Outer membrane porins are important in maintenance of the surface structure of Escherichia coli cells.

Escherichia coli cells lacking the OmpF and OmpC proteins, porin proteins of the outer membrane, are often unstable and easily revert to strains which either have regained one or both of these proteins or contain a new outer membrane protein. The structural importance of porin proteins in the cell surface was studied in the present work. Tris-hydrochloride buffer at a concentration of 120 mM caused deformation of the cell surface of a strain lacking these porins; the undulated appearance of the negatively stained cell surface changed to a smooth and expanded form. The Tris-induced deformation was seldom observed with either the wild-type strain or a pseudorevertant that possessed the OmpF protein. The role of the OmpF protein in stabilizing the cell surface against Tris treatment could be slightly taken over by the LamB protein, which shares a number of unique properties with the former proteins. The deformation of the cell surface by Tris-hydrochloride buffer was accompanied by a loss of viability, the lethal damage being especially significant when the cells lacked porins. Upon induction with maltose, cells with the undulated appearance could absorb lambda phages, whereas the deformed cells could not. These results suggest that the instability of cells lacking porins is primarily due to a structural defect of the outer membrane.     

The Effect of Putrescine on the Apoplast Ultrastructure in the Leaf Mesophyll of Mesembryanthemum crystallinum under Salinity Stress

The effects of NaCl, putrescine (Put), and the combination of two agents on the contents of free polyamines (PA), peroxidase activity, and the ultrastructure of the mesophyll apoplast were studied in the third leaf pair of Mesembryanthemum crystallinum L. plants. The NaCl solution was added to soil daily for three days (100 ml of the 100 mM solution), and plants were sprayed with the 1 mM Put solution twice per day for a six-day period. The accumulation of Put and especially spermidine (Spd) by day 3 of salinization was followed by a dramatic drop in Put and Spd contents by day 6. In contrast, the activities of soluble and ion-bound peroxidases increased following a long lag-period. Treatments with Put and NaCl plus Put considerably enhanced this rise in two peroxidase activities. An electron microscopic examination of cell walls in the control and stressed plants demonstrated that a gap developed at the middle lamella with pockets filled with amorphous polysaccharides (AP), presumably pectins. At the maximum gap width, the pockets fused with the intercellular spaces, and, in this case, the intercellular spaces also contained AP. Following salinization, AP in the apoplast swelled and expanded. Apparently the genetic determination of high AP content in M. crystallinum plants is the basis for the ability of juvenile plants to bind Na⁺ and Cl^– ions and excess PA and also to accumulate water. In the plants treated with NaCl plus Put, the number of pockets and their volume increased, and the surface of some cell walls became plated with suberin, thus providing an additional barrier for ion transport from the pockets and intercellular spaces into cells. The formation of suberin plates was correlated with the high activity of ion-bound peroxidase essential for suberin deposition. The authors presume that H₂O₂ results from PA oxidation and, in its turn, induces the activity of peroxidase involved in the suberin plate formation.     

Ultrastructure of Aeluropus littoralis leaf salt glands under NaCl stress

The effects of salt uptake on the morphology and ultrastructure of leaf salt glands were investigated in Aeluropus littoralis plants grown for two months in the presence of 400 mM NaCl. The salt gland is composed of two linked cells, as observed in some other studied Poaceae species. The cap cell, which protrudes from the leaf surface, is smaller than the basal cell, which is embedded in the leaf mesophyll tissues and bears the former. The cuticle over the cap cell is frequently separated from the cell wall to form a cavity where salts accumulate prior to excretion. The basal cell cytoplasm contains an extensive intricate or partitioning membrane system that is probably involved in the excretion process, which is absent from the cap cell. The intricate membrane system seems to be elongated and heavily loaded with salt. The presence of 400 mM NaCl induced the disappearance of the collecting chamber over the glands and an increase in the number of vacuoles and their size in both gland cells. In the basal cell, salt greatly increased both the density and size of the intricate membrane system. The electron density of both gland cells observed under salt treatment reflects a high activity. All these changes probably constitute special adaptations for dealing with salt accumulation in the leaves. Despite the high salt concentration used, no serious damage occurred in A. littoralis salt gland ultrastructure, which consolidates the assumption that they are naturally designated for this purpose.     

Survival of biological cells deformed in a narrow gap

Recent studies show that during slow freezing of biological cells, the cells may be also injured by not only chemical damage but also mechanical damage induced by ice crystal compression. A new experimental procedure is developed to quantify cell destruction by deformation with two parallel surfaces. The viability of cells (prostatic carcinoma cells, 17.5 microns in mean diameter) is measured as a function of gap size ranging from 3.5 microns to 16.2 microns at 0 degree C, 23 degrees C and 37 degrees C. The viability at a smaller gap size is significantly lower at 37 degrees C than at 23 degrees C, while the difference between 0 degree C and 23 degrees C is much smaller. This suggests that deformation damage is related to the deformation of the cytoskeleton rather than the mechanical properties of the lipid membrane.     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na⁺, K⁺ and Cl⁻ concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na⁺ and Cl⁻ accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K⁺ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.     

Studies of excitable membrane formed on the surface of protoplasmic drops isolated from Nitella II. Tension at the surface of protoplasmic drops

A protoplasmic drop isolated from an internodal cell of Nitella became electrically excitable in a solution containing 0.5 mM NaCl, 0.5 mM KNO₃, 1mM Ca(NO₃)₂ and 2mM Mg(NO₃)₂. A thermodynamic property of the excitable membrane was characterized in terms of tension at the surface of the protoplasmic drop. This was determined by the compression method and/or by the sessile-drop method. The surface tension of the membrane was obtained as a function of the composition of the salts in the external solution, and the time during the formative period of the excitable surface membrane. The results are summarized as follows:

1. 1. The surface of the protoplasmic drop increased with time starting from 0.003 dyne/cm and approached a steady value of about 0.1 dyne/cm within 1 h after the drop was placed in the test solution described above. The membrane became electrically excitable when the surface tension attained the steady value.

2. 2. Increase of concentration of either Na⁺ or K⁺ in the solution induced a sudden decrease of the surface tension, which followed a suppression of the excitability. The critical concentration of Na⁺ or K⁺ was about 10 mM.

3. 3. The surface tension remained constant at about 0.1 dyne/cm in a Ca²⁺ concentration ranging between about 0.1 and 10 mM. At this concentration the drop was excitable. Below and above this range of Ca²⁺ concentration, the surface tension changed sharply with concentration, and the excitability disappeared. At about 0.1 mM Ca²⁺ concentration a discrete variation of the surface tension was observed.

4. 4. The surface tension of the drop stayed constant at 0.1 dyne/cm in the range between 1 and 10 mM of Mg²⁺ concentration. Above and below this range of Mg²⁺ concentration, the surface tension increased sharply with the variation of Mg²⁺ concentration.

These results indicate that the protoplasmic drop retains its excitability in a limited range of salt composition in the external solution. This implies that the excitable membrane of the drop must be very labile in its structure against external perturbations such as electrical stimulus and/or slight variation of salt composition in the solution.     

Physiological and proteomic analysis of salinity tolerance of the halotolerant cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp

The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na⁺ content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K⁺ content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K⁺/Na⁺ ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K⁺/Na⁺. Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF–MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na⁺ content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na⁺ ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the halotolerant cyanobacterium Anabaena sp is grown under low and high salinity levels.     

Changes in animal cell natural aggregates in suspended batch cultures

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical aissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1. 2 · 10⁶ cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 · 10⁸ cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells.     

Glucosylglycerol,a compatible solute,sustains cell division under salt stress

The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress. Although the molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated, the role of GG in protection against salt stress remains poorly understood. In our study of the role of GG in the tolerance to salt stress, we found that salt stress due to 450 mM NaCl inhibited cell division and significantly increased cell size in DeltaggpS mutant cells, whereas the inhibition of cell division and increase in cell size were observed in wild-type cells at high concentrations of NaCl, such as 800 mM. Electron microscopy revealed that, in DeltaggpS cells, separation of daughter cells was incomplete, and aborted division could be recognized by the presence of a structure that resembled a division ring. The addition of GG to the culture medium protected DeltaggpS cells against salt stress and reversed the adverse effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions.     

Development of the Casparian strip in primary roots of maize under salt stress

The Casparian strip in the endodermis of vascular plant roots appears to play an important role in preventing the influx of salts into the stele through the apoplast under salt stress. The effects of salinity on the development and morphology of the Casparian strip in primary roots of maize (Zea mays L.) were studied. Compared to the controls, the strip matured closer to the root tip with increase in the ambient concentration of NaCl. During growth in 200 mM NaCl, the number and the length of the endodermal cells in the region between the root tip and the lowest position of the endodermal strip decreased, as did the apparent rate of production of cells in single files of endodermal cells (the rate of cell formation being equal to the rate at which cells are lost from the meristem). The estimated time required for an individual cell to complete the formation of the strip after generation of the cell in the presence of 200 mM NaCl was not very different from that required in controls. Thus, salinity did not substantially affect the actual process of formation of the strip in individual cells. The radial width of the Casparian strip, a morphological parameter that should be related to the effectiveness of the strip as a barrier, increased in the presence of 200 mM NaCl. The mean width of the lignified region was 0.92 m in distilled water and 1.33 m in 200 mM NaCl at the lowest position of the strip. The mean width of the strip relative to that of the radial wall at this position was significantly greater after growth in the presence of 200 mM NaCl than in the controls, namely, 20.5% in distilled water and 33.9% in 200 mM NaCl. These observations suggest that the function of the strip is enhanced under salt stress.     

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.     

Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperatures above 0 degrees C

Cell injury due to osmotic dehydration, which is regarded as a major cause of injury during freeze-thaw processes, was examined closely using a perfusion microscope. Human prostatic adenocarcinoma cells (PC-3), which were put in a chamber, were subjected to hyperosmotic stresses by perfusing NaCl solutions of varying concentrations into the chamber. Cells were exposed to 2.5 and 4.5M NaCl solutions for 1-60 min by changing the concentrations at 0.2, 1, and 10 M/min. Decrease in cell viability was biphasic: the viability decreased first after the increase in NaCl concentration due to dehydration and then after return to isotonic conditions due to rehydration. Rehydration was substantially more responsible for cell injury than dehydration, which was marked at lower NaCl concentrations and lower temperatures. Injury resulting from contraction was negligible at the 2.5 M NaCl solution. While the hypertonic cell survival, which was determined without a return to isotonic conditions, was almost independent of time of exposure to hyperosmotic concentrations, the post-hypertonic survival after returning to isotonic conditions decreased with increasing exposure time, suggesting that the rehydration-induced injury was a consequence of time-dependent alteration of the plasma membrane. The post-hypertonic survival was lower for higher NaCl concentrations and higher temperatures, which was qualitatively consistent with previous studies. Effects of the rate of concentration change on the post-hypertonic cell survival were observed at 4.5 M; the highest rate of survival was obtained by slower increase and faster decrease in the NaCl concentration. However, the effect was negligible at 2.5 M.     

Anatomical Changes in Prosopis tamarugo Phil. Seedlings Growing at Different Levels of NaCl Salinity

Seedlings of Prosopis tamarugo were grown in artificial substrateswith additions, of 200, 400 and 600 mM NaCl, and without salttreatment. Salinity induced anatomical changes in the roots,stems and leaflets. The diameters of the roots of seedlingsgrown in the increasing salt concentrations (up to 400 mM) wereprogressively smaller and differentiation of the stelar tissueswas delayed. At an NaCl concentration of 600 mM, the root structurewas strongly altered. On the contrary, stem diameter increasedas salinity rose. In the stems of seedlings grown in a concentrationof 200 mM NaCl, secondary xylem differentiation appeared earlierthan in the controls. At a concentration of 400 mM NaCl, disorganizationof the vascular cylinder of the stem was evident. Leaflets ofseedlings grown in 200 mM NaCl showed a delay in structuraldifferentiation: no water-storage cells or ‘special cells’could be seen. The leaflets from plants grown in 400 mM NaCl,had larger numbers of intercellular air spaces; probably anindication of the beginning of tissue disorganization. A progressivedecrease in cell size of leaflets as salinity rose was alsodemonstrated. Prosopis tamarugo Phil., seedling, salinity, anatomical changes     

Sodium chloride affects propidium monoazide action to distinguish viable cells

Propidium monoazide (PMA) is a DNA-intercalating agent used to selectively detect DNA from viable cells by polymerase chain reaction (PCR). Here, we report that high concentrations (>5%) of sodium chloride (NaCl) prevents PMA from inhibiting DNA amplification from dead cells. Moreover, Halobacterium salinarum was unable to maintain cell integrity in solutions containing less than 15% NaCl, indicating that extreme halophilic microorganisms may not resist the concentration range in which PMA fully acts. We conclude that NaCl, but not pH, directly affects the efficiency of PMA treatment, limiting its use for cell viability assessment of halophiles and in hypersaline samples.     

Ionic homeostasis disturbance is involved in tomato cell death induced by NaCl and salicylic acid

The ability of salicylic acid and NaCl to induce programmed cell death by disturbing ionic homeostasis was investigated using tomato suspension culture cells. NaCl (300?mM) and salicylic acid (1?mM) inhibited cell growth and caused cell death within 1?wk of exposure. Treatment with NaCl increased the production of reactive oxygen species and the permeability of plasma membrane, but it also led to a reduction in the pH of the culture medium and resulted in a disturbance in ionic homeostasis of the cells. Salicylic acid-induced cell death in tomato suspension culture was also accompanied by production of reactive oxygen species and increases in both electrolyte leakage and pH of the culture media. However, reactive oxygen species production was not significantly different in cultures treated with a lethal salicylic acid concentration and 100?mM NaCl, in which most of the cells survived. A decrease in the K⁺/Na⁺ ratio was observed only in those cell cultures in which the salicylic acid treatment induced the death of cells. These results suggest that the decrease of the intracellular K⁺ concentration and K⁺/Na⁺ ratio is a common phenomenon in triggering programmed cell death by lethal concentrations of salicylic acid and NaCl.     

Chemokinetic behavior of the infective third-stage larvae of Strongyloides ratti on a sodium chloride gradient.

The movements of the infective third-stage larvae (L3) of a rodent parasitic nematode Strongyloides ratti were examined on a sodium chloride (NaCl) gradient set up on agarose plates. The movements of larvae were followed by observing their tracks on the surface of the agarose. The direction of movement depended on the NaCl concentration at the point of their initial placement on the gradient. Larvae placed at between 230 and 370 mM NaCl tended to migrate towards areas of lower concentration. On the other hand, when placed at concentrations less than 20 mM NaCl, larvae tended to migrate initially towards higher concentrations but did not linger in areas where the concentration was over approximately 80 mM NaCl. It seems that S. ratti L3, tested in vitro, prefer regions with a concentration of NaCl below 80 mM NaCl. Two typical chemokinetic behaviors are seen; a unidirectional avoidance movement when initially placed in unfavorable environmental conditions and a random dispersal movement when placed within an area of favorable conditions. Track patterns were straight in the avoidance movement but included multiple changes of direction and loops in the dispersal movement. This study introduces an assay system suitable for studying chemokinetic behavior of larvae of Strongyloides ratti.     

Elemental composition of extremely alkaliphilic anaerobic bacteria

The contents of several chemical elements were assessed in the haloalkaliphilic acetogenic bacterium Natroniella acetigena and the alkaliphilic sulfate-reducing bacterium Desulfonatronum lacustre using X-ray microanalysis, stereoscanning microscopy, and mass spectrometry. The organisms were found to differ significantly in their relative contents of S, K, P, and Cl. The P/S ratio in cells of the alkaliphilic bacteria studied grown on mineral media at different pH was pH-dependent. With a pH increase from 9 to 10, potassium extrusion from cells was observed, suggesting that secondary K+/H+ antiport activity accounts for the homeostasis of cytosolic pH. Deenergization of bacterial cells in the presence of inhibitors and ionophores results in specific changes in the P/S ratio, which may be considered an indicator of the cell energetic state. In Natroniella acetigena, the content of intracellular Cl was directly proportional to the NaCl concentration in the medium. Some metals were shown to be necessary for the N. acetigena viability; the requirement for Ni and Co was absolute. Although little demand for Mg was characteristic of the bacteria studied, their growth was stimulated by an increase in Mg concentration, and the cell resistance to lysis was enhanced.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.