Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Anesthetics alter outer membrane architecture and temperature range of growth of Escherichia coli K12

The addition of local anesthetics procaine and 2-phenylethanol during cell growth and membrane isolation lowered the phase transition temperature of purified outer membranes of $\text{Escherichia}\text{coli}$. Furthermore, when added to growth media, these anesthetics lowered to an equal extent the maximum temperature of growth without affecting growth at low temperatures. The phase transition of the cytoplasmic membrane was not affected by the presence of the drugs. These data substantiate the hypothesis that the temperature range over which the cell can maintain the outer membrane in a mixed (gel + liquid crystalline) lipid state determines the temperature range over which growth can occur.     

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

Effects of pressure-induced membrane phase transitions on inactivation of HorA, an ATP-dependent multidrug resistance transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37 degrees C and pressure treated at 15 degrees C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15 degrees C and pressure treated at 37 degrees C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.     

Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.     

Lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The thermotropic behaviour of fatty acid-homogeneous membranes of Acholeplasma laidlawii B was investigated by Fourier transform infrared spectroscopy. The organism was grown at 37°C in the presence of avidin, an inhibitor of fatty acid synthesis, in a medium supplemented with pentadecanoic acid-d₂₉; the enrichment of the membranes with this fatty acid was 95%. The temperature-dependent phase behaviour of the membranes was studied via the C–D stretching vibrational modes of the membrane lipids and was compared with that of the lipid extract. The high level of fatty acid homogeneity results in a sharp (for natural membranes) gel to liquid crystalline phase transition. The transition, in both the membranes and extracted lipids, is centered at about 6°C above the growth temperature. During the transition two principal liquid states are evident, one being more conformationally ordered than the other. The effect of proteins on the principal lipid phase transition is minimal. However, in the intact membranes there is evident a weaker, lower temperature transition, which is not evident in the extracted lipids.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling

Transfer of phosphatidylcholine molecules between different membrane fractions of Tetrahymena pyriformis cells grown at 15, 27 and 39.5°C was studied by electron spin resonance (ESR). Microsomes were labeled densely with a phosphatidylcholine spin label and the spin-labeled microsomes were incubated with non-labeled cilia, pellicles or microsomes. The transfer of the phosphatidylcholine spin labels was measured by decrease in the exchange broadening of the electron spin resonance spectrum. In one experiment, the lipid transfer was measured between ³²P-labeled microsomes and non-labeled pellicles by use of their radioactivity. The result was in good agreement with that by ESR. The fluidity of the membrane was estimated using a fatty-acid spin label incorporated into the membranes. Transfer between lipid vesicles was also studied. The results obtained were as follows: (1) The transfer between sonicated vesicles of egg- or dipalmitoyl phosphatidylcholine occurred rapidly in the liquid crystalline phase, with an activation energy of 20 kcal/mol, whereas it hardly occurred in the solid crystalline phase. (2) The transfer rate between microsomal membranes increased with temperature, and an activation energy of the reaction was 17.8 kcal/mol. (3) The transfer from the spin-labeled microsomes to subcellular membranes of the cells grown at 15°C was larger than that to the membranes of the cells grown at 39.5°C. The membrane fluidity was larger for the cells grown at lower temperature. (4) Similar tendency was observed for the transfer between microsomal lipid vesicles prepared from the cells grown at 15°C and at 39.5°C. (5) The transfer from microsomes to various membrane fractions increased in the order, cilia < pellicles < microsomes. The order of increase in the membrane fluidity was cilia < microsomes < pellicles, although the difference between microsomes and pellicles was slight. These results indicate a crucial role of the membrane fluidity in the transfer reaction. (6) Some evidence supported the idea that the lipid transfer between these organelles occurred through the lipid exchange rather than through the fusion.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Molecular order in acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids

The first application of deuterium magnetic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37°C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.     

Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli

We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W 3110. While these proteins were distributed randomly at the growth temperature (37 °C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 °C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes.The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction.Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while d-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes.These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.     

The relationships between growth temperature,fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37°C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37°C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73–76%), phosphatidylglycerol (9–11%) and cardiolipin (11–13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37°C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at ?13, ?9 and 1°C for membranes from cells grown at 5, 22 and 37°C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5°C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.     

Membrane phase behavior of a psychrotrophic and mesophilic bacillus

The phase properties of membranes isolated from the psychrotrophBacillus psychrophilus and the mesophileB. megaterium were examined using wide-angle X-ray diffraction. The temperature at which the transition from liquid-crystalline to crystalline (gel) phase occurred was below −30°C for both microorganisms, regardless of the temperature at which the microbial cells were grown. Thus the membranes for both microorganisms were exclusively liquid-crystalline over the entire growth temperature range. Indeed, the membrane was completely fluid at temperatures where growth of the psychrotroph ceases, thus indicating that the phase transition temperature is not the determinant of the minimum growth temperature.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and anlyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance.

The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy. The L51 strain of E. coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids. The cytoplasmic and outer membranes were separated using standard techniques. The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes. At low temperatures, the shape and width of the spectrum were characteristic of the gel phase. The relative intensities of the liquid-crystalline and gel features varied systematically with temperature. A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments. The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C. It is clear from the results that most of the phospholipid molecules participate in the phase transition.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Temperature-induced vertical shift of proteins in membranes

Thermotropic changes in the transverse order of microsomal membranes isolated from Tetrahymena are revealed by low-angle X-ray diffraction. These are correlated with the lateral order of the membrane lipids by wide-angle X-ray diffraction. Upon lowering the temperature from 28 °C to 2 °C, the Bragg period of the membrane stack reveals an abrupt increase of ~3.0 nm at ~19 °C, which is reversible upon reheating to 28 °C. This is coupled with an alteration in the electron density profile, revealing a shift of mass from the hydrophobic core towards one of the two hydrophilic surfaces. Between 35 °C and 0 °C, the membrane lipids undergo a broad, thermotropic “two-stage” liquid crystalline ? crystalline phase separation with a “breakpoint” at ~18 °C. This breakpoint signals an abrupt lipid redistribution, presumably due to a change in the composition of the two coexisting liquid crystalline and crystalline lipid phases. We conclude: (1) the temperature-induced mass shift reflects a shift in the transverse partition of proteins in membranes; (2) this is triggered by an abrupt lipid redistribution occurring during a broad liquid crystalline ? crystalline phase separation.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.