Effect of glucose on the biosynthesis of the membranes of bacillus

In the presence of glucose in complex media, the following changes in the characters of the membranes of Bacillus subtilis and Bacillus cereus were observed. (1) The activity of succinate dehydrogenase and the amount of cytochromes of the membranes were greatly reduced. (2) The ratio of lipid to protein in the membranes was decreased and a membrane subfraction, which had a density of around 1.2 (B. subtilis) and 1.24 g/cm³ (B. cereus), was newly formed (B. subtilis) or increased (B. cereus). (3) The phospholipid and diglyceride contents in the membranes were reduced.Polyacrylamide gel electrophoresis of proteins of the two types (plus and minus glucose) of the membranes show that the patterns were very different between the two types of membranes, though the lost or newly formed membrane protein components were not observed.The cytochrome content was not increased when the cells were grown in glucose medium supplemented with haemin, therefore, glucose or its metabolite may not be involved in the inhibition of haem biosynthesis.     

Membrane tubulin

Tubulin has been identified as a membrane component of nerve synaptosomes and myelin, plasma membranes of platelets, thyroid, and tissue culture cells, brain and liver coated vesicles, mitochondria, and in cilia but not flagella of certain molluscs. Membrane tubulin can differ from cytoplasmic forms in isoelectric point, non-polar amino acid substitutions, lack of carboxy-terminal tyrosine, carbohydrate content, and selective ability to reassociate with lipids. This tubulin may function as an attachment site for binding vesicles or plasma membranes to cytoplasmic microtubules, as a source of precursor tubulin at the growing tips of axonemes, or as a component of signal transduction in sensory cilia.     

Formate protects stationary-phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide

For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary-phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability-increasing protein (BPI). BPI-derived peptide P2 rapidly halted oxygen consumption by stationary-phase cells preincubated with glucose, pyruvate or malate and caused a 109-fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary-phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh-N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.     

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.Abbreviations used IM inner membrane - OM outer membrane - KDO 2-keto-3-deoxyoctonate - EDTA ethylenediaminetetraacetic acid - SDH succinate dehydrogenase     

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

The effect of transfer from low to high light intensity on electron transport in Rhodospirillum rubrum membranes

The effects of transfer from low to high ligh intensity on membrane bound electrontransport reactions of Rhodospirillum rubrum were investigated. The experiments were performed with cultures which did not form bacteriochlorophyll (Bchl) for about two cell mass doublings during the initial phase of adaptation to high light intensity. Lack of Bchl synthesis causes a decrease of Bchl contents of cells and membranes. Also, the cellular amounts of photosynthetically active intracytoplasmic membranes decrease.In crude membrane fractions containing both cytoplasmic and intracytoplasmic membranes the initial activities of NADH oxidizing reactions increase only slightly (about 1.2 times) per protein, but the initial activities of succinate oxidizing reactions decrease (multiplied by a factor of 0.7). On a Bchl basis activities of NADH oxidizing reactions increase 3.4 times while activities of succinate dependent reactions increase 1.9 times. With isolated intracytoplasmic membranes activities of NADH as well as succinate dependent reactions increase to a comparable extent on a Bchl basis (about 1.8 times) and stay nearly constant on a protein basis. Cytochrome c oxidase responds like succinate dependent reactions. The data indicate that in cells growing under the conditions applied NADH oxidizing electron transport systems are incorporated into both, cytoplasmic and intracytoplasmic membranes, while incorporation of succinate oxidizing systems is confined to intracytoplasmic membranes only.Activities of photophosphorylation and succinate dependent NAD⁺ reduction in the light increase per Bchl about 1.8 times. On a Bchl basis increases of the fast light induced on reactions at 422 nm and increases of soluble cytochrome c ₂ levels are comparable to increases of photophosphorylations and succinate dependent activities. But increases of slow light off reactions at 428 nm and of b-type cytochrome levels become three times greater then increases of cytochrome c ₂ reactions and levels. These results infer that although electrontransport reactions of intracytoplasmic membranes change correlated to each other, Bchl, cytochrome c ₂ and b-type cytochromes cellular levels are independent of each other. Furthermore, the data indicate that cytochrome c ₂ rather than b-type cytochrome is involved with steps rate limiting for photophosphorylation.Abbreviations Bchl bacteriochlorophyll - DCIP 2,6-dichlorophenolindophenol     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

Primary distribution of n-alkane through the structures of yeast cells]

When n-alkane enters the cells as a result of molecular sorption and diffusion, it is distributed within all membrane structures of the cell (microsomes, mitochondria, membranes, vacuoles, cytoplasmic membranes, and cell walls). Sorption of n-alkane by all these structures in vivo comes at equilibrium after incubation of the cells with n-alkane during 3-4 min. Accumulation of the hydrocarbon in the morphological fractions of the cell depends on its concentration in the incubation medium. Isotherms of the sorption are convex curves. Sorption power, maximum sorption capacity, affinity and strength of the bonds with the hydrocarbon differ among the membrane structures of the cell. The maximum capacity of sorption of n-alkane by the structures does not correlate with the content of lipids and phospholipids in the structures. Sorption of n-alkanes is presumed to depend on the structural organization of lipids in the morphological fractions.     

Production of formate,acetate, and succinate by anaerobic fermentation of Lactobacillus pentosus in the presence of citrate

Summary The formation of acetate, formate and succinate was studied in Lactobacillus pentosus. These compounds were produced in addition to lactic acid when cells were exposed to anaerobic growth conditions with limited carbohydrates and in the presence of citrate. Citrate was metabolised via oxalacetate serving as an H-acceptor in a joint process together with lactate. The metabolism of citrate resulted in stoichiometric amounts of succinate and acetate. Lactate was degraded to formate and acetate in a reaction catalysed by pyruvate formate lyase. These fermentation products can potentially affect the flavour of fermented food but ecological factors in fermenting meat, e.g. the presence of glucose, nitrate or nitrite prevent this reaction. Offprint requests to: G. Wolf     

Physiological characterization ofPseudomonas pseudoflava GA3

Pseudomonas pseudoflava GA3, an aerobic hydrogen-oxidizing bacterium, was characterized physiologically. The doubling time of autotrophically growing cells was 2.5 h for cells growing heterotrophically on sucrose it was 1.5 h; and for cells growing heterotrophically on fructose, succinate, lactate, glutamate, treahalose, glucose, and pyruvate it was about 1.6 h. Glucose and fructose were degraded via the Entner-Doudoroff pathway. 6-Phosphogluconate dehydrogenases (NAD or NADP-dependent) were not detected. Hydrogen did not suppress or inhibit the utilization of organic substrates. The specific hydrogenase activity was influenced by the growth conditions. An oxygen concentration of 20% (vol/vol) in the gas atmosphere provided conditions for continued exponential growth but diminished the specific hydrogenase activity. Cells contained a single membrane-bound hydrogenase; intact membranes were able to reduce menadion, ubiquinone Q10, methylene blue, phenazine methosulfate, and dichlorophenolindophenol. The hydrogenase was solubilized from the membrane fraction by treatment with 0.2% Triton X-100 and 10% sucrose, resulting in a yield of about 60% of total activity.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.     

Study of the action of nystatin on the plasmatic membranes of the liver of rabbits]

The effect of mystatin on the plasmic membranes of the rabbit liver after intravenous administration of the antibiotic to the animals in a dose of 5 mg/kg was studied. It was found that intravenous administration of nystatin had no effect on the quantitative content of protein, lipids and nucleic acids in the plasmic membranes of the liver. The method of electrophoresis in polyacrylamide gel revealed significant changes in the composition of the liver membrane protein due to the treatment with nystatin. The effect of nystatin on the composition of lipids and fatty acids contained in the membrane lipids was also investigated. The data of the thin layer chromatography showed that nystatin did not affect the qualitative composition and the content of separate lipid fractions in the lipids of the liver plasmic membranes. However, the fatty acid analysis of the membrane lipids after intravenous administration of nystatin revealed a number of qualitative and quantitative differences in the composition of the lipid fatty acids of the membranes tested. The results showed that nystatin affected the membrane structures of the rabbit liver cells.     

Membrane lipids and soluble sugars dynamics of the alkaliphilic fungus Sodiomyces tronii in response to ambient pH

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4–10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4–7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

     

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.     

A confirmation of the phase behavior of Escherichia coli cytoplasmic membrane lipids by X-ray diffraction.

The lipid fatty acid composition of the cytoplasmic membranes of Escherichia coli can be varied by growing an unsaturated fatty acid auxotroph in the presence of different fatty acid supplements. Electron spin resonance (ESR) studies of spin-label partitioning into the cytoplasmic membranes of different lipid fatty acid compositions as a function of temperature have been interpreted as indicating a broad order-to-disorder transition in the membrane lipids, the end points of the transition depending upon the fatty acid composition. We have utilized x-ray diffraction to confirm the ESR studies for three different fatty acid supplements (oleic, elaidic, and bromostearic). We found that the characteristic end-point temperatures detected by ESR were indeed the end-point temperatures of a broad order-to-disorder transition of the cytoplasmic membrane lipids. In addition, Patterson functions calculated from lamellar x-ray diffraction from partially oriented cytoplasmic membranes indicate a decrease in average membrane thickness upon fatty acid chain melting.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.