Si (as potassium silicate). Compared with the plants treated with salt alone, superoxide dismutase (SOD) activity in plant leaves and H⁺-ATPase activity in plant roots increased, and malondialdehyde (MDA) concentration in plant leaves decreased significantly for both cultivars when treated with salt and Si. The addition of Si was also found to reduce sodium but increase potassium concentrations in shoots and roots of salt-stressed barley. Sodium uptake and transport into shoots from roots was greatly inhibited by added Si under salt stress conditions. However, Si addition exhibited little effect on calcium concentrations in shoots of salt-stressed barley. Thus, Si-enhanced salt tolerance is attributed to selective uptake and transport of potassium and sodium by plants. The results of the present study suggest that Si is involved in the metabolic or physiological changes in plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Intracellular salt and solute concentrations in Ectothiorhodospira marismortui: glycine betaine and Nα-carbamoyl glutamineamide as osmotic solutes

Ectothiorhodospira marismortui, a moderately halophilic purple sulfur bacterium from a hypersaline sulfur spring, contains glycine betaine and N-carbamoyl glutamineamide (CGA) as the main intracellular osmotic solutes, with sucrose as a minor component. The concentration of glycine betaine was found to increase with increasing salt concentration of the medium, from 0.47 M to 1.29 M in cells grown from 0.85 to 2.56 M NaCl, while the estimated CGA concentration rose from about 0.2 M to 0.5 M. The concentration of sucrose remained constant at a value of around 0.05 M. Intracellular sodium and potassium concentrations were relatively low (around 0.5 and 0.3 M, respectively, at an external NaCl concentration of 1.8 M). The concentration of the novel compound N-carbamoyl glutamineamide was enhanced when l-glutamine was added to the growth medium, suggesting that glutamine served as a precursor for the synthesis of the compound.Abbreviations CGA N-carbamoyl glutamineamide     

The leaf tonoplast V-H(+)-Atpase activity of a C3 halophyte Suaeda salsa is enhanced by salt stress in a Ca-dependent mode

Suaeda salsa seedlings grown in Hoagland nutrient solution were treated with different concentrations of NaCl combined with two levels of Ca2+ (0 and 20 mmol/L) to study the effect of Ca2+ nutrition on the growth and activity of leaf tonoplast V-H(+)-ATPase. Increase of Ca2+ concentration in the solution markedly increased the relative growth quantity of S. salsa seedlings and Ca2+ and K+ concentration in the leaf cell sap under NaCl stress. The leaf V-H(+)-ATPase activity was significantly increased with increasing NaCl concentration under high Ca2+ application (20 mmol/L), but little changed under Ca2+ starvation (0 mmol/L). Western blot analysis showed that the leaf V-H(+)-ATPase of S. salsa was at least composed of A, B, D and c subunits, and their protein amounts were not affected by NaCl treatments under Ca2+ starvation (0 mmol/ L) with an exception of 100 mmol/L NaCl, but increased under high Ca2+ application (20 mmol/L). There was a positive correlation between activity of V-H(+)-ATPase and the protein amounts of the subunits. The results suggest that Ca2+ nutrition played an important role in the salt tolerance of S. salsa, and that enhancement of V-H(+)-ATPase activity under salt stress was Ca2(+)-dependent.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Physiological response of the unicellular green alga Chlorococcum submarinum to rapid changes in salinity

Cell volumes and intracellular concentrations of major solutes of Chlorococcum submarinum were determined before and after salinity shocks. Cells were found to shrink in size by about 30% following changes from 0.1 to 0.5 M NaCl, there was a transitory increase in sodium concentration and more permanent increases in concentrations of potassium, proline and glycerol (the major osmolyte). Conversely, cells doubled in size after the reciprocal downshock, there was rapid loss of about 70% of the cells' glycerol to the medium, a much smaller loss of cellular potassium and a steady disappearance of proline from the cells. The respiratory and photosynthetic responses to salinity fluctuations were also studied. Salinity downshocks stimulated respiration by 30% and inhibited photosynthesis by 16% within 5 min, but within 2 h these rates were identical to control rates. Upshocks caused a slight inhibition of respiration, but decreased photosynthesis by 40% within 5 min and recovery took 2 h. Downshocks had little effect on chlorophyll fluorescence, however, Fo strongly increased and both Fm and Fv/Fm declined within 5 min of salinity increases. This is consistent with a decrease in efficiency of PS2. Ecological and metabolic implications of the results are discussed.Abbreviations DMSO dimethyl sulphoxide - Hepes N-[2-hydroxyethyl]piperazine-N-2-ethane sulphonic acid - TCA trichloroacetic acid - Tris tris[hydroxymethyl]aminoethane     

23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311

We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.     

Involvement of Long-Distance Na<Superscript>+</Superscript> Transport in Maintaining Water Potential Gradient in the Medium-Root-Leaf System of a Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

The aim of this study was to determine the range of NaCl concentrations in the nutrient solution that allow Suaeda altissima (L.) Pall., a salt-accumulating halophyte, to maintain the upward gradient of water potential in the “medium-root-leaf” system. We evaluated the contribution of Na⁺ ions in the formation of water potential gradient and demonstrated that Na⁺ loading into the xylem is involved in this process. Plants were grown in water culture at NaCl concentrations ranging from zero to 1 M. The water potential of leaf and root cells was measured with the method of isopiestic thermocouple psychrometry. When NaCl concentration in the growth medium was raised in the range of 0–500 mM (the medium water potential was lowered accordingly), the root and leaf cells of S. altissima decreased their water potential, thus promoting the maintenance of the upward water potential gradient in the medium-root-leaf system. Growing S. altissima at NaCl concentrations f 750 mM and 1 M disordered water homeostasis and abolished the upward gradient of water potential between roots and leaves. At NaCl concentrations of 0–250 mM, the detached roots of S. altissima were capable of producing the xylem exudate. The concentration of Na⁺ in the exudate was 1.3 to 1.6 times higher than in the nutrient medium; the exudate pH was acidic and was lowered from 5.5 to 4.5 with the rise in the salt concentration. The results indicate that the long-distance Na⁺ transport and, especially, the mechanism of Na⁺ loading into the xylem play a substantial role in the formation of water potential gradient in S. altissima. The accumulation of Na⁺ in the xylem and acidic pH values of the xylem sap suggest that Na⁺ loading into the xylem is carried out by the Na⁺/H⁺ antiporter of the plasma membrane in parenchymal cells of the root stele.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 549–557.Original Russian Text Copyright © 2005 by Balnokin, Kotov, Myasoedov, Khailova, Kurkova, Lun’kov, Kotova.     

Effects of CaSO4 and NaCl on growth and nitrogen fixation ofLeucaena leucocephala

Effects and interactions of varying CaSO₄ and NaCl levels on growth and nitrogen fixation ofLeucaena leucocephala K8 were examined. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM) in randomized blocks with five replications. While NaCl significantly reduced plant growth, additions of CaSO₄ increased plant height, leaf number, and biomass of salt treated plants. For the nonsaline treatments, high CaSO₄ levels slightly depressed growth, which contradicts suggestions that Leucaena has a high calcium requirement. A significant calcium/sodium interaction was not seen for nodule number or weight. Nodule number was significantly depressed by 100 mM NaCl and nodule weight of the salt stressed plants significantly increased as CaSO₄ concentration increased from 0.5 to 2.5 mM. Effects of NaCl and CaSO₄ on nitrogen content of plant parts were inconclusive. The promotion of Leucaena salinity tolerance by addition of CaSO₄ may be attributed to the effect of calcium in maintaing the selective permeability of membranes.     

Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola.

Vibrio costicola grown in the presence of different NaCl concentrations contains cell-associated Na+ and K+ ions whose sum is equal to or greater than the external Na+ concentration. In the presence of 0.5 M NaCl, virtually no in vitro protein is synthesized in extracts of cells grown in 1.0 M NaCl. However, we report here that active in vitro protein synthesis occurred in 0.6 M or higher concentrations of Na2SO4, sodium formate, sodium acetate, sodium aspartate, or sodium glutamate, whereas 0.6 M NaF, NaCl, or NaBr completely inhibited protein synthesis as measured by polyuridylic acid-directed incorporation of [14C]phenylalanine. Sodium glutamate, sodium aspartate, and betaine (0.3 M) counteracted the inhibitory action of 0.6 M NaCl. The cell-associated Cl- concentration was 0.22 mol/kg in cells grown in 1.0 M NaCl. Of this, the free intracellular Cl- concentration was only 0.02 mol/kg. Cells contained 0.11 mol of glutamate per kg and small concentrations of other amino acids. All of the negative counterions for cell-associated Na+ and K+ have not yet been determined. In vitro protein synthesis by Escherichia coli was inhibited by sodium glutamate. Hybridization experiments with ribosomes and the soluble (S-100) fractions from extracts of E. coli and V. costicola showed that the glutamate-sensitive fraction was found in the soluble, not the ribosomal, part of the system. The phenylalanyl-tRNA synthetase of V. costicola was not inhibited by 0.5 M or higher concentrations of NaCl; it was slightly more sensitive to high concentrations of sodium glutamate. Therefore, this enzyme was not responsible for the salt response of the V. costicola in vitro protein-synthesizing system.