Journal of Bacteriology, October 2001, p. 6144-6147, Vol. 183, No. 20
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.20.6144-6147.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Escherichia coli Minicell Membranes
Are Enriched in Cardiolipin
Cecile-Marie
Koppelman,1
Tanneke
Den
Blaauwen,1
Marc C.
Duursma,2
Ron M. A.
Heeren,2 and
Nanne
Nanninga1,*
Swammerdam Institute for Life Sciences,
BioCentrum Amsterdam, University of Amsterdam,1
and FOM-Institute for Atomic and Molecular
Physics,2 Amsterdam, The Netherlands
Received 8 June 2001/Accepted 20 July 2001
 |
ABSTRACT |
The phospholipid composition of Escherichia coli
minicells has been studied as a model for the cell division site.
Minicells appeared to be enriched in cardiolipin at the expense of
phosphatidylglycerol. Mass spectrometry showed no differences between
the gross acyl chain compositions of minicells and wild-type cells.
| |
TEXT |
Escherichia coli cell
division is initiated by formation of the FtsZ ring in the middle of
the cell. Subsequently, other cell division proteins localize to this
ring and together they form the divisome, which carries out the fission
process. Although localization of the cell division proteins has been
studied intensively, it is still not known how they find the correct
position (16, 22). Presumably, divisomal proteins
recognize a certain protein and lipid environment. The invagination
process, as such, might also require a specific local phospholipid
composition to facilitate membrane curvature. One aspect of the process
with respect to membrane curvature might be the shape of the
phospholipids, i.e., whether they are cones, inverted cones, or
cylinders. For instance, cardiolipin (CL) has a cylindrical shape,
whereas its Ca2+ form is cone shaped (for a
review, see reference 9).
To gain more insight into whether specific phospholipids or specific
lipid combinations are involved in the division process, we compared
the phospholipid compositions of so-called minicells from mutant and
wild-type cells. Minicells are formed after cell division at the cell
pole in min mutants (1) (Fig.
1A).
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FIG. 1.
Minicell formation. (A) Schematic representation of
normal cell division and minicell formation. (B) Microscopic images of
the minicell-producing strain LMC1088. The upper panel shows a
phase-contrast image, and the lower panel shows the corresponding DAPI
fluorescence image. Note that the minicells lack DNA and therefore do
not show DAPI fluorescence. Bar = 1 µm. (C) Phase-contrast image
of isolated minicells.
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Isolation of minicells.
E. coli LMC500
lysA (25) was used as the wild-type strain, and
LMC1088 was used as a minicell-forming mutant (constructed by E. Mulder
[21]). Cells were grown to steady state (7) with a doubling time of 80 min at 28°C in glucose minimal medium. Minicells were isolated from a 5-liter cell culture of LMC1088. All
steps were performed at 0 to 4°C. After harvest at an optical density
at 450 nm of 0.2, the cells were resuspended in 100 ml of BSG (1.5 M
NaCl, 20 mM KH2PO4, 50 mM
Na2HPO4 · 2H2O, 0.1% [wt/vol] gelatin [pH 7.7]). Large
cells were removed by centrifugation at low speed (5 min at 500 × g), and the remaining cells were subsequently pelleted by
centrifugation for 10 min at 15,000 × g. After
resuspension in 5 ml of BSG, the cell suspension was layered on a
sucrose gradient prepared in BSG consisting of a 3-ml 20%, a 12-ml
10%, and an 18-ml 5% (wt/vol) sucrose top layer. Centrifugation was
done in a swing-out rotor (Beckman JS-13) for 10 min at 2,500 × g. The minicell fraction was collected from the 5% sucrose
layer and was washed with and then resuspended in a small volume of
membrane buffer (50 mM
KH2PO4/K2HPO4
[pH 7.2], 5 mM MgSO4, 0.5 mM Pefablock
[Boehringer Mannheim, Mannheim, Germany]) and stored at 
70°C. The
purity was checked by DAPI (4',6'-diamidino-2-phenylindole)
fluorescence microscopy (Fig. 1B and C). Typically, the yield of
minicells isolated from a 5-liter culture was 3 to 5 mg on a protein
basis. If the minicell suspension contained more than 5%
DNA-containing cells, the sucrose gradient centrifugation step and wash
step were repeated.
Phospholipid head group analysis.
Phospholipids from 3-mg
samples (based on protein content) were extracted (2) and
separated by two-dimensional thin-layer chromatography
(6) on boric acid-impregnated plates (thin-layer chromatography plates with silica gel 60; Merck, Germany) with chloroform-methanol-water-ammonia (120:75:6:2, vol/vol/vol/vol) as the
first developer and chloroform-methanol-acetic acid (65:25:10, vol/vol/vol) for the second direction. The spots were visualized with
iodine vapor. After being scraped from the plate, the lipid phosphorus
of each spot was determined (8).
Phospholipid head group normal-cell and minicell composition.
Exponentially growing wild-type cells were found to contain 81.1 mol%
phosphatidylethanolamine (PE) (range, 78.4 to 86.7 mol%), 17.1 mol%
phosphatidylglycerol (PG) (range, 11.4 to 19.9 mol%), and 1.8 mol% CL
(range, 1.5 to 2.1 mol%) (n = 4) (Fig.
2), values which are in good agreement
with those found in the literature (4). The phospholipid
compositions of cells from the minicell-producing strain LMC1088 were
more or less the same as those of the wild-type cells (Fig. 2).
However, when the phospholipid composition of minicell extracts
(n = 6) was compared with that of LMC1088
(n = 6), it was found that the amount of CL was
increased from 1.9 mol% (range, 1.2 to 2.4 mol%) to 6.5 mol% (range,
3.1 to 9.9 mol%). By contrast, the amount of PG was decreased from
18.4 mol% (range, 17.4 to 19.5 mol%) to 12.8 mol% (range, 10.0 to
16.6 mol%). The amounts of PE appeared to be the same, 81.1 mol%
(range, 79.4 to 86.7 mol%) in LMC1088 and 80.7 mol% (range, 76.4 to
84.7 mol%) in minicells.
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FIG. 2.
Comparison of phospholipid (PL) head group compositions
of E. coli LMC500 (wild type), LMC1088 (mutant), and
minicells. Values are averages from four, six, and six duplicate
experiments with LMC500, LMC1088, and minicells, respectively.
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The phospholipid domains in E. coli have been visualized
previously with the CL-specific fluorescent dye 10-N-nonyl
acridine orange (19). Their polar localizations are
compatible with our biochemical data. However, our data differ from
those of Goodell and coworkers (10), who found a relative
increase in the level of PG in minicells. We have no explanation for
this discrepancy.
ESI-FTICR-MS.
Electrospray ionization-Fourier transform ion
cyclotron resonance mass spectrometry (ESI-FTICR-MS) was performed on a
modified Bruker APEX 7.0e FTICR-MS (11, 13).
High-resolution mass spectra of both wild-type and minicell
phospholipid extracts were taken. Expanded mass spectral regions of
wild-type extracts are shown in Fig. 3.
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FIG. 3.
Positive-ion ESI-FTICR-MS analysis of E.
coli phospholipids. A total phospholipid extract from wild-type
cells was dissolved in dichloromethane-ethanol (7:3, vol/vol) and
sprayed with 10 mM ammonium acetate. Expanded regions from broad-band
mass spectra obtained after Fourier transformation of the ion cyclotron
signal are shown. Individual phospholipid species are indicated by head
group and overall acyl chain composition (expressed as
x:y, where x is the number
of carbon atoms and y is the number of unsaturated
bonds). (A) Region between an m/z of 680 and an
m/z of 800, containing mass spectral peaks derived from
PE [M + H]+ ( ), PG [M + H]+ ( ), PG
[M + NH4]+ ( ), and PA [M + H]+ ( ). (B) Region between an m/z of
1,350 and an m/z of 1,500, containing mass spectral
peaks derived from CL [M + H]+ ( ).
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The first region of interest ranges from a mass-to-charge ratio
(m/z) of 650 to an m/z of 800 (Fig. 3A), and it
contains predominantly the singly charged PE, PG, and phosphatidic acid
(PA) mass spectral peaks. The PE peaks are most abundant, as expected
from the chemical phospholipid composition analysis. The PG species
were observed chiefly as [M + NH4]+ ions. The total acyl
chain lengths of the PG species varied between 30 and 36 carbon atoms,
and they contained 0 to 2 unsaturated bonds. Only one species of PA (PA
36:0) was found in the mass spectra, which is not surprising since PA
occurs in E. coli as only a minor phospholipid.
The second region of interest is that between an m/z of
1,350 and an m/z of 1,500 (Fig. 3B), and this region
contains the singly charged CL ions. The most abundant ion series were
[M + H]+ ions, with a gross acyl chain
composition ranging from 66 to 72 carbon atoms and containing 0 to 3 unsaturated bonds. In addition to the saturated and unsaturated CL
species with an even number of carbon atoms, two species with odd
numbers of C atoms were also found (CL 67:0 and CL 69:1). All peaks
derived from phospholipids were found in the spectra of wild-type and
minicell extracts. Thus, a qualitative comparison indicated no
clear-cut differences.
Lipids and cell division.
Kikuchi and coworkers
(12) constructed a pgsA null mutant which was
able to grow and divide despite the fact that PG and CL were not
detectable (molar percentages of <0.001 in each case). The highly
increased level of PA in the null mutant suggested that the absence of
the anionic phospholipids PG and CL was functionally compensated for,
at least in part, by PA (12). Thus, neither CL nor PG
seems to be essential for growth (12; for a review, see
reference 17). Although anionic phospholipids are
necessary for cell viability, the exact phospholipid compositions may
differ (see also reference 23).
One role of anionic phospholipids may be in the recruitment of cell
division proteins by electrostatic interactions (for reviews, see
references 17 and 18). For instance, SecA
protein-dependent translocation of proteins across the inner membrane
has been shown to be dependent on anionic phospholipids (3, 5,
14, 15). Since many membrane-anchored divisomal proteins have
large periplasmic domains, it appears likely that membrane insertion
also requires the presence of anionic phospholipids.
PE-deficient cells are incapable of division, and the filaments do not
show visible constrictions (20; also reference
24 and references therein). However, in cells containing a
pss-93 null mutation (pss encodes
phosphatidylserine synthase), the essential cell division proteins
FtsZ, FtsA, and ZipA were still able to localize to potential division
sites (20). This suggests that PE is needed in the
constriction process and less so in the positioning of the cytokinetic ring.
Despite the absence of a specific role for CL in cell division, one
might speculate that the increase in the level of CL in minicells
reflects the requirement of CL that proteins in the cell division
complex (divisome) be either stabilized, activated, or both. A second
possible role, as mentioned in the introduction, relates to the
establishment of membrane curvature, as required for the initiation of
cell constriction. Whether this polymorphic property is relevant for
E. coli cell division remains to be established.
| |
ACKNOWLEDGMENTS |
This work was supported in part by ALW-NWO (C.-M.K. and N.N.;
program number 805-33-220-P) and by FOM-NWO (M.C.D. and R.M.A.H.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Swammerdam
Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands. Phone: 31-20-525 5194. Fax:
31-20-525 6271. E-mail: nanninga{at}science.uva.nl.
| |
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Journal of Bacteriology, October 2001, p. 6144-6147, Vol. 183, No. 20
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.20.6144-6147.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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