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.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 Ca 2ϩ 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).Isolation of minicells. E. coli LMC500 lysA (25) was used as the wild-type strain, and LMC1088 was used as a minicellforming 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 KH 2 PO 4 , 50 mM Na 2 HPO 4 ⅐ 2H 2 O, 0.1% [wt/vol] gelatin [pH 7.7]). Large cells were removed by centrifugation at low speed (5 min at 500 ϫ g), and the remaining cells were subsequently pelleted by centrifugation for 10 min at 15,000 ϫ 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 ϫ 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 KH 2 PO 4 /K 2 HPO 4 [pH 7.2], 5 mM MgSO 4 , 0.5 mM Pefablock [Boehringer Mannheim, Mannheim, Germany]) and stored at Ϫ70°C. The purity was checked by DAPI (4Ј,6Ј-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...

SummaryWe investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP). FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ. FtsZ-R174D could also localize in an FtsZ depletion strain. However, in contrast to wildtype FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl 2 . This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.