Relationships between the osmoadaptation strategy, amino acid composition of total cellular protein, and properties of certain enzymes of haloalkaliphilic bacteria

Haloalkaliphilic microorganisms isolated from soda lakes were compared in terms of the amino acid composition of total cellular protein and the reaction of a number of key enzymes to salts and pH of the medium. In the extremely halophilic bacterium Natroniella acetigena (salt-inside osmoadaptation strategy), acidic amino acids (glutamic and aspartic) made up 30.91 mol % of the total of cellular protein amino acids. In the moderate haloalkaliphiles Tindallia magadiensis, Halomonas campisalis, and Halomonas sp. AIR-1 (compatible-solutes osmoadaptation strategy), the proportion of acidic amino acids (24.36, 23.15, and 23.58 mol %, respectively) was lower than in N. acetigena but higher than in the freshwater Acetobacterium paludosum (20.77 mol %). The excess of acidic amino acids over basic amino acids (lysine and arginine) increased with the degree of halophily. The enzymes of haloalkaliphiles proved to be tolerant to salts and high pH values, although the degree of tolerance varied. The activity of N. acetigena CO dehydrogenase was maximum in the presence of 0.7 M NaCl, but it was virtually independent of the NaHCO3 concentration. The hydrogenase and CO dehydrogenase of T. magadiensis exhibited maximum activity in the absence of NaCl; the CO dehydrogenase was most active at 0.25 M NaHCO3, and hydrogenase activity was only weakly dependent on NaHCO3 in the concentration range of 0-1.2 M. The nitrate reductases of H. campisalis and Halomonas sp. AIR-2 were active in broad ranges of NaCl and KCl concentrations; the activity maxima were recorded at moderate concentrations of these salts. The pH optima of most of the studied enzymes of haloalkaliphiles were in the alkaline zone. Thus, it was shown that the amino acid composition of total cellular protein is determined by the osmoadaptation strategy employed by the bacterium. A correlation was found between the salt tolerance of enzymes and the proportion of acidic amino acids in the total cellular protein. The ability of enzymes to function at high pH values is one of the mechanisms of adaptation of microorganisms to high pH values.     

Osmoadaptation in Representatives of Haloalkaliphilic Bacteria from Soda Lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K⁺, Na⁺, and Cl^? ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na⁺ in the medium were revealed in this organism. At a concentration of 1.91 M Na⁺ in the medium, N. acetigena accumulated 0.83 M K⁺, 0.91 M Na⁺, and 0.29 M Cl^? in cells, and, with an increase in the Na⁺ content in the medium to 2.59 M, it accumulated 0.94 M K⁺, 1.98 M Na⁺, and 0.89 M Cl^?, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Isolation and Characterization of Halotolerant Alkaliphilic Methanotrophic Bacteria from Tuva Soda Lakes

Two strains (5Z and 20Z) of halotolerant alkaliphilic obligate methanotrophic bacteria were first isolated from moderately saline soda lakes in Tuva (Central Asia). The strains grow fastest at pH 9.0–9.5 and much more slowly at pH 7.0. No growth occurred at pH ≤ 6.8. They require NaHCO₃ or NaCl for growth in alkaline medium. Gram-negative, motile rods with ordered cup-shaped cell wall structures and Type I intracytoplasmic membranes assimilate methane and methanol via the ribulose monophosphate pathway. The G + C content of strains 5Z and 20Z are 47.6 and 47.9 mol%, respectively. Based on their alkaliphilic physiology, both strains were referred to as Methylobacter alcaliphilus sp. nov. The changes in cell phospholipids, fatty acids, and amino acids have been observed upon varying salinity and pH of the medium, thus suggesting structure-function osmoadaptation of the strains studied. Whole-cell experiments revealed the salt- and pH-dependence of CH₄ oxidation and assimilation rates. Cell motility was also Na⁺ dependent and sensitive to some energy uncouplers and ionophores. Received: 7 March 1997 / Accepted: 14 April 1997     

Comparative effects of NaCl and NaHCO3 stresses on respiratory metabolism,antioxidant system,nutritional status,and organic acid metabolism in tomato roots

Physiological responses of tomato roots to NaCl and NaHCO₃ stresses were investigated in a hydroponic setting. The relative growth rate of tomato plants was significantly reduced in both NaCl and NaHCO₃ treatments, especially under NaHCO₃ stress. Tomato root respiration increased under low concentrations of NaCl and NaHCO₃ stresses. However, high concentrations of both NaCl and NaHCO₃ significantly inhibited respiration, especially in the NaHCO₃ treatment. With increasing concentration of NaCl and NaHCO₃ treatment, root Na accumulation increased, while accumulation of N, P, K, Fe, and Mg was significantly lower. Compared to NaCl, NaHCO₃ treatment resulted in more dramatic changes in these nutrients. All organic acids investigated were increased by NaHCO₃ after 5 days of treatment, but only oxalate, tartrate and malate were induced by NaCl. This implies that global regulation of organic acids might play an important role in tomato’s alkali stress tolerance. Compared to NaCl treatments, NaHCO₃ treatments induced much higher levels of reactive oxygen species (ROS) and lipid peroxidation after 5 days of treatment, which was accompanied by higher activities of antioxidant enzymes and higher concentrations of ascorbate–glutathione. However, after 10 days of treatment, 100 mM NaHCO₃ stress led to lower accumulation of ROS, antioxidant enzyme activities, and ascorbate–glutathione content. This may have been because root metabolism had almost completely stopped, as indicated by lower root respiration and activity.     

Salt responses of enzymes from species differing in salt tolerance

Enzymes which are affected by the addition of inorganic salts during in vitro assay were extracted from salt-sensitive Phaseolus vulgaris, salt-tolerant Atriplex spongiosa, and Salicornia australis and tested for sensitivity to NaCl. In each case malate dehydrogenase, aspartate transaminase, glucose 6-phosphate dehydrogenase, and isocitrate dehydrogenase showed NaCl responses similar to those found for commercially available crystalline enzymes from other organisms. Enzymes extracted from plants grown in saline cultures showed no important changes in specific activity or salt sensitivity. Interaction of pH optima and NaCl concentrations suggests that enzymes may differ in the way they respond to salt treatment.     

Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that is found in alkaline soil areas in northeast China and is highly tolerant to carbonate stress. We constructed a cDNA library from C. virgata seedlings treated with NaHCO₃, and isolated a type1 metallothionein (MT1) gene (ChlMT1: AB294238) from the library. The amino acid sequence of ChlMT1 contained 12 cysteine residues that constituted the Cys-X-Cys (X = amino acid except Cys) motifs in the N- and C-terminal regions. Northern hybridization showed that expression of ChlMT1 was induced by several abiotic stresses, from salts (NaCl and NaHCO₃), a ROS inducer (paraquat), and metals (CuSO₄, ZnSO₄, and CoCl₂). ChlMT1 expression in leaf was induced by 200 mM NaCl and 100 mM NaHCO₃. About 5 μM Paraquat, 500 μM Zn²⁺, and 500 μM Co²⁺ also induced expression of ChlMT1 in leaf after 6 h, and 100 μM Cu²⁺ induced it after 24 h. Saccharomyces cerevisiae when transformed with the ChlMT1 gene had dramatically increased tolerances to salts (NaCl and NaHCO₃) and ROS.     

The Effect of Sodium Salts and pH on the Hydrogenase Activity of Haloalkaliphilic Sulfate-Reducing Bacteria

Hydrogenase is the main catabolic enzyme of hydrogen-utilizing sulfate-reducing bacteria. In haloalkaliphilic sulfate reducers, hydrogenase, particularly if it is periplasmic, functions at high concentrations of Na⁺ ions and low concentrations of H⁺ ions. The hydrogenases of the newly isolated sulfate-reducing bacteria Desulfonatronum thiodismutans, D. lacustre, and Desulfonatronovibrio hydrogenovorans exhibit different sensitivity to Na⁺ ions and remain active at NaCl concentrations between 0 and 4.3 M and NaHCO₃ concentrations between 0 and 1.2 M. The hydrogenases of D. lacustre and D. thiodismutans remain active at pH values between 6 and 12. The optimum pH for the hydrogenase of D. thiodismutans is 9.5. The optimum pH for the cytoplasmic and periplasmic hydrogenases of D. lacustre is 10. Thus, the hydrogenases of D. thiodismutans, D. lacustre, and Dv. hydrogenovorans are tolerant to high concentrations of sodium salts and extremely tolerant to high pH values, which makes them unique objects for biochemical studies and biotechnological applications.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 460–465.Original Russian Text Copyright © 2005 by Detkova, Soboleva, Pikuta, Pusheva.     

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.     

A possible mechanism of ammonium ion regulation of photosynthetic carbon flow in higher plants

Addition of NH₄⁺ to the photosynthesizing leaf cells of Dolichos lab lab L. var. Lignosis Prain and leaf discs of Vigna sinensis L. savi ex Hassk caused a significant increase in the flow of photosynthetic carbon toward amino acids with a concomitant decrease toward sugars without affecting the over-all photosynthetic rate. Similar diversion of photosynthetic carbon away from sugars was also observed in the photosynthesizing isolated chloroplasts of V. sinensis, but the latter differed in that they accumulated organic acids rather than amino acids. In an effort to understand the mechanism of NH₄⁺-mediated regulation, the specific and total activities of NAD(P)-glutamate dehydrogenase, glutamine synthetase, pyruvate kinase, alkaline fructose 1,6-bisphosphatase, and NAD(P)-glyceraldehyde-3-phosphate dehydrogenase of the cells of D. lab lab were checked but none was affected by the added ammonium salts even after prolonged incubation. At certain concentrations, ammonium ions abolished the light activation of NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose 1,6-bisphosphatase in isolated chloroplasts from dark-adapted Vigna leaves without interfering with the basal dark activity of these enzymes. Based on these observations, a possible mechanism of action of NH₄⁺ in regulating the photosynthetic carbon flow is postulated.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Tissue-Specific and Cation/Anion-Specific DNA Methylation Variations Occurred in C. virgata in Response to Salinity Stress

Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na₂SO₄, NaHCO₃, Na₂CO₃) and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na₂CO₃> NaHCO₃> Na₂SO₄> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.     

Germination and Photosynthetic Responses to Salinity and Alkalinity in <i>Avicenna marina</i> Propagules

Avicenna marina (Forssk.) Vierh is a halophytic mangrove. The reproductive unit is green and has photosynthetic propagules. Mangroves are naturally exposed to fluctuations in some abiotic factors at the soil surface, including salinity and alkalinity. The objective of this study was to determine the effects of two salts including NaCl and NaHCO₃ on germination processes and discuss the relationships between cotyledon photosynthesis and embryo axis growth in A. marina propagules. These propagules came from Al Birk, located on the shoreline of the Saudi Red Sea. The results showed that the studied salts did not affect neither the final germination percentage nor the embryo axis growth. However, rooting and root growth were delayed by both salts at 300 mM and were strongly inhibited by 600 mM NaHCO₃. Both NaCl and NaHCO₃ reduced the photosynthetic activity. These two salts did not affect the other photosynthetic parameters, including stomatal conductance, net transpiration, and intercellular CO₂. Thereafter, the reduction in net photosynthesis was not related to any limitation of stomatal conductance. The early germination phase was independent of cotyledon photosynthesis, whereas rooting and root growth may be limited by reduced photosynthesis under NaCl and NaHCO₃.     

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H₂-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H₂ uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH₃, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.     

Salt Stress Control of Intracellular Solutes in Streptomycetes Indigenous to Saline Soils

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (−3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K⁺ salts sharply increased.     

燕麦对松嫩草地三种主要盐分胁迫的生理适应策略

为明确燕麦幼苗对松嫩盐碱草地3种主要盐分Na Cl、Na HCO_3和Na_2CO_3的适应机制,设定不同浓度梯度(48—144 mmol/L)的胁迫处理液,测定燕麦幼苗的生长与生理指标变化。结果表明,尽管试验设定的Na Cl浓度并不影响幼苗的存活率,但在各组胁迫处理下,随着浓度的增加,燕麦幼苗的分蘖数、植株高度、茎叶与根系的生物量均呈下降趋势,下降幅度为Na_2CO_3Na HCO_3Na Cl。另外,与Na Cl胁迫相比,Na_2CO_3与Na HCO_3胁迫下茎叶与根中积累了更多的有毒Na~+,同时K~+下降幅度也更大,并且根系中含有更高的Na~+与更低的K~+以及更高的Na~+/K~+。在Na Cl胁迫下,燕麦幼苗积累大量的无机Cl~-和脯氨酸来维持细胞内的渗透与离子平衡,而Na HCO_3与Na_2CO_3胁迫造成了燕麦幼苗体内阴离子的亏缺,此时幼苗主要通过积累大量的有机酸和更多的脯氨酸来维持渗透与离子平衡。上述结果表明,碱性盐Na_2CO_3与Na HCO_3对植物的胁迫伤害程度大于中性盐Na Cl,并且Na_2CO_3的毒害效应最强,而燕麦幼苗对不同的盐分胁迫伤害也有会产生不同的生理适应策略。     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

GmHKT1;4, a novel soybean gene regulating Na+/K+ ratio in roots enhances salt tolerance in transgenic plants

Salt is an important factor affecting the growth and development of soybean in saline or alkaline soil. The aims of the present study were to identify and functionally analyse the soybean GmHKTs gene family, and to explore their roles under NaHCO₃ and NaCl stresses. The GmHKTs gene family were isolated from soybean using genome sequence information. The GmHKTs gene family were further analysed for the structure and phylogenetic relationship. The expression patterns of soybean GmHKTs genes under NaHCO₃ and NaCl stresses were analysed via quantitative real-time PCR. As a result, the expression level of GmHKT1;4 was extremely up regulated in root under each treatment. Overexpression of GmHKT1;4 significantly enhanced the tolerance of transgenic tobacco plants to NaHCO₃ and NaCl stresses, compared with null plants. The overexpressed transgenic plants of this gene accomulated more K⁺ and less Na⁺ under salt stress, compaired with null plants. Our findings suggest that GmHKT1;4 plays an important role for regulation Na⁺/K⁺ ratio in roots under alkaline (NaHCO₃) and saline (NaCl) stresses.     

Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO₂, and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K_m values for both NAD⁺ (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from ~7.5 to ~6.4 when the MgCl₂ and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl₂ concentration. MgCl₂ was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate.     

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