(Received for publication, November 7, 1995; and in revised form, January 9, 1996)
From the
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.
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), ()and the monoacylated derivatives of MGlcDAG and
diglucosyldiacylglycerol (DGlcDAG) (
)(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.
The acyl chain compositions were determined by gas chromatography using the methyl esters of the fatty acids (Rilfors, 1985).
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 H NMR spectra were recorded as described in Lindblom et al.(1993).
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.
The phase
behaviors of IOM and IM phospholipid extracts hydrated with 20 and 50
weight % H
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 %
H
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 %
H
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)
H
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 %
H
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 %
H
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 %
H
O,
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 % (
) or 50
weight % (
)
H
O. b, IOM lipid
extracts (filled symbols) and IM lipid extracts (unfilled
symbols) hydrated with 20 weight %
H
O, and
IM lipid extracts hydrated with 50 weight %
H
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 %
H
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
8.5
10
m
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 + k
+ l
)
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 %
H
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
, where a
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 %
H
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 %
H
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 %
H
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 %
H
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 % H
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.
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(
)(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
(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
) 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
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.
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. T
values (a) and T
values (b) for synthetic diacyl-PE species having two saturated
(
) or two cis-9-monounsaturated acyl chains (
),
and for a synthetic 16:0/18:1c9-PE species (
). The T
and 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).
denotes the growth temperature of the cells, and
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 T
values and T
values for the synthetic PE species are taken
from Koynova and Caffrey(1994).
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.
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 value of the membrane lipids;
however, the concomitant increase in the acyl chain length raises the T
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
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
value
of natural lipids can be calculated from a proper combination of the T
values of synthetic lipids. Fig. 5shows
the calculated T
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
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
value. Fig. 5a also shows
that the calculated T
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 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
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
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).