A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M_r cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.     

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens

Geobacter sulfurreducens is a δ-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.     

Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Basic Properties of the p38 Signaling Pathway in Response to Hyperosmotic Shock

Some properties of signaling systems, like ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none responses at a single cell level, are important to understand the regulation of irreversible processes. Xenopus oocytes are a suitable cell model to study these properties. The p38 MAPK (mitogen-activated protein kinase) pathway is activated by different stress stimuli, including osmostress, and regulates multiple biological processes, from immune response to cell cycle. Recently, we have reported that activation of p38 and JNK regulate osmostress-induced apoptosis in Xenopus oocytes and that sustained activation of p38 accelerates cytochrome c release and caspase-3 activation. However, the signaling properties of p38 in response to hyperosmotic shock have not been studied. Here we show, using Xenopus oocytes as a cell model, that hyperosmotic shock activates the p38 signaling pathway with an ultrasensitive and bimodal response in a time-dependent manner, and with low hysteresis. At a single cell level, p38 activation is not well correlated with cytochrome c release 2 h after hyperosmotic shock, but a good correlation is observed at 4 h after treatment. Interestingly, cytochrome c microinjection induces p38 phosphorylation through caspase-3 activation, and caspase inhibition reduces p38 activation induced by osmostress, indicating that a positive feedback loop is engaged by hyperosmotic shock. To know the properties of the stress protein kinases activated by hyperosmotic shock will facilitate the design of computational models to predict cellular responses in human diseases caused by perturbations in fluid osmolarity.     

Regulation of glycerol synthesis in response to osmotic changes in dunaliella

Changes in phosphometabolites, following osmotic shock, were analyzed by two-dimensional thin layer chromatography, in extracts of the halotolerant alga Dunaliella salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphorylcholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in Dunaliella. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in Dunaliella is discussed.     

Aquaporin AqpZ Is Involved in Cell Volume Regulation and Sensitivity to Osmotic Stress in Synechocystis sp. Strain PCC 6803

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.     

Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress

During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca²⁺/calmodulin–dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P₂ phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P₂-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca²⁺/calcineurin and PI(4,5)P₂ signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.     

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.     

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.     

Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1⁻ cells undergo mitotic delay at elevated temperatures and G₂ arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1⁻ cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24⁻ does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.     

PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: Evidence for the involvement of oxidative stress

Hyperosmotic shock elicits a stress response in mammalian cells and can lead to apoptotic cell death. In the present study, we report that hyperosmotic shock can induce activation of a 36 kDa kinase detected by an in-gel kinase assay in several cell types, including mouse Balb/c 3T3 fibroblasts, and human Hep 3B and A431 cells. This 36 kDa kinase can be recognized by an antibody against the C-terminal region of a family of p21^Cdc42/Rac-activated kinases (PAKs) on immunoblot. Further studies with this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-terminal catalytic fragment in cells. The cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in hyperosmotically shocked cells. Furthermore, pretreating the cells with two caspase inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all these hyperosmotic shock-induced changes (i.e., activation of caspase-3, cleavage/activation of PAK2, and DNA fragmentation) in cells could be blocked by antioxidants such as ascorbic acid (vitamine C), α-tocopherol (vitamine E), dithiothreitol, β-mercaptoethanol, and glutathione. Taken together, our results show that PAK2 is cleaved and activated via a caspase-dependent mechanism during hyperosmotic shock–induced apoptosis and suggest the involvement of antioxidant-preventable oxidative stress in inducing this process. J. Cell. Physiol. 178:397–408, 1999. © 1999 Wiley-Liss, Inc.     

Influence of hyperosmolar basal media on hybridoma cell growth and antibody production

To investigate the influence of hyperosmolar basal media on hybridoma response, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in a batch mode using hyperosmolar basal media resulting from additional sodium chloride supplementation. The basal media used in this study were IMDM, DMEM, and RPMI 1640, all of which are widely used for hybridoma cell culture. In IMDM, two hybridomas showed different responses to hyperosmotic stress regarding specific MAb productivity (q _MAb), though they showed similar depression of cell growth in hyperosmolar media. Unlike S3H5/γ2bA2 hybridoma, the q _MAb of DB9G8 hybridoma was not enhanced significantly around 390 mOsm kg^?1. The variation of basal media influenced DB9G8 hybridoma response to hyperosmotic stress regarding q _MAb. In IMDM, the q _MAb of DB9G8 hybridoma was increased by more than 200% when the osmolality increased from 281 to 440 mOsm/kg. However, in RPMI 1640 and DMEM, similar amplitude of osmolality increase resulted in less than 100% increase in q _MAb. The variation of basal media also influenced the cell growth in hyperosmolar medium. Both hybridomas were more tolerant against hyperosmotic stress in DMEM than in IMDM, which was found to be due to the high osmolality of standard DMEM. The osmolalities of standard IMDM and DMEM used for inocula preparation were 281 and 316 mOsm kg^?1, respectively. Thus, when the cells were cultivated at 440 mOsm kg^?1, the cells in IMDM experienced higher osmotic shock than in DMEM. By using the inoculum prepared at 317 mOsm kg^?1 in IMDM, S3H5/γ2bA2 cell growth at 440 mOsm kg^?1 in IMDM was comparable to that in DMEM. Taken together, the results obtained from this study show that the selection of basal media is an important factor for MAb production by employing hyperosmotic stress.     

Hyperosmotic Shock Engages Two Positive Feedback Loops through Caspase-3-dependent Proteolysis of JNK1-2 and Bid

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-x_L markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-x_L co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.     

The actin-binding protein comitin (p24) is a component of the Golgi apparatus

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.     

Protein kinases involved in mating and osmotic stress in the yeast Kluyveromyces lactis

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Gα and Gβ, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.     

Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network

Hyperosmotic stress can be encountered by the kidney and the skin, as well as during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells detect hyperosmolarity and how the cell chooses between cell cycle arrest or death remains to be established. It has been proposed that hyperosmolarity is detected directly by growth factor receptor protein tyrosine kinases. To investigate this, we tested whether growth factors and osmotic stress cooperate in the activation of signaling pathways. Receptors responded normally to the presence of growth factors, and we observed normal levels of GTP-bound Ras under hyperosmotic conditions. In contrast, activation of Raf, Akt, ERK1, ERK2, and c-Jun NH₂-terminal kinase was strongly reduced. These observations suggest that hyperosmotic conditions block signaling directly downstream of active Ras. It is thought that apoptotic cell death due to environmental stress is initiated by cytochrome c release from the mitochondria. Visualization of cytochrome c using immunofluorescence showed that hypertonic conditions result in a breakup of the mitochondrial network, which is reestablished within 1 h after hypertonic medium is replaced with isotonic medium. When we carried out live imaging, we observed that the mitochondrial membrane potential disappeared immediately after the onset of hyperosmotic shock. Our observations provide new insights into the hypertonic stress response pathway. In addition, they show that signaling downstream of Ras and mitochondrial dynamics can easily be manipulated by the exposure of cells to hyperosmotic conditions. protein tyrosine kinases; Ras; mitogen-activated protein kinase; hyperosmotic shock     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.