©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Wild-type Escherichia coli Cells Regulate the Membrane Lipid Composition in a Window between Gel and Non-lamellar Structures (*)

(Received for publication, November 7, 1995; and in revised form, January 9, 1996)

Sven Morein Ann-Sofie Andersson Leif Rilfors (§) Göran Lindblom

From the Department of Physical Chemistry, Umeå University, S-901 87 Umeå, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Escherichia coli strain K12 was grown at 17, 27, and 37 °C. The acyl chain composition of the membrane lipids varied with the growth temperature; the fraction of cis-vaccenoyl chains decreased, and the fraction of palmitoyl chains increased, when the growth temperature was increased. However, the polar head group composition did not change significantly. The equilibria between lamellar and reversed non-lamellar phases of lipids extracted from the inner membrane (IM), and from both the membranes (IOM), were studied with NMR and x-ray diffraction. At temperatures above the growth temperature the lipid extracts formed a reversed hexagonal phase, or a bicontinuous cubic phase, depending on the degree of hydration of the lipids. It was observed that: 1) at equal elevations above the growth temperature, IM lipid extracts, as well as IOM lipid extracts, have a nearly equal ability to form non-lamellar phases; 2) IM extracts have a stronger tendency than IOM extracts to form non-lamellar phases; 3) non-lamellar phases are formed under conditions that are relatively close to the physiological ones; the membrane lipid monolayers are thus ``frustrated''; and 4) as a consequence of the change of the acyl chain structures, the temperature for the lamellar gel to liquid crystalline phase transition is changed simultaneously, and in the same direction, as the temperature for the lamellar to non-lamellar phase transition. With a too large fraction of saturated acyl chains the membrane lipids enter a gel state, and with a too large fraction of unsaturated acyl chains the lipids transform to non-lamellar phases. It is thus concluded that the regulation of the acyl chain composition in wild-type cells of E. coli is necessary for the organism to be able to grow in a ``window'' between a lamellar gel phase and reversed non-lamellar phases.


INTRODUCTION

Practically all membranes contain significant amounts of at least one lipid species that forms non-lamellar phases under physiological conditions (Ansell et al., 1973; Goldfine, 1982). Furthermore, many membrane lipids can transform from a lamellar to a non-lamellar phase by changes in biologically relevant conditions such as the pH value, the concentration of divalent cations, the temperature, and the hydration (for a review, see Lindblom and Rilfors (1992)). It is well documented that all kinds of organisms adapt their membrane lipid composition to the prevailing environmental and physiological conditions. Three strategies seem to be utilized: changes in the acyl chain structure, changes in the polar head group structure, and reshuffling of acyl chains to form new lipid molecular species without changing the average acyl chain composition (Hazel and Williams, 1990; Suutari and Laakso, 1994). The regulation of the membrane lipid composition by the cell wall-less bacterium Acholeplasma laidlawii strain A-EF22 has been intensively studied in our laboratory. The organism can be grown under conditions where the regulatory changes occur predominantly in the polar head group structures. The conclusion has been drawn that the cells strive to maintain a certain balance between the lipids constituting a bilayer structure and those forming reversed non-lamellar structures (Lindblom et al., 1986, 1993; Rilfors et al., 1993, 1994; Österberg et al., 1995). A. laidlawii strain A-EF22 synthesizes eight membrane lipids of which three are able to form non-lamellar phases: monoglucosyldiacylglycerol (MGlcDAG), (^1)and the monoacylated derivatives of MGlcDAG and diglucosyldiacylglycerol (DGlcDAG) (^2)(Lindblom et al., 1993; Rilfors et al., 1993). Moreover, four of these lipids are uncommon, namely the lipids having an acyl group or a glycerophosphoryl group linked to the head groups of MGlcDAG and DGlcDAG (Hauksson et al., 1994a, 1994b, 1995). The Gram-positive bacterium Clostridium butyricum has also been used to investigate the regulation of the membrane lipid composition and, in resemblance with A. laidlawii, these cells can be grown under conditions where the regulation of the composition is primarily confined to the structures of the polar head groups (Goldfine et al., 1987a, 1987b). The purpose of the lipid regulation in C. butyricum was suggested to be the same as in A. laidlawii, although most of the membrane lipids differ between the two organisms. C. butyricum synthesizes at least eight membrane lipids. Phosphatidylethanolamine (PE) and its plasmalogen form, plasmenylethanolamine, form non-lamellar phases under physiological conditions, and two of the lipids are uncommon (glycerol acetal and phosphatidylglycerol acetal derivatives of plasmenylethanolamine) (Goldfine et al., 1987b; Johnston and Goldfine, 1988).

In the present work we have studied the metabolic regulation and the phase equilibria of the membrane lipids from wild-type cells of the Gram-negative bacterium Escherichia coli. The reasons for this are manifold: E. coli is recognized as one of the foremost prokaryotic model organisms; the bacterium has only three main membrane phospholipids which occur frequently in prokaryotic as well as eukaryotic organisms; and the regulation of the lipid composition in wild-type cells is brought about by changes in the acyl chain structure, above all in the degree of unsaturation of the acyl chains (Marr and Ingraham, 1962), which is a very common response to changes in the environmental temperature among a variety of organisms (Hazel and Williams, 1990). A large number of biochemical and physico-chemical studies have been carried out on E. coli membranes and membrane lipids (Cronan et al., 1987; Cronan and Rock, 1987 and references therein). A lipid biosynthetic mutant of E. coli was recently used to study the polymorphic regulation of the membrane lipid composition (Rietveld et al., 1993, 1994). This mutant lacks the ability to synthesize PE which constitutes about 75-80 mol % of the membrane lipids in wild-type E. coli cells and has the ability to form non-lamellar phases (Cullis and de Kruijff, 1978; Rilfors et al., 1984). It was suggested that one of the remaining lipids, diphosphatidylglycerol (DPG), together with divalent cations can replace the role of PE and that E. coli also exhibits a polymorphic regulation of its membrane lipid composition (Killian et al., 1994; Rietveld et al., 1993, 1994). No studies have so far been undertaken to elucidate if there exists a relationship between the regulation of the lipid composition, and the transition between lamellar and non-lamellar phases, in wild-type cells of E. coli grown at different temperatures. Moreover, the presence of an outer and an inner membrane in E. coli, having different chemical compositions and functions, raises the question whether this phase equilibrium differs between the membranes. Therefore, in this work we have studied the lamellar to non-lamellar phase transitions of lipids extracted from the inner membrane and from both the membranes of E. coli.


MATERIALS AND METHODS

Cell Growth

The E. coli wild-type strain K12, kindly provided from the Department of Microbiology at the Umeå University, was used in all experiments. Cells were grown at aerobic conditions in Luria broth (LB medium consisting of 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/liter distilled water, pH 7.5) at 17, 27, and 37 °C. The growth medium was inoculated with 1% (v/v) of an overnight culture grown at the same temperature as the main culture, and the cells were grown under vigorous agitation in Erlenmeyer flasks (200 ml of medium in 2-liter flasks; 200 rpm) or in an Electrolux fermentor (6-8 liters of medium; 20 liters of air/min; 800 rpm). In the fermentor the pH value was kept constant at 7.5 by using aqueous solutions of 2 M HCl and 5 M NaOH, and a 15% (v/v) dispersion of silicon oil in water was used as an antifoam agent. Normally about 40 ml of HCl, 20 ml of NaOH, and 5-10 ml of antifoam agent were consumed for a 6-liter culture in the fermentor. The cells were grown until late log phase (A = 0.8 or 1.8 in Erlenmeyer flasks and fermentor, respectively), harvested by a centrifugation at 11,300 times g(max) for 15 min at 4 °C, washed once in buffer (20 mM Tris-HCl and 2 mM EDTA, pH 7.0), and finally suspended in the same buffer. Portions of the cell pellet were taken aside for preparation of the inner plus outer membrane (IOM) lipid extracts before disruption in a French press.

Separation and Isolation of Inner and Outer Membranes

All the subsequent steps were carried out on ice or at 4 °C if not otherwise mentioned. The cells were lysed by disruption in a French press, and the outer and inner membranes were separated in Percoll gradients as described by Morein et al.(1994). The purity of the inner membrane (IM) fractions was checked by the NADH oxidase activity (Osborn and Munson, 1974) and the 2-keto-3-deoxyoctonate content (Karkhanis et al., 1978) as described in Morein et al.(1994). The membrane material was collected, and the remaining Percoll was removed by three centrifugation steps: one at 50,000 rpm in a Ti70 rotor in a Beckman LE-70 preparative ultracentrifuge and two at 70,000 rpm in a TLA100.3 rotor in a Beckman bench top TL-100 ultracentrifuge. During these centrifugation steps the membrane material sediments as a layer on top of a clear Percoll pellet. After the last centrifugation, no Percoll pellet could be seen.

Isolation and Purification of Lipids

The cells or the inner membranes were extracted twice with chloroform/methanol (2:1, v/v), and once with methanol, in an ice water bath on a magnetic stirrer. Cell debris was removed by centrifugation, and non-lipid contaminants were removed by chromatography on Sephadex G-25 Fine (Wells and Dittmer, 1963). The lipids were then applied to a silicic acid column (Bio-Sil HA minus 325 mesh, Bio-Rad). The column was first eluted with chloroform to remove the neutral lipids (mainly free fatty acids and small amounts of monoacylglycerols, diacylglycerols, and pigments), and next the phospholipids were eluted with chloroform/methanol (1:1, v/v). The phospholipids were then converted to their sodium salts by a modified version of the procedure by Smaal et al.(1985) as described by Rilfors et al.(1994).

Determination of Polar Head Group and Acyl Chain Compositions

The polar head group compositions of the IOM and IM lipid extracts were determined by first separating the lipids by thin layer chromatography (TLC) on glass plates precoated with a 0.25-mm-thick layer of Silica gel 60 (Merck, Darmstadt, Germany); the chromatograms were developed with chloroform/methanol/acetic acid (65:25:10, v/v). The spots were visualized by iodine vapor, and the phospholipids were identified with known reference lipids. The phosphate content in each spot was determined according to Chen et al.(1956) with the following changes. The reagent mixture contained 1 ml of concentrated sulfuric acid, 1 ml of 10% ascorbic acid in water, and 1 ml of 2.5% ammonium molybdate in water, per 10 ml. After the ashing procedure, 4 ml of the reagent mixture was added to each test tube. The color was developed at 65 °C for 6 min in a water bath.

The acyl chain compositions were determined by gas chromatography using the methyl esters of the fatty acids (Rilfors, 1985).

NMR Sample Preparation

The lipids, dissolved in chloroform, were first dried to a film with a stream of N(2) in an 8-mm glass tube and then dried to constant weight in vacuum. After addition of ^2H(2)O, the tubes were flame-sealed. The lipids were pelleted in the bottom of the tube by centrifugation with a Beckman J2-YY centrifuge in a JA20.1 rotor at 12,900 times g(max) for 60 min. All samples were freeze-thawed repeatedly and equilibrated at room temperature in darkness over night before the NMR measurements. The samples contained approximately 15-20 mg of IM lipids or approximately 35-50 mg of IOM lipids.

NMR Spectroscopy

P NMR spectra were obtained with a Bruker ACP-250 spectrometer at 101.27 MHz. A phase cycled Hahn echo sequence (Rance and Byrd, 1983) with high power proton decoupling was used. The 90° pulse was 10 µs, and the spectral width was 100 kHz. A relaxation delay of 1 s was used. A variable temperature unit was used to control the air flow around the sample in the spectrometer, and the temperature in the probe was measured with a calibrated thermistor. During the temperature dependence measurements the samples were allowed to equilibrate for 30 min at each temperature before recording the spectra. All experimental free induction decays (FIDs) were transformed from Bruker to Felix format by the routines in Felix 2.0, which was used to process all spectra. A line broadening of 50 Hz was applied before Fourier transformation.

The lipid diffusion coefficient in a cubic phase formed by a total lipid extract prepared from E. coli was determined by the pulsed magnetic field gradient technique (Lindblom and Orädd, 1994), and the ^1H NMR spectra were recorded as described in Lindblom et al.(1993).

Low Angle X-ray Diffraction

X-ray investigations of the cubic phase mentioned in the previous section were performed at Station 8.2 at the Daresbury Laboratory (Cheshire, United Kingdom) using a monochromatic beam of wavelength 1.5 Å. The experiments were performed at 76 °C.


RESULTS

Membrane Lipid Composition

The IOM lipid extracts all contained about 75-80 mol % PE, 15-20 mol % phosphatidylglycerol (PG), 2-6 mol % DPG (Table 1) and trace amounts (less than 1 mol %) of a lipid that probably is phosphatidic acid. The IM lipid extracts contained the same phospholipids, but with slightly less PE (70-75 mol %) and a larger fraction of PG and DPG (18-22 and 6-8 mol %, respectively) than in the IOM lipid extracts (Table 1). The phospholipid head group composition did only change marginally as a function of the growth temperature. The small differences noted are not larger than the variation between different extracts obtained from the same growth temperature.



The acyl chain compositions of the different lipid extracts are summarized in Table 2. Palmitoyl (16:0), palmitoleoyl (16:1c9), and cis-vaccenoyl (18:1c11) chains are the dominating acyl chains in all lipid extracts. The fraction of unsaturated acyl chains, above all 18:1c11, was decreased when the growth temperature was increased. In the IOM phospholipid extracts from E. coli grown at 17 °C there was 60 mol % unsaturated acyl chains compared with 45 mol % in the corresponding extracts from E. coli grown at 37 °C, while E. coli grown at 27 °C had an intermediate fraction of unsaturated acyl chains. It is also evident that the average acyl chain length decreased when the growth temperature was raised (Table 2). Finally, it was found that the acyl chains of the IM lipids have a higher degree of unsaturation and are slightly longer. From a comparison of our gas chromatograms with those obtained by Cronan and Rock(1994) we suggest that the unidentified acyl chains probably consist mainly of 9,10-methylene-hexadecanoyl chains and a small fraction of 11,12-methylene-octadecanoyl chains.



Phase Behavior of the Phospholipid Extracts

P NMR was used to investigate the phase behavior of the lipid-water systems. The line shape of the P NMR signal depends on the symmetry of the phospholipid aggregates (Brentel et al., 1987; Cullis and de Kruijff, 1976; Lindblom et al., 1986; McLaughlin et al., 1975). A lamellar liquid crystalline (L) phase gives rise to a spectrum with a high field peak and a low field shoulder. Lipids in a reversed hexagonal (H) phase cause a spectrum with a low field peak and a high field shoulder. The measured chemical shift anisotropy (CSA) for the H phase is smaller, and of reversed sign, as compared with that obtained for the L phase. Isotropic solution phases (e.g. micellar phases) and cubic liquid crystals usually cause a single, relative narrow P NMR signal. Multiphase systems cause a superposition of the line shapes characteristic of each phase present.

The phase behaviors of IOM and IM phospholipid extracts hydrated with 20 and 50 weight % ^2H(2)O were investigated in the temperature range from 25 to 65 °C. The IOM phospholipid extract from E. coli grown at 37 °C, and hydrated with 20 weight % ^2H(2)O, formed an L phase up to above 55 °C (Fig. 1A). At 65 °C a low field peak can be seen at 5 ppm, most probably due to the formation of an H phase. The CSA of the spectra is approximately -45 ppm within the whole temperature range. A corresponding lipid extract from E. coli grown at 27 °C formed an L phase up to 45 °C; an H phase started to form at 55 °C, and the fraction of this phase increased when the temperature was raised further (Fig. 1B). The CSA of a spectrum recorded at 45 °C is approximately -50 ppm. Finally, the IOM lipid extract from E. coli grown at 17 °C, and hydrated with 20 weight % ^2H(2)O, showed an L phase up to 35 °C (Fig. 1C). For this lipid extract the transition from an L to an H phase is apparent already at 45 °C, and at 65 °C the spectral component from the H phase dominates the spectrum. The CSA of a spectrum recorded at 45 °C is approximately -50 ppm. P NMR spectra were also recorded for all samples after the temperature had been lowered to 35 °C (Fig. 1, A-C), and the H phase had almost disappeared after 30 min equilibration at this temperature. Moreover, all samples were repeatedly stored at -20 °C, freeze-thawed, and equilibrated as described under ``Materials and Methods,'' and a new series of P NMR spectra were recorded between 25 and 65 °C; nearly identical spectra were obtained each time. From Fig. 1, A-C it is concluded that the fraction of lipids in the H phase was approximately the same in the three IOM lipid extracts when spectra were recorded at equal elevations above the growth temperature.


Figure 1: P NMR spectra showing the phase behavior, as a function of temperature, for IOM lipid extracts isolated from E. coli K12. The cells were grown at 37 °C (A), 27 °C (B), and 17 °C (C and D). The membrane lipids were extracted, the neutral lipids were removed, and the lipid extracts were hydrated with 20 weight % (A, B, and C) or 50 weight % (D) ^2H(2)O. The spectra shown in the top of the figure were recorded after the temperature had been lowered from 65 to 35 °C, and the samples had been equilibrated at 35 °C for 30 min.



When the water content in the IOM lipid extracts was increased from 20 to 50 weight %, a change in the phase behavior was observed. P NMR spectra recorded from the lipid extract isolated from E. coli grown at 17 °C, and hydrated with 50 weight % ^2H(2)O, are shown in Fig. 1D. An L phase was observed up to 45 °C, and by raising the temperature further, there was a transition from an L to an isotropic phase as seen by the appearance of a narrow symmetrical signal in the spectra. An isotropic phase was formed also in the IOM lipid extracts isolated from E. coli grown at 27 and 37 °C and hydrated with 50 weight % ^2H(2)O; in these extracts the isotropic phase appeared at temperatures above approximately 60 and 70 °C, respectively (data not shown). Fig. 2a summarizes the fraction of non-lamellar phases obtained at 65 °C for the different IOM lipid extracts. For each phospholipid extract a decrease in the fraction of non-lamellar phases is observed when the water content is increased from 20 to 50 weight %. It is striking that the fraction of non-lamellar phases in the lipid extracts, hydrated with 20 weight % HO, increases from 0-10% to 90-100% as a result of the modulation of the acyl chain composition when the growth temperature of the E. coli cells is decreased from 37 to 17 °C.


Figure 2: The fraction of non-lamellar phase obtained in different membrane lipid extracts isolated from E. coli K12 grown at 17, 27, and 37 °C. The neutral lipids had been removed from the extracts. The fraction of non-lamellar phase was calculated by simulation of P NMR spectra recorded at 65 °C; simulation of the spectra was performed as described in Eriksson et al.(1985). a, IOM lipid extracts hydrated with 20 weight % (bullet) or 50 weight % (circle) ^2H(2)O. b, IOM lipid extracts (filled symbols) and IM lipid extracts (unfilled symbols) hydrated with 20 weight % ^2H(2)O, and IM lipid extracts hydrated with 50 weight % ^2H(2)O (unfilled symbols with star). Symbols with the same shape at a certain growth temperature denote that the IOM and IM lipid extracts were prepared from the same batch of cells. Lipid extracts hydrated with 20 and 50 weight % ^2H(2)O form an H phase and a bicontinuous cubic phase belonging to the space group Pn3m, respectively.



The isotropic phase obtained in the IOM lipid extracts isolated from E. coli grown at 17 °C (Fig. 1D) was investigated by x-ray diffraction. Ten separate reflections were obtained which were shown to originate from a cubic liquid crystalline phase belonging to the space group Pn3m (Fig. 3). From the plot shown in Fig. 3it can be calculated that the length of the unit cell is 148.1 Å. A P NMR spectrum recorded after the temperature had been lowered from 65 to 35 °C for a sample forming the cubic phase, and after the sample had been equilibrated at this temperature for 30 min, shows that the fraction of the cubic phase had not decreased (Fig. 1D). This hysteretic behavior is a characteristic feature of bicontinuous cubic phases (Lindblom and Rilfors, 1989). The presence of a bicontinuous cubic phase was established by measurements of the lipid diffusion coefficient with the pulsed magnetic field gradient technique (Lindblom and Orädd, 1994). A diffusion coefficient of 8.5bullet10 m^2 s was obtained at 60 °C, which is close to the value obtained at 65 °C in a bicontinuous cubic phase formed by PE isolated from Bacillus megaterium (Rilfors et al., 1982), and is of the same order of magnitude as the lateral diffusion coefficient observed for lipids in bilayers (Lindblom and Orädd, 1994).


Figure 3: Plot of 1/d versus (h^2 + k^2 + l^2) of the reflections obtained in an x-ray diffractogram recorded from the cubic phase formed by an IOM lipid extract isolated from E. coli K12 grown at 17 °C. The neutral lipids had been removed from the extract. The sample contained 50 weight % ^2H(2)O, and the diffractogram was recorded at 76 °C. For the correct choice of space group, this plot gives a straight line passing through the origin and having the slope of 1/a(0), where a(0) is the length of the unit cell.



P NMR spectra recorded from IM lipid extracts isolated from E. coli cells grown at 37, 27, and 17 °C are shown in Fig. 4. The samples were hydrated with 20 weight % ^2H(2)O and can therefore be compared with the corresponding spectra recorded from the IOM lipid extracts (Fig. 1, A-C). The IM lipid extracts all transformed from an L to an H phase when the temperature was raised; however, the H phase appeared at temperatures approximately 10 °C below the temperatures found for the corresponding IOM lipid extracts (Fig. 4, A-C). It is concluded also for the three IM lipid extracts that the fraction of lipids in the H phase was approximately the same when P NMR spectra were recorded at equal elevations above the growth temperature. A comparison of the phase equilibria exhibited by IOM and IM lipid extracts, hydrated with 20 weight % ^2H(2)O, is shown in Fig. 2b. At each growth temperature, there is a larger fraction of non-lamellar phases at 65 °C in the IM extracts than in the IOM extracts isolated from the same batch of cells. This difference is clearly seen for the lipid extracts isolated from cells grown at 27 and 37 °C. The difference in phase behavior for the lipid extracts isolated from cells grown at 17 °C is masked at this high temperature. However, the different tendencies of these extracts to form non-lamellar phases are evident in P NMR spectra recorded at 45 and 55 °C. Moreover, by comparing Fig. 2a with Fig. 2b it is seen that the IM lipid extract (17 °C) has a greater ability to form non-lamellar phases than the IOM lipid extract (17 °C) when they are hydrated also with 50 weight % ^2H(2)O. When the water content was increased in the IM lipid extracts, a decrease in the fraction of non-lamellar phases was observed (Fig. 2b), in similarity with the IOM lipid extracts.


Figure 4: P NMR spectra showing the phase behavior, as a function of temperature, for IM lipid extracts isolated from E. coli K12. The cells were grown at 37 °C (A), 27 °C (B), and 17 °C (C). The neutral lipids had been removed from the extracts, and the lipid extracts were hydrated with 20 weight % ^2H(2)O.



Some investigations were also performed on IOM lipid extracts in which the neutral lipids were still present (see ``Materials and Methods''). These lipid extracts were generally more prone to adopt non-lamellar phases. It is noteworthy that IOM lipid extracts (containing the neutral lipids) isolated from all the three growth temperatures, and hydrated with 70 weight % ^2H(2)O, were able to form an isotropic phase; the lipids isolated from cells grown at 37 °C started to form the isotropic phase between 50 and 60 °C, while this phase appeared between 40 and 50 °C in the lipids isolated from cells grown at 27 and 17 °C.

When the E. coli cells were grown in the fermentor, a 15% (v/v) dispersion of silicon oil in water was used as antifoam agent. To check whether the silicon oil could have any influence on the phase behavior of the isolated lipid extracts, cell cultures were grown in LB in Erlenmayer flasks with and without the antifoam agent, but otherwise under identical conditions. The IOM phospholipids were extracted as described under ``Materials and Methods.'' However, P NMR measurements on these lipid extracts did not show any difference in the phase behavior due to the presence of the antifoam agent in the growth medium.


DISCUSSION

Together with water most isolated membrane lipids form lamellar phases, which are built up by bilayer aggregates. However, during the last 30 years it has been shown that membrane lipids can form several other aggregate structures and phase structures. The H and cubic phases are examples of such phases that have been paid a great deal of attention (Lindblom and Rilfors, 1989; Seddon, 1990); the former phase is built up by reversed cylindrical aggregates arranged in a hexagonal lattice, and the latter phases are built up by lamellar, cylindrical, or spherical aggregates arranged in three-dimensional lattices. PE is an example of a commonly occurring lipid that is able to form these reversed non-lamellar phases (vide infra). The ability of membrane lipids, and of amphiphilic molecules in general, to self-assemble and form different aggregate structures can be described by two different theoretical models. In the model by Israelachvili and colleagues(1976, 1980, 1992) the lipid molecules are dealt with explicitly and are schematically ascribed to have a simple shape. Helfrich(1973), and Gruner and colleagues(1985, 1988), have developed a theory that is based on a phenomenological model of material physics, where it is assumed that the lipid monolayers in the aggregates are characterized by spontaneous radii of curvature. A short summary of the theoretical models can be found in the review by Lindblom and Rilfors (1989).

The two theories for lipid self-assembly constitute decisive bases for the model we have presented concerning the regulation of the membrane lipid composition in A. laidlawii strain A-EF22 (Lindblom et al., 1986, 1993; Rilfors et al., 1993; Wieslander et al., 1980). The organism can synthesize only saturated fatty acids (Rilfors et al., 1993). Seven different polar head group structures occur in the membrane lipids (Hauksson et al., 1994a, 1994b, 1995); three of these lipids are able to form reversed non-lamellar phases, three form only lamellar phases, while one lipid can form even a micellar solution phase(^3)(Hauksson et al., 1995; Lindblom et al., 1993; Rilfors et al., 1993). The molar fractions of all these lipids are varied in relation to the structure of the fatty acids that are either synthesized endogenously, or taken up by the cells from the growth medium, and then covalently incorporated into the lipids. The two most important conclusions that have been drawn from a combination of biochemical and physico-chemical studies of the A. laidlawii membrane lipids are: 1) the proportions of all the lipids are carefully balanced in relation to the prevailing growth conditions^2 (Rilfors et al., 1993; Wieslander et al., 1995). The consequence of this balanced lipid synthesis is that the tendency of total lipid extracts to form reversed non-lamellar phases is maintained within narrow limits (Lindblom et al., 1986; Lindblom and Rilfors, 1989; Rilfors et al., 1994; Österberg et al., 1995); and 2) total lipid extracts have an ability to form reversed non-bilayer aggregates and non-lamellar phases under conditions that are relatively close to the physiological ones (Lindblom et al., 1986; Rilfors et al., 1994; Österberg et al., 1995).

A regulation of the temperature for the transition between L and L phases (T(m)) of the membrane lipids can be excluded in many cases in A. laidlawii strain A-EF22 (Rilfors et al., 1993). In one of the commonly employed models for membrane lipid regulation it is asserted that such a regulation is the reason why numerous organisms change the proportion between saturated and unsaturated acyl chains in relation to the surrounding temperature (Hazel and Williams, 1990; McElhaney, 1984). However, it should be recognized that a lipid bilayer being completely in a liquid crystalline state surely has the best prerequisites for maintaining the integrity and the permeability barrier of the membrane and to support full activity of membrane-associated enzymes and transport proteins. It has consistently been shown that living cells prefer to grow with their membrane lipids exclusively, or nearly exclusively, in the liquid crystalline state (McElhaney, 1984). Therefore, one aim with the adjustment of the membrane lipid composition may be to avoid the occurrence of gel state lipids in the membrane or at least to reduce the fraction of gel state lipids as much as possible. The regulation of the lipid composition in A. laidlawii strain A-EF22 can also in a few cases be interpreted to be an adjustment of the T(m) value of the lipids (Rilfors et al., 1993). In the following sections it will be shown that similar conclusions as those presented for A. laidlawii can be drawn also from the present study of wild-type cells of E. coli, although this bacterium synthesizes other membrane lipids than A. laidlawii. The approach chosen in the present work, namely to study the balance between L and non-lamellar liquid crystalline phases in lipid extracts isolated from E. coli cells grown at different temperatures, has not been taken before.

Composition of E. coli Lipid Extracts

The polar head group and acyl chain compositions of the membrane lipids of the wild-type strain K12 used in this investigation are very similar to those reported earlier (Aibara et al., 1972; Cronan and Rock, 1987; Ishinaga et al., 1979; Lugtenberg and Peters, 1976; Marr and Ingraham, 1962; Suutari and Laakso, 1994). The cells responded to a higher growth temperature by synthesizing lipids with shorter and more saturated acyl chains. PE is enriched in the outer membrane, while the acyl chains of the IM lipids have a higher degree of unsaturation and are slightly longer. The sole enzyme responsible for the thermal regulation of the acyl chain composition in E. coli is beta-ketoacyl-acyl carrier protein synthase II (Magnusson et al., 1993). A decrease in the fraction of unsaturated acyl chains is a very common response to increases in the environmental temperature among both prokaryotes and eukaryotes (Hazel and Williams, 1990; Suutari and Laakso, 1994), and a study of the equilibria between L and non-lamellar liquid crystalline phases in total lipid extracts with varying degrees of acyl chain unsaturation is therefore of general relevance for investigations of the regulation of membrane lipid composition.

Phase Equilibria of Individual E. coli Lipids

Two of the major lipids in E. coli, the anionic lipids PG and DPG, form only an L phase up to 55 °C at water contents between 20 and 95 weight % and with the lengths and degrees of unsaturation of the acyl chains that are relevant to the present study (Lindblom et al., 1991). Thus, although the shape of the lipid molecules, and the values of the spontaneous radii of curvature for monolayers formed by these phospholipids, will change with longer and more unsaturated acyl chains, the change is not large enough to make these molecules form non-bilayer aggregates under physiological conditions. However, the situation is quite different for the zwitterionic lipid PE. This lipid has a comparatively small polar head group and depending on the structure of its acyl chains it can form L, reversed cubic, and H phases under conditions relevant for E. coli cells (Koynova and Caffrey, 1994; Rilfors et al., 1994). It is illustrative to compare the T(m) values, and the temperatures for the transition between L and H phases (T), for some fully hydrated diacyl-PE species (Fig. 5). When the growth temperature was raised for the E. coli cells, the major change in the acyl chain composition was the replacement of 18:1c11 by 16:0 (Table 2). The T(m) and T values of dipalmitoyl-PE and dioleoyl-PE differ by 69 and 113 °C, respectively (Fig. 5). These data are highly relevant even though E. coli synthesizes 18:1c11 instead of oleic acid (18:1c9), since the T(m) value for lipids having 18:1c11 or 18:1c9 is practically the same (Barton and Gunstone, 1975), which in turn implies that the T values for PE containing these acyl chains most probably are very similar (Lewis et al., 1989). It is noteworthy that the T(m) values for PE having saturated acyl chains is above the maximum growth temperature of E. coli cells and that the T values of PE containing cis-9-monounsaturated acyl chains coincide with the temperature interval of growth for this organism. These data convincingly show that, among the three major membrane lipids in E. coli, PE has the strongest propensity to form reversed non-lamellar phases, and that this propensity is profoundly influenced by the length and the degree of unsaturation of the acyl chains.


Figure 5: Calculated T and T values for PE species having the acyl chain compositions of the IM lipid extracts isolated from E. coli K12 grown at 17, 27, and 37 °C. Tvalues (a) and T values (b) for synthetic diacyl-PE species having two saturated (box) or two cis-9-monounsaturated acyl chains (up triangle), and for a synthetic 16:0/18:1c9-PE species (down triangle). The Tand T values for the synthetic PE species were used to calculate, through interpolation, these values for PE species containing acyl chains with the average lengths and degrees of unsaturation determined for the IM lipids (Table 2). circle denotes the growth temperature of the cells, and bullet denotes the interpolated T values (a) or T values (b). The 20 °C decrease in the growth temperature is calculated to decrease the T and T values by 12 and 19 °C, respectively. The T values are 10-18 °C below, and the T values are 43-44 °C above, the growth temperatures. The Tvalues and T values for the synthetic PE species are taken from Koynova and Caffrey(1994).



Phase Equilibria of E. coli Lipid Extracts

IM and IOM lipid extracts prepared from wild-type E. coli cells grown at 17, 27, and 37 °C were found to be able to form H and reversed cubic phases (Fig. 1, Fig. 3, and Fig. 4). It has been observed earlier that an H phase or an isotropic phase can be formed by such lipid extracts isolated from wild-type E. coli strains (Burnell et al., 1980; Killian et al., 1992) or from E. coli strains defective in fatty acid synthesis and/or fatty acid degradation (Fabrie et al., 1994; Ianzini et al., 1990; Killian et al., 1992; Ranck et al., 1984), grown at 37 °C. The type of non-lamellar phase that is formed, and the temperature at which it appears, differ between the studies. However, this is not surprising, since the composition of the lipid-water samples studied varies substantially between the different works: 1) the neutral lipids are present or removed; 2) the water content varies; 3) a buffer component, NaCl, and EDTA are present in some samples; and 4) the mutant strains were grown on, and thus forced to incorporate, different fatty acids. The influence of the first three factors on the phase equilibria was studied in the present work. IOM lipid extracts with the neutral lipids present had a stronger tendency to form reversed non-lamellar phases than the corresponding lipid extracts with the neutral lipids removed. The neutral lipids consist mainly of free fatty acids, and of small amounts of monoacylglycerols, diacylglycerols and pigments (data not shown). The three former constituents shift the phase equilibria of membrane lipid-water mixtures toward reversed non-lamellar phases (Das and Rand, 1986; Gutman et al., 1984; Seddon and Cevc, 1993; Siegel et al., 1989), and this fact most probably explains the observed difference. The degree of hydration of the IM and IOM lipid extracts affected the phase equilibria ( Fig. 1and Fig. 2a). At higher temperatures an H phase was formed with 20 weight % water, while a bicontinuous cubic phase belonging to the space group Pn3m was formed with 50 weight % water. It has been observed earlier that total lipid extracts, or lipid fractions, from animal and plant sources can form an H phase with water contents up to 20 weight % and transform to an L phase at higher water contents (see Rilfors et al.(1984)). Cubic phases of membrane lipids often form between L and H phases (Lindblom and Rilfors, 1989), and it is therefore reasonable that a cubic phase can form at higher water contents. Moreover, the total polar membrane lipids isolated from the bacterium Sulfolobus solfataricus form a cubic phase belonging to the space group Pn3m when the water content is 40 weight % (Gulik et al., 1985). Generally, we studied the E. coli lipid extracts in the absence of a buffer component, NaCl, and EDTA. However, in a control sample 0.1 MNaCl (the concentration commonly used) was added to an IOM lipid extract prepared from E. coli grown at 37 °C, and hydrated with 50 weight % water. This additive lowered the temperature at which the cubic phase appeared by 15-20 °C. The fact that previously studied samples of IOM lipid extracts, isolated from wild-type E. coli cells grown at 37 °C, transform to an isotropic phase at lower temperatures than the corresponding extracts studied by us is thus most probably explained by the presence of NaCl (Killian et al., 1992) or of NaCl and neutral lipids (Burnell et al., 1980), in the former lipid extracts.

When lipid extracts prepared from the same cell batch were compared, the IM extracts had a stronger tendency than the IOM extracts to adopt non-lamellar phases (Fig. 2). The acyl chain compositions of the IM lipids make them more prone to form reversed non-lamellar phases, and this effect seems to outweigh the effect of having a larger fraction of PE in the IOM lipids. A physiological explanation for the difference in phase equilibria can possibly be found in the different roles and structures for the inner and outer membranes. The highly specialized outer membrane with its outer monolayer of lipopolysaccharides functions as a barrier to hydrophobic compounds and as a sieve to hydrophilic compounds (Nikaido and Vaara, 1987). All membrane-associated metabolism in E. coli takes place in the inner membrane. This membrane must constitute a permeability barrier to most hydrophilic molecules, which instead are transported over the membrane by specific proteins (Cronan et al., 1987). The IM lipids contain PE species with a more pronounced tendency to form non-lamellar phases. This results in a denser packing and higher molecular order of the acyl chains in a bilayer (Thurmond and Lindblom, 1995), which in turn decreases the passive permeability over the membrane.

Lipid Regulation and Phase Equilibria in E. coli

All lipid extracts formed an L phase at the temperature corresponding to the growth temperature of the cells from which the lipids were derived. In samples containing 20 weight % water, a transition from an L to an H phase was observed 10-15 and 20-25 °C above the growth temperature in the IM and IOM lipid extracts, respectively (Fig. 1, Fig. 2, and Fig. 4). Generally, an increased temperature shifts membrane lipid phase equilibria from lamellar toward reversed cubic and/or H phases (Rilfors et al., 1984). Wild-type E. coli cells respond to higher growth temperatures by incorporating shorter and more saturated acyl chains into their membrane lipids. These changes decrease the ability of PE to form reversed non-lamellar phases (vide supra), thus counteracting the increase in temperature. Consequently, the lipid extracts have a nearly equal ability to form non-lamellar phases at equal elevations above the growth temperature.

The increase in the degree of acyl chain unsaturation when E. coli is grown at lower temperatures can also be a striving of the cells to lower the T(m) value of the membrane lipids; however, the concomitant increase in the acyl chain length raises the T(m) value (Fig. 5a). Results obtained from previous studies of wild-type E. coli membranes, and extracted membrane lipids, show that the L to L phase transition is completed approximately 7-17 °C below the temperature of growth (Jackson and Cronan, 1978; Nakayama et al., 1980). E. coli cells subjected to a significant decrease in the growth temperature therefore have to incorporate a larger fraction of unsaturated acyl chains into its membrane lipids in order to avoid the deleterious effects of having a mixture of lipids in the gel and liquid crystalline states. The effect of changes of the acyl chain structure on the T(m) and T values of E. coli lipid extracts can be estimated by using the values from various synthetic PE species (Fig. 5). The validity of such calculations is justified by the study of Pluschke and Overath(1981). They concluded that the midpoint temperature of the L to L phase transition of E. coli lipid extracts is mainly determined by PE and that changes in the T(m) value of natural lipids can be calculated from a proper combination of the T(m) values of synthetic lipids. Fig. 5shows the calculated T(m) and T values for PE molecules having the acyl chain compositions of the IM lipids obtained in the present investigation (Table 2). It is seen that a 20 °C decrease in the growth temperature is predicted to decrease the T(m) and T values by 12 and 19 °C, respectively. The explanation to the more pronounced compensation of the T value is that the increased length of the acyl chains at the lower growth temperature decreases the T value but increases the T(m) value. Fig. 5a also shows that the calculated T(m) values of PE are 10-18 °C below the growth temperature of E. coli, which is in very good agreement with the experimental results obtained from E. coli membranes (Jackson and Cronan, 1978; Nakayama et al., 1980). On the other hand, the calculated T values of PE are about 45 °C above the growth temperature (Fig. 5b), which is appreciably higher than the phase transition temperatures found experimentally (Fig. 4). However, 20 weight % water is below the maximum hydration for PE (Rilfors et al., 1994) as well as for PG and DPG (Lindblom et al., 1991) having the acyl chain compositions in question, and this condition reduces the T value (Rilfors et al., 1994). When the lipid extracts were hydrated with 50 weight % water, a cubic phase was formed instead of an H phase; as mentioned previously, cubic phases of membrane lipids often form between L and H phases, and this explains why the cubic phase is formed at lower temperatures than the H phase.

From the arguments put forward it now appears that two of the conclusions drawn from the studies of the regulation of the membrane lipid composition in A. laidlawii strain A-EF22 also hold for E. coli: 1) IM, as well as IOM, lipid extracts have a nearly equal ability to form reversed non-lamellar phases at equal elevations above the growth temperature of the organism, as a result of the regulation of the acyl chain structures; and 2) Reversed non-lamellar phases start to form under conditions that are relatively close to the physiological ones. The two lipid monolayers of the E. coli inner membrane are thus ``frustrated'' (Lindblom and Rilfors, 1989) and have a negative spontaneous radius of curvature. Furthermore, the T(m) and T values are regulated simultaneously, and in the same direction, when the growth temperature of wild-type cells of E. coli is varied, since PE is the major lipid and the predominant regulation is the change in the degree of unsaturation of the acyl chains. However, it should be pointed out that the accompanying change in the length of the acyl chains affects the two phase transition temperatures in opposite directions and in fact makes the calculated T values of PE more closely follow the growth temperature than does the calculated T(m) values (Fig. 5).

Some arguments favor our conclusion that the presence of PE, and the tendency to form non-lamellar phases, are of crucial importance for E. coli cells. A mutant of E. coli lacking the ability to synthesize PE increases the fraction of PG and DPG and has an absolute growth requirement for high concentrations of Mg, Ca, and Sr (Rietveld et al., 1993, 1994), which make DPG form cubic and H phases (de Kruijff et al., 1982; Rand and Sengupta, 1972); the fraction of DPG in the mutant is also inversely related to the potency of the different divalent cations to promote the formation of an H phase (Killian et al., 1994). Another argument can be given; suppose that E. coli synthesized phosphatidylcholine (PC) or PG instead of PE. The tendency to form non-lamellar phases under physiological conditions would then have been completely lost (Lindblom et al., 1991; Sjölund et al., 1989), and there would be practically no need for a regulation of the acyl chain structure; calculations using the acyl chain composition obtained in the present study at 37 °C show that the T(m) value of PC and PG would be 30 °C below the growth temperature (Silvius, 1982). Most probably, it is the presence of PE in the E. coli membranes that necessitates the careful regulation of the acyl chain structure. The growth of E. coli cells, like all living cells, is restricted by two kinds of membrane lipid phase transitions: the transition to an L phase and the transition to reversed non-lamellar phases. If the degree of acyl chain unsaturation becomes too low a large fraction of the lipids may enter the gel state (Fig. 5a), and if the degree of acyl chain unsaturation becomes too high, the tendencies to form non-lamellar phases will be too large, and there is a risk for the bilayer to disrupt (Fig. 5b). Thus, our model for the regulation of membrane lipid composition most probably explains the observations that lysis of E. coli cells occurs when the lipids contain a very large fraction of long, saturated acyl chains, and that the passive permeability of the cells increases when the fraction of 18:1c11 exceeds a certain value (Cronan and Rock, 1987; Davis and Silbert, 1974). Our model also likely explains why a mutant of E. coli was unable to grow although essentially all of its lipids were in the liquid crystalline state (Pluschke and Overath, 1981); the average acyl chain length was only 13.8 and PE with such short acyl chains has lost its ability to form an H phase (Koynova and Caffrey, 1994).


FOOTNOTES

*
This work was supported by The Swedish Natural Science Research Council and The Knut and Alice Wallenberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-90-16-6343; Fax: 46-90-16-7779.

(^1)
The abbreviations used are: MGlcDAG, monoglucosyldiacylglycerol; DGlcDAG, diglucosyldiacylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PC, phosphatidylcholine; L, lamellar gel phase; L, lamellar liquid crystalline phase; H, reversed hexagonal phase; CSA, chemical shift anisotropy; IOM, inner plus outer membrane; IM, inner membrane.

(^2)
A.-S. Andersson, L. Rilfors, M. H. J. Bergqvist, and G. Lindblom, submitted for publication.

(^3)
D. Danino, A. Kaplun, G. Lindblom, L. Rilfors, G. Orädd, J. B. Hauksson, and Y. Talmon, submitted for publication.


ACKNOWLEDGEMENTS

We thank Greger Orädd for performing the x-ray diffraction experiments and Stefan Persson for determining the lipid diffusion coefficient in the cubic phase.


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