Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures.

Listeria monocytogenes is a food-borne pathogen capable of growth at refrigeration temperatures. Membrane lipid fatty acids are major determinants of a sufficiently fluid membrane state to allow growth at low temperatures. L. monocytogenes was characterized by a fatty acid profile dominated to an unusual extent (> 95%) by branched-chain fatty acids, with the major fatty acids being anteiso-C15:0, anteiso-C17:0, and iso-C15:0 in cultures grown in complex or defined media at 37 degrees C. Determination of the fatty acid composition of L. monocytogenes 10403S and SLCC 53 grown over the temperature range 45 to 5 degrees C revealed two modes of adaptation of fatty acid composition to lower growth temperatures: (i) shortening of fatty acid chain length and (ii) alteration of branching from iso to anteiso. Two transposon Tn917-induced cold-sensitive mutants incapable of growth at low temperatures had dramatically altered fatty acid compositions with low levels of i-C15:0, a-C15:0, and a-C17:0 and high levels of i-C14:0, C14:0, i-C16:0, and C16:0. The levels of a-C15:0 and a-C17:0 and the ability to grow at low temperatures were restored by supplementing media with 2-methylbutyric acid, presumably because it acted as a precursor of methylbutyryl coenzyme A, the primer for synthesis of anteiso odd-numbered fatty acids. When mid-exponential-phase 10403S cells grown at 37 degrees C were temperature down-shocked to 5 degrees C they were able, for the most part, to reinitiate growth before the membrane fatty acid composition had reset to a composition more typical for low-temperature growth. No obvious evidence was found for a role for fatty acid unsaturation in adaptation of L. monocytogenes to cold temperature. The switch to a fatty acid profile dominated by a-C15:0 at low temperatures and the association of cold sensitivity with deficiency of a-C15:0 focus attention on the critical role of this fatty acid in growth of L. monocytogenes in the cold, presumably through its physical properties and their effects, in maintaining a fluid, liquid-crystalline state of the membrane lipids.     

Adaptational changes of fatty acid composition and the physical state of membrane lipids following the change of growth temperature in Yersinia enterocolitica.

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.     

The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus. Composition-structure-function relationships

The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var. nondiastaticus as test-organism. The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures. Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C. Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found. Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains. The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms. Unsaturated fatty acids were not produced by cells grown at 65 degrees C. In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C. The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures. Completion of the lipid melting occurred always at temperatures below those employed for growth. A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile. The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature. The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated. The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively. This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus.     

Adaptation of the psychrotroph Arthrobacter chlorophenolicus A6 to growth temperature and the presence of phenols by changes in the anteiso/iso ratio of branched fatty acids

Arthrobacter chlorophenolicus is a previously described Gram-positive bacterium capable of degrading high concentrations of several phenolic compounds under optimal mesophilic (28 degrees C) as well as psychrophilic (5 degrees C) conditions. However, the exact mechanisms by which this organism is able to tolerate such extremes in temperature and high levels of toxic compounds are currently not known. In this study, we monitored changes in the fatty acid composition of the cell membrane under different extreme growth conditions. Arthrobacter chlorophenolicus adapts to differences in temperature and phenol concentrations by altering the anteiso/iso ratio of fatty acids in the cell membrane to different extents. According to the different physico-chemical properties of those two species of branched fatty acids, the bacteria showed an increased amount of anteiso fatty acids when grown under psychrophilic conditions to decrease the viscosity of their membranes. On the other hand, at higher growth temperatures as well as in the presence of toxic concentrations of phenol, 4-chlorophenol and 4-nitrophenol, the cells adapted their membrane by a dose-dependent decrease in the anteiso/iso ratio, leading to a more rigid membrane and counteracting the fluidity increase caused by the higher temperature and the organic solvents.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

The relationship between environmental temperature, cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus.

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes. Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might directly be determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

The relationship between environmental temperature,cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes.Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might be directly determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO.

Growth of Pseudomonas aeruginosa PAO1 at 15 to 45 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharides (LPSs), and outer membrane proteins of the cells. Cells grown at 15 degrees C contained, relative to those cultivated at 45 degrees C, increased levels of the phospholipid fatty acids hexadecenoate and octadecenoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation with decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-hydroxydecanoate and 2-hydroxdodecanoate as the growth temperature decreased. In addition, LPS extracted from cells cultivated at the lower temperatures contained a higher content of long-chain S-form molecules than that isolated from cells grown at higher temperatures. On the other hand, the percentage of LPS cores substituted with side-chain material decreased from 37.6 mol% at 45 degrees C to 19.3 mol% at 15 degrees C. The outer membrane protein profiles indicated that at low growth temperatures there was an increase in a polypeptide with an apparent molecular weight of 43,000 and decreases in the content of 21,000 (protein H1)- and 27,500-molecular-weight proteins.     

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.     

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

Phase Transition of Thylakoid Membranes Modulates Photoinhibition in the Cyanobacterium Anabaena siamensis

A shift of the growth temperature from 40 degrees C to 18 degrees C promoted an increase in the degree of fatty acids unsaturation and a decrease, from 26 degrees C to 0 degrees C, of the phase transition temperature of thylakoid membranes in Anabaena siamensis. The pattern of photoinhibition of photosynthesis at distinct temperatures varied as a function of the phase transition temperature. In the absence of streptomycin, a pronounced photoinhibition at temperatures near the phase transition (26 degrees C) was observed in cells grown at 40 degrees C, while protection from photodamage was observed at chilling temperatures (15 degrees C to 5 degrees C). In this same range of temperature, such a protection was not verified if cells were grown at 18 degrees C. In both types of cells, however, the rate of photoinactivation in the presence of streptomycin was progressively decreased by lowering the temperature of photoinhibition. When recovery from photoinhibition was followed at the respective temperature in which cells were grown, the restoration profile of the photosynthetic O(2) evolution to initial levels was essentially the same in both types of cells. The protective effect of low temperatures against photoinhibition was attributed to a decreased solubility and diffusion of oxygen in the thylakoid membranes due to an increase of the membrane viscosity that would avoid the photogeneration of reactive oxygen species around PS II.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

The effects of unsaturated fatty acid depletion on the proton permeability and energetic functions of yeast mitochondria

1. The fatty acid composition of the ole-1 and ole-1 petite mutants of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of defined supplements of Tween 80 or by allowing cells that had first been grown in the presence of Tween 80 to deplete their unsaturated fatty acids by sequent growth in the absence of Tween 80. 2. The transition temperature of Arrhenius plots of mitochondrial ATPase (adenosine triphosphatase) increases as the unsaturated fatty acid content is lowered. 3. Cells require larger amounts of unsaturated fatty acids to grow on ethanol at lower temperatures. 4. Cells that stop growing owing to unsaturated fatty acid depletion at low temperatures are induced to grow further by raising the temperature and this results in a further depletion of unsaturated acids. This is due to a higher rate, but not a greater efficiency, of mitochondrial ATP synthesis. 5. Arrhenius plots of the passive permeability of mitochondria to protons between 4 and 37 degrees C are linear. The rate and the Arrhenius activation energy of proton entry increase greatly as the unsaturated fatty acid content is lowered. 6. Unsaturated fatty acid depletion has the same effects on the proton permeability of ole-1 petite mitochondria, indicating that the mitochondrially synthesized subunits of the ATPase are not involved in the enhanced rates of proton entry. 7. The adenylate energy charge of depleted ole-1 cells is greatly decreased by growth on ethanol medium. 8. The adenylate energy charge of isolated mitochondria is also lowered by unsaturated fatty acid depletion. 9. The results confirm that unsaturated fatty acid depletion uncouples oxidative phosphorylation in yeast both in vivo and in vitro, and is a consequence of changes in the lipid part of the membrane.     

Low temperature-induced fatty acid desaturation in Brassica napus: thermal deactivation and reactivation of the process.

When Brassica napus plants are grown at low temperatures (e.g., 5 degrees C) the rate of desaturation in leaves of newly formed fatty acids in both chloroplastic (MGDG) and cytosolic (PC) diacylglycerols is higher or more rapid than in plants grown at higher temperatures (e.g., 30 degrees C). This low temperature-induced increase in the rate of desaturation is lost within hours if plants are transferred to higher temperatures. However, if plants are then returned to low temperatures they regain the ability to rapidly desaturate fatty acids. This process is restored relatively slowly (over days) in contrast to the more rapid loss at high temperatures. This has important physiological consequences on the level of unsaturated fatty acids in plant membranes and the process of temperature control of the fatty acid composition of membrane lipids.     

Membrane lipids of a psychrophilic and barophilic deep-sea bacterium

In a psychrophilic and barophilic marine bacterial isolate of the genusAlteromonas, the ratio of total unsaturated versus saturated fatty acids in the membrane lipids increased when the organism was grown at increasing hydrostatic pressures and decreasing temperatures. This regulatory capacity, as well as the presence of relatively large amounts of 20:5 polyunsaturated fatty acid, appear to be functional in maintaining membrane fluidity within a range of pressures distinctly below and above the specific optimum and at typical deep sea temperatures.     

Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.