Osmotic Behavior of Bacterial Protoplasts: Temperature Effects

Among protoplasts released from cells of Bacillus megaterium grown at 20, 30, or 37 C, osmotic swelling in NaCl solution at a given external osmotic pressure was greatest for protoplasts from cells grown at 20 C and least for protoplasts from cells grown at 37 C. Protoplasts from cells grown at lower temperaturs were also less stable to osmotic shock and lysed at higher external osmotic pressures than did protoplasts from cells grown at higher temperatures. But for cells grown at any one temperature, osmotic stabilization was itself temperature dependent so that the higher the ambient incubation temperature, the higher the osmotic pressure needed to prevent lysis of a given fraction of the input protoplast population. However, comparison of the osmotic stability of protoplasts from cells grown at different temperatures at various ambient incubation temperatures revealed that, except at 5 C where no differences were discerned, protoplasts from cells grown at lower temperatures still lysed at higher osmotic pressures than did those from cells grown at higher temperatures. The apparent internal osmolality (28 to 31 atm) did not vary significantly among whole cells from the three growth temperatures. Therefore, the observed differences in osmotic behavior could not be attributed to changes in internal osmotic pressure. Rather, it seemed likely that the differences were due to changes in membrane properties.     

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

     

Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.     

Turgor and net ion flux responses to activation of the osmotic MAP kinase cascade by fludioxonil in the filamentous fungus Neurospora crassa

The internal hydrostatic pressure (turgor) of the filamentous fungus Neurospora crassa is regulated at about 400–500 kiloPascals, primarily by an osmotic MAP kinase cascade which activates ion uptake from the extracellular medium and glycerol synthesis. In the absence of hyperosmotic stress, the phenylpyrrole fungicide fludioxonil activates the osmotic MAP kinase cascade, resulting in cell death. Turgor, the electrical potential and net ion fluxes were measured after treatment with fludioxonil. In wildtype, fludioxonil causes a hyperpolarization of the plasma membrane and net H⁺ efflux from the cell, consistent with activation of the H⁺-ATPase. At the same time, net K⁺ uptake occurs, and turgor increases (about 2-fold above normal levels). None of these changes are observed in the os–2 mutant (which lacks a functional MAP kinase, the last of the three kinases in the osmotic MAP kinase cascade). Tip growth ceases as hyperpolarization, net ion flux changes, and turgor increases begin. The inappropriate turgor increase is the probable cause of eventual lysis and death. The results corroborate a multi-pathway response to hyperosmotic stress that includes activation of plasma membrane transport. The relation to cell expansion (tip growth) is not direct. Increases in turgor due to ion transport might be expected to increase growth rate, but this does not occur. Instead, there must be a complex regulatory interplay between the growth and the turgor driving force, possibly mediated by regulation of cell wall extensibility.     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative α-cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade β-glucan of their cell walls.     

Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae.

Physiological states associated with inositol starvation of spheroplasts of Saccharomyces cerevisiae were investigated and compared with conditions preceding death of starved whole cells. In the absence of synthesis of inositol-containing lipids, cell surface expansion terminated after one doubling of whole cells. In spheroplasts, cessation of membrane expansion was apparently followed by rapid development of an osmotic imbalance, causing lysis. Continued synthesis and accumulation of cytoplasmic constituents within the limited cell volume were implicated as a cause of the osmotic imbalance. In whole cells, an increase in internal osmotic pressure also follows termination of membrane and cell wall expansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmic osmotic pressure to persist in the period preceding onset of inositol-less death.     

Expansins are involved in cell growth mediated by abscisic acid and indole-3-acetic acid under drought stress in wheat

Expansin protein is a component of the cell wall generally accepted to be the key regulator of cell wall extension during plant growth. Plant hormones regulate expansin gene expression as well as plant growth during drought stress. However, the relationship between expansin and plant hormone is far from clear. Here, we studied the involvement of expansin in plant cell growth mediated by the hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) under osmotic stress which was induced by polyethylene glycol (PEG)-6000. Wheat coleoptiles from a drought-resistant cultivar HF9703 and a drought-sensitive cultivar 921842 were used to evaluate cell growth and expansin activity. Osmotic stress induced the accumulation of ABA. ABA induced expansin activity mainly by enhancing expansin expression, since ABA induced cell wall basification via decreasing plasma membrane H⁺-ATPase activity, which was unfavorable for expansin activity. Although ABA induced expansin activity and cell wall extension, treatment with exogenous ABA and/or fluridone (FLU, an ABA inhibitor) suggested that ABA was involved in the coleoptile growth inhibition during osmotic stress. IAA application to detached coleoptiles also enhanced coleoptile growth and increased expansin activity, but unlike ABA, IAA-induced expansin activity was mainly due to the decrease of cell wall pH by increasing plasma membrane H⁺-ATPase activity. Compared with drought-sensitive cultivar, the drought-resistant cultivar could maintain greater expansin activity and cell wall extension, which was contributive to its resultant faster growth under water stress.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Effect of glucose and sucrose on the survival in batch culture of Streptococcus mutans C67-1 and a non-cariogenic mutant C67-25. Morphological studies.

This study comprised an ultrastructural examination of a cariogenic strain of Streptococcus mutans, C67-1, and a non-cariogenic mutant of that strain, C67-25. The aim of the work was to define more clearly the relationship between S. mutans and dental caries and, more specifically, to elicit ultrastructural evidence for the conclusion from a previous investigation that the greater survival of the parent strain in sucrose broth at uncontrolled pH was related partly to the production in this medium of abundant extracellular polysaccharide (EPS). The strains were grown as previously in 5% (w/v) glucose or sucrose broths, the pH being either allowed to fall or maintained above 6.0, and processed by the thiosemicarbazide technique for election microscopy. It was confirmed that EPS was most abundant in the sucrose broth culture of the parent strain at uncontrolled pH. While the presence of abundant EPS relates to the greater survival of the parent strain in sucrose broth at uncontrolled pH, this organism possesses at least one other mechanism of survival in acid media, possibly dependent on cell wall properties, in view of its greater cell wall thickness and increased survival in pH-uncontrolled glucose broth in the absence of detectable EPS production. It is postulated that intracellular and extracellular polysaccharide formation, cell wall thickening and reduced viability were indicators of unfavourable growth conditions in the test media. Cariogenic strains of S. mutans appear to be able to survive better under such conditions and hence the prevalence of this and other polysaccharide-producing organisms in stagnant sites in natural dental plaques.     

Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation

The effect of growth temperature on the loss of virulence of the fish pathogen Aeromonas salmonicida was investigated. Three virulent strains were grown in Trypticase soy broth at temperatures ranging from 22 to 30 degrees C. Growth at a higher-than-optimal temperature (26 to 27 degrees C for the three strains studied) resulted in the selection of spontaneous attenuated derivatives in the initial bacterial population. For example, virulent bacteria represented less than 10% of the population of a culture grown at 30 degrees C, and attenuated derivatives were easily isolated by streaking the culture on solid medium and picking single colonies. Virulent strains autoaggregated during growth and possessed a cell wall layer (A-layer) external to the outer membrane, as previously described. Attenuated strains did not autoaggregate and did not possess the A-layer. The A-layer apparently shielded bacteriophage receptors and a mannose-specific yeast agglutinin located in the outer membrane. Thus, virulent strains exhibited impaired adsorption of phages, whereas attenuated strains were phage sensitive. Furthermore, attenuated strains agglutinated yeast cells but virulent strains did not. The attenuated strains had higher maximum growth temperatures than their virulent parent strains, and this accounts for their selection at high temperatures. It is proposed that the A-layer contributes significantly to the physical properties of the A. salmonicida cell envelope and that these physical properties of the A. salmonicida cell envelope and that these physical change upon loss of the A-layer to permit growth at a higher-than-usual temperature.     

TRANSFORMATION OF ROD-SHAPED ALGAL CELLS INTO SPHERICAL CELLS

Transformation of the rod-shaped algal cells, which before the conversion could be considered as belonging to the genus Stichococcus, into spherical cells was achieved by the action of the tryptic soy broth and some other organic media. Transformation did not involve degradation of the cell wall, and the resulting spheres were not sensitive to osmotic shock. The transformed cells could be propagated as spheres for several months but eventually reverted to the original rod shape. The reversion could be hastened by transferring spherical cells back into inorganic medium and growing the cells autotrophically. For a long time after reversion, the cells maintained characteristics different from those of the original rod-shaped cells which did not go through the conversion and reversion processes. The reverted cells had a higher rate of heterotrophic growth, a lower rate of autotrophic growth, and a lower capacity for being again transformed into spheres under the influence of tryptic soy broth.     

THE INITIAL STRUCTURAL LESION OF PENICILLIN ACTION IN BACILLUS MEGATERIUM

The effect of penicillin on the structure of Bacillus megaterium cells was followed in media with and without osmotic stabilization. In peptone without osmotic support the cells showed a distortion of the normal membrane-wall relationship by 20 minutes. This appeared to be a combination of both membrane distortion and cytoplasmic leakage. Lytic changes quickly followed. With osmotic support a clean-cut lesion at the transverse-septal site developed by 10 minutes' growth in penicillin. The membrane lost its normal relationship to the cell wall and formed a pocket which was filled with a fibrous material which appeared to be unorganized wall mucopeptide. The pocket of fibers enlarged until the cell either lysed or formed a protoplast.     

Effects of growth temperature on protoplast membrane properties in Bacillus megaterium.

Changes in the protoplast membrane of the KM strain of Bacillus megaterium were assessed after growth at 20, 30, or 37 degrees, C. Although the overall membrane concentrations of lipids and proteins were virtually unchanged, increased culture temperature resulted in cells with membranes that contained relatively more unbranched and long-chain fatty acids and more acidic phospholipids, as well as different proportions and numbers of individual proteins. Electrophoretic analysis revealed 23, 31, or 29 protein bands, respectively, in membranes from cells grown at the three temperatures. Protoplasts from cells grown at higher temperatures were considerably less susceptible to lysis by shearing forces. As judged by passive leakage at 30 degrees C, intact cells from cultures grown at 37 degrees C were the least permeable to erythritol. Relatively low ambient concentrations of Ca2+ or Mg2+ protected protoplasts from osmotic lysis but even much higher concentrations left erythritol leakage virtually unaffected. Thus, growth temperature affected not only membrane lipis but also membrane proteins and these changes resulted in membranes with altered mechanical properties and permeabilities.     

Induction of Enterococcal L-Forms by the Action of Lysozyme

Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.     

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.     

Assessment of cellular damage during cooling to −196 °C using radiochemical markers

The release of ten radiochemical markers from MRC-5 and CHO cells after cooling at various rates and thawing from temperatures in the range of 0 to ?196 °C was measured. Many of these radiochemicals had specific sites of attachment on or within the cell and the aim was to determine the effect of freeze-thaw stresses on various parts of the cell. Cell death during cooling and thawing was, in most instances, accompanied by osmotic damage and loss of cytoplasmic constituents. Significant damage to the cell membrane occurred only after the cell was already dead and was related to the disruption of cells killed at higher temperatures and to osmotic stress during rewarming. The release of cations and other cytoplasmic markers was correlated to cell shrinkage and dehydration. The data were used to assess the relative effects of some of the proposed damaging factors in freeze-thaw injury (thermal shock, ice damage, dilution shock, etc.). CHO cells showed a much higher survival rate and release of cations after fast cooling than MRC-5 cells. This, and additional circumstantial information, indicated that CHO cells survived freeze-thaw cycles better than MRC-5 cells because they are able to dehydrate more readily, even at fast cooling rates.     

Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.     

Locus of the Action of Serum and the Role of Lysozyme in the Serum Bactericidal Reaction

The mechanism of the lethal action of human serum on a rough strain of Escherichia coli was investigated by use of serum with and without lysozyme, in medium of low and high osmotic pressure, with cells radioactively labeled in the peptidoglycan polymer, and by electron microscopy. The results suggested that there are two separate components in the bacterial cell wall that afford structural support for the cell. Lysozyme attacked one of these, the peptidoglycan polymer. Serum damaged the other, which is probably the peripherally located lipopolysaccharide-phospholipid complex. The cell wall damage caused by lysozyme-free serum promptly resulted in cell death under usual conditions. In plasmolyzed cells, however, the wall damage was not lethal, presumably because the membrane of the plasmolyzed cell was protected from secondary lethal changes which otherwise occur.     

Loss of function of KRE5 suppresses temperature sensitivity of mutants lacking mitochondrial anionic lipids

Disruption of PGS1, which encodes the enzyme that catalyzes the committed step of cardiolipin (CL) synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol (PG) and CL. The pgs1Delta mutant exhibits severe growth defects at 37 degrees C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Delta that grew at 37 degrees C. One of the suppressors has a loss of function mutation in KRE5, which is involved in cell wall biogenesis. The cell wall of pgs1Delta contained markedly reduced beta-1,3-glucan, which was restored in the suppressor. Stabilization of the cell wall with osmotic support alleviated the cell wall defects of pgs1Delta and suppressed the temperature sensitivity of all CL-deficient mutants. Evidence is presented suggesting that the previously reported inability of pgs1Delta to grow in the presence of ethidium bromide was due to defective cell wall integrity, not from "petite lethality." These findings demonstrated that mitochondrial anionic lipids are required for cellular functions that are essential in cell wall biogenesis, the maintenance of cell integrity, and survival at elevated temperature.     

Plasma membrane expansion terminates in Saccharomyces cerevisiae secretion-defective mutants while phospholipid synthesis continues.

Phospholipid synthesis activity and plasma membrane growth have been studied in the Saccharomyces cerevisiae temperature-sensitive, secretion-defective mutants isolated by Novick and Schekman (Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979; Novick et al., Cell 21:205-215, 1980). The mutants, sec1 through sec23, do not grow at 37 degrees C and exhibit lower rates of phospholipid synthesis than does the wild-type strain X2180. None of the mutants exhibits a decline in lipid synthesis rapid enough to explain secretion failure. Plasma membrane growth was assessed indirectly by examining the osmotic sensitivity of spheroplasts derived from cultures transferred from 24 to 37 degrees C. Spheroplasts from the normal-growing strain X2180 exhibited a small rapid increase in osmotic sensitivity and stabilized at a more sensitive state. Spheroplasts from the sec mutants exposed to the same temperature shift exhibited progressively increasing osmotic sensitivity. Cycloheximide treatment prevented progressive increases in osmotic fragility. These data are compatible with the hypothesis that plasma membrane expansion is restricted in the sec mutants. During incubation at 37 degrees C, the accumulation of intracellular materials within the no-longer expanding plasma membrane exerts osmotic stress on the membrane, increasing with time. The gene products defective in Novick and Schekman's sec mutants appear to be required for both extracellular protein secretion and plasma membrane growth in yeast cells.