PROTEIN AND AMINO ACID COMPOSITION OF THE OBLIGATE HALOPHILE APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

The production of extracellular and intracellular free amino acids during aerated fermentation of glucose by Baker’s yeast (Saccharomyces cerevisiae)

During a study of the effects of a high level of NaCl on the content of free intracellular amino acids in baker’s yeast grown in aerated fermentation of glucose it was found (Malaneyet al. 1988, 1989; Malaney and Tanner 1988) that 0.6 mol/L exogenous NaCl significantly increased the content of free intracellular citrulline, glutamine, ornithine, arginine and lysine (all basic amino acids) over that observed at zero mol/L exogenous NaCl. (Exogenous is defined as salt added beyond that present in the mineral salts in the culture medium.) This paper describes the production and relative relationships of both extracellular and free intracellular amino acids byS. cerevisiae under conditions of high NaCl content in the growth medium at pH 5 and 32 °C. For early culture times (6 h), the production of glutamine, citrulline, valine, isoleucine, ornithine, lysine and histidine were all enhanced by the addition of NaCl. For late times (24 h), except for ornithine, the early-time-enhanced amino acids continued to be enhanced by the addition of NaCl. In addition, the yields of several other amino acids also were increased by exogenous salt at this late time. These include aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine. Deceased, August 12, 1988. This study was supported by theNational Science Foundation (Grant No. CPE8209945) in conjunction with the NSF United States — Taiwan Cooperative Science Program.     

Effect of nystatin on the release of glycerol from salt-stressed cells of the salt-tolerant yeast Zygosaccharomyces rouxii

The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 microM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.     

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.     

Salt stress in cultured rice cells: effects of proline and abscisic acid

Abstract. The presence of 1 and 10 mol m⁻³ proline in media containing 100 and 200 mol m⁻³ of NaCl, had little effect on the growth of salt-adapted callus of rice. However, in such callus proline accumulation was stimulated by 10 mol m⁻³ proline in the presence of 100 mol m⁻³ NaCl. On the other hand, with 100 mol m⁻³ NaCl, both 1 and 10 mol m⁻³ proline significantly increased both the growth and proline content of salt-unadapted callus. On replacing NaCl with KCl (100 and 200 mol m⁻³), growth of saltadapted as well as unadapted callus was inhibited, but the presence of 10 mol m⁻³ proline had an ameliorating effect. Abscisic acid (ABA) supressed the growth of both salt-adapted and unadapted callus of rice in the absence of salt stress. ABA inhibited the growth of callus adapted to and grown in 100 and 200 mol m⁻³ of NaCl or when it was replaced by equimolar concentrations of KCl. Growth of 100 mol m⁻³ NaCl adapted cells was inhibited when they were transferred to a medium containing 200 mol m⁻³ of NaCl, but in the presence of ABA it was stimulated. ABA increased the growth of unadapted cells when subjected to different salts. Also, ABA accelerated the adaptation of cells exposed to salt but not to water deficits imposed by nonionic solutes.     

Effect of Salt Stress on Callus Cultures of Oryza sativa L.

Kavi Kishor, P.B 1988. Effect of salt stress on callus culturesof Oryza sativa L.—. exp. Bot 39 235–240 Callus cultures of rice adapted to grow under increasing NaClstress were found to accumulate considerable amounts of freeproline, compared with unadapted cells. Salt-adapted cells grownfor 10 passages (25 d each) on NaCl-free medium accumulatedproline on re-exposure to salt as did cells which were growncontinuously on NaCl. On replacing NaCl (100 mol m^–3)with 100 mol m^–3 of KC1, fresh and dry weights as wellas free proline content of salt-adapted callus declined comparedto that attained on 100 mol m^–3 NaCl medium. However,equimolar concentrations of NaCl and KC1 (when added together)produced an increase in growth and free proline accumulationin salt adapted callus. Omission of Ca²⁺ from the growth mediuminhibited the growth of salt-adapted cells in the presence ofNaCl, while it had little effect on the growth of non-adaptedcells in the presence of NaCl. ABA increased the fresh and dryweights of the non-adapted callus only in the presence of 200mol m^–3 of NaCl but not in the absence of NaCl. ABA failedto evoke the same response in salt adapted cells in the presenceof the salt. Tissues exhibited good growth under inhibitorylevels of NaCl (500 mol m^–3) only when glycine betaine,choline and proline were added to the medium but showed no growthin the presence of sarcosine, glycine and dimethylglycine. Key words: Oryza saliva, callus cultures, NaCl stress     

Fermenting Escherichia coli Is Able to Grow in Media of High Osmolarity, But Is Sensitive to the Presence of Sodium Ion

Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na⁺ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na⁺ were shown to extrude Na⁺ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231–237). The growth rate and Na⁺ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na⁺. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na⁺ extrusion activity. Received: 18 September 1998 / Accepted: 2 April 1999     

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。     

Effects of accumulation of 3-O-Methylglucose on levels of intracellular glycerol inDunaliella tertiolecta

Regulation of the concentration of osmotic solute was studied inDunaliella tertiolecta grown at an external salinity ranging between 0.5 and 1.5 mol/L NaCl. The total solute content of the cells was increased by applying 3-O-methylglucose (8 mmol/L), which was not metabolized, but accumulated at concentrations ranging between 7.5 and 12.5 μmol per mg dry mass within 2 h after its addition to the medium. 3-O-Methylglucose uptake resulted in a decreased concentration of glycerol, the solute mainly responsible for adaptation ofD. tertiolecta to high external salinity. 3-O-Methylglucose had no direct effect on the pathway of glycerol synthesis or degradation after external salinity increased or decreased, respectively. Thus, 3-O-methylglucose had no direct effects on glycerol metabolism, and it can bo assumed that it acts solely as an inert osmotic solute with the cells. 3-O-Methylglueose accumulation increased the respiration rate, as expected from an active transport.     

Alteration of cell-wall composition ofFusarium oxysporum by copper stress

A strain ofFusarium oxysporum tolerated copper in the growth medium at concentrations up to 600 mg/L. The optimum growth was obtained at 200 mg Cu/L. The mycelium acquired a blue color in the presence of copper. The copper content of isolated cell walls obtained from mycelium grown in the presence of 600 mg Cu/L was 1.5 times higher than that of cell walls obtained from mycelium grown at 200 mg Cu/L and it contained 2.2 and 3.3% copper at 200 and 600 mg Cu/L, respectively. The amount of protein and total sugars increased in both the mycelium and its isolated cell walls in the presence of copper in the growth medium, chitin was also increased in the cell wall, reaching its maximum amount at 200 mg Cu/L— about 2.4 times higher than without copper. Most of amino acid concentrations in the cell wall were increased in the presence of 200 mg Cu/L and decreased above this concentration. Isoleucine, leucine, tyrosine, phenylalanine, and arginine showed the highest increase at this concentration. The altered cell walls obtained from mycelium grown at 200 and 400 mg Cu/L could rebind individual metals more than the control cell walls could. Rebinding of individual metals was in the order Zn>Fe>Ni>Cu>Co. Rebinding of copper by isolated cell walls depended on pH and temperature.     

Separation and Purification of Molecular Species of Galactolipids by High Performance Liquid Chromatography

The NaCl concentration of the growth medium affected hydrogen production by Lyngbya sp. (No. 108) strain. Cells grown in medium containing 3% NaCl produced the most hydrogen. The carbohydrate content of this strain also increased with increasing NaCl concentration of the growth medium up to 720 fig/mg cells at 5 % NaCl. In the presence of 20 finlol/ml MFA (monofluoroacetic acid), inhibition of hydrogen production was observed. We extracted the glycogen from this nonheterocystous filamentous cyanobacterium, Lyngbya sp. (No. 108), and observed that glycogen and carbohydrate consumption of this strain is coincident with hydrogen production.

These results led us to the conclusion that the reserve glycogen or other carbohydrate were used as sources of electron donors for hydrogen production, and that the NaCl concentration of the medium affected the hydrogen production by this strain.     

Phosphorus uptake and translocation in salt-stressed melon plants

Melon seedlings (Cucumis melo L. cv.Galia) were grown hydroponically to study the effect of salinity (80 mmol/LNaCl) on phosphate (Pi) uptake and translocation at two levels of Pi (25 μmol/L and 1 mmol/L). Net uptake rates of Pi were determined by depletionof the medium and by plant content. Salinity decreased Pi uptake at low Pi (high affinity uptake mechanism), 25 μmol/L, although no specific competitive inhibition of Pi uptake by Cl⁻ was observed. When plants were grown with high Pi (1 mmol/L), the uptake of Pi through the low affinity system was increased by 80 mmol/L NaCl. Salinity also reduced the phosphorus flux, as Pi, through the xylem. It is hypothesised that high levels of NaCl decrease the mobility of Pi stored in vacuoles, and as a result, inhibit export from this storage compartment to other parts of the plant.     

Effect of nitrogen source and concentration to produce proteins in mass cultures of the microalgae <i>Chaetoceros muelleri</i>

Proteins are one of the major metabolites in biomass from microalgae that constitute the diet of marine organisms grown in aquaculture, and are essential for their growth. The quantity of this component is influenced by nutrients, temperature and light intensity, among others. We examined the growth, biomass production and protein of Chaetoceros muelleri with two sources of nitrogen (nitrate and urea) at three concentrations, using the medium f/2 (0.88 mol/L) (nitrates) as control. The treatments were the medium 2f (3.53 mol/L) and 4f (7.05 mol/L) with NO_3^-, and the medium f/2 (0.88 mol/L), 2f (3.53mol/L) and 4f (7.05 mol/L) with urea. In general, the productive parameters were greater using urea than nitrate in the media. Higher cell concentrations (2.83 x 10⁶ cell/mL), average and cumulative growth rates (1.50 div/day and 6.01 divisions), dry weight (0.0044 g/L), and proportion of proteins (23.74%) were found when urea was used as the N source. However, most of the bands on the electrophoretic profile were present in the mediums with NO_3^- (~6.5 to 90 kDa).     

Effect of osmotic stress onAspergillus chevalieri respiratory system

The osmotolerant fungusAspergillus chevalieri tolerates up to 80% sucrose concentration in the growth medium. At 50% sucrose the growth rate is 1.5-fold higher than in control (3% sucrose), at 80% sucrose it drops to 30% of the control level. Total proteins and lipids in the mitochondrial fractions obtained from the mycelium rise with increasing sucrose concentration during growth (2.6 and 2.1 times at 80% sucrose). The rate of respiration by whole cells and mitochondrial fractions increases with increased sucrose level in the growth medium. The activity of respiratory enzymes, such as succinate dehydrogenase, cytochrome oxidase, NADH oxidase and succinate oxidase, were also higher in cells growth in the presence of elevated sucrose concentrations. The largest increase was observed with NADH dehydrogenase.A. chevalieri cells grown at high osmotic stress exhibited enlarged mitochondria. The mean mitochondrial diameter at 50 and 80% sucrose was approximately 2.9- and 2.6-fold larger than in the control, respectively. Agarose gel electrophoresis of nucleic acids revealed the presence of high-density bands of RNA in mitochondrial fractions from cells grown at elevated sucrose levels.     

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     

Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum

 The effect of the addition of oleuropein (OLP) and NaCl on the growth and the DL-lactic acid production of Lactobacillus plantarum DSM 10492 has been investigated by using an unconventional medium. The growth of L. plantarum was not inhibited by the addition of increasing amounts of untreated OLP in the presence or absence of glucose. However, bacterial cells grew in quantity slightly with OLP alone. The increased addition of NaCl was associated with a delay in growth. Moreover, there was no growth with 8% NaCl. The addition of both NaCl and OLP resulted in growth inhibition, and the survival of cells decreased strongly. The main fermentation product was DL-lactic acid, but acetic acid was also detected after a prolonged incubation. L. plantarum produced DL-lactic acid in the presence of OLP alone but its formation decreased with increasing levels of OLP. On the other hand, heat-treated OLP had a bactericidal effect. Received: 16 October 1995/Received last revision: 5 February 1996/Accepted: 12 February 1996     

Uptake and incorporation of the products of proteolysis by the rumen bacteriumBacteroides ruminicola R8/4

The polypeptides of the proteolytic rumen bacteriumBacteroides ruminicola R8/4 grown in the presence of either leaf Fraction 1 protein, bovine serum albumin, or Bactocasitone as sole nitrogen source were separated by SDS-polyacrylamide gel electrophoresis. Over 40 polypeptides were resolved; the pattern for organisms grown on Fraction 1 protein was similar but not identical to that of the serum albumin and Bactocasitone-grown bacteria. All the bacterial polypeptides were distinguishable from the polypeptides of Fraction 1 protein (and serum albumin). The stained pattern was the same for organisms sampled at intervals during the growth of a batch culture. After incubation of the growing organisms with [¹⁴C]-Fraction 1 protein, all the bacterial polypeptides were labeled. Bacteria grown in the presence of nonlabeled Fraction 1 protein and a mixture of [^14C]-labeled amino acids incorporated label into all the polypeptides; the bacteria did not grow in the absence of intact protein, and then virtually no label was incorporated from the amino acid mixture.     

Intracytoplasmic membrane production inEscherichia coli O111a1: Nutritional parameters

Escherichia coli O111a₁ ceased growth prematurely and accumulated intracytoplasmic membrane at 42°C in an amino acids-mineral salts medium. The amount of membrane formed appeared to be proportional to the concentration of amino acids in the medium—the greater the concentration of amino acids in the medium, the greater the membrane production.E. coli O111a₁, did not grow at 42°C in glucose-, glycerol- or acetate-mineral salts medium, but mesosome-like structures were produced in glucose-grown cells and some intracytoplasmic membrane in cells grown on glycerol and acetate. Supplementation of the glucose medium with pantothenate and/or thiamine permitted normal growth. The vitamins did not restore growth of the mutant in glycerol or acetate, but intracytoplasmic membrane production was increased, especially in glycerol. Amino acids plus glucose supported normal growth with no membrane production. Glycerol and acetate had no effect on the growth in the amino acids medium, but stimulated the accumulation of membrane.