Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol
2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês
10.1074/jbc.m805189200
ISSN1083-351X
AutoresEugenia Mileykovskaya, Andrea C. Ryan, Xi Mo, Chun‐Chieh Lin, Khaled Khalaf, William Dowhan, Teresa A. Garrett,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface. The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface. A unique lipid composition and lipid-protein interactions appear to exist at the transient membrane domain that defines the division site in bacterial cells (1Mileykovskaya E. Dowhan W. Curr. Opin. Microbiol... 2005; 8: 135-142Google Scholar). Using the cardiolipin (CL) 4The abbreviations used are: CL, cardiolipin; DAPI, 4′,6′-diamidino-2-phenylindole; DIC, differential interference contrast; ESI-MS, electrospray ionization time of flight mass spectrometry; GFP, green fluorescent protein; LC-MS, liquid chromatography MS; MS/MS, mass and collision-induced decomposition MS; N-acyl-PE, N-acylphosphatidylethanolamine; NAO, 10-N-nonyl acridine orange; PBS, phosphate buffered saline; PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.-specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2Mileykovskaya E. Dowhan W. J. Bacteriol... 2000; 182: 1172-1175Google Scholar). Subsequently others reported similar CL domains in Bacillus subtilis (3Kawai F. Shoda M. Harashima R. Sadaie Y. Hara H. Matsumoto K. J. Bacteriol... 2004; 186: 1475-1483Google Scholar) and Pseudomonas putida (4Bernal P. Munoz-Rojas J. Hurtado A. Ramos J.L. Segura A. Environ. Microbiol... 2007; 9: 1135-1145Google Scholar). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ΔminCDE mutant (5Koppelman C.M. Den Blaauwen T. Duursma M.C. Heeren R.M. Nanninga N. J. Bacteriol... 2001; 183: 6144-6147Google Scholar). We suggested that formation of CL domains at cell pole/division sites plays an important role in selection and recognition of the division site by amphitropic cell cycle and cell division proteins, such as DnaA (initiation of DNA replication at oriC), MinD (a part of MinCDE system preventing positioning of the divisome at cell poles in E. coli), and FtsA (bacterial actin, which is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin), responsible for targeting the Z-ring to the mid-cell membrane domain). They interact directly with membrane phospholipids through specific amphipathic motifs enriched in basic amino acids, which confers the preference for anionic lipids (for references see Ref. 1Mileykovskaya E. Dowhan W. Curr. Opin. Microbiol... 2005; 8: 135-142Google Scholar). In E. coli the ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP bound to MinD resulting in release of MinD, and consequently MinC, from the membrane into the cytoplasm. As a result, all three proteins oscillate between the cell poles maintaining the maximum concentration of the inhibitor MinC at the cell poles and its minimum concentration at the cell center. Pole-to-pole oscillation of Min proteins occurs by dynamic redistribution of the proteins within a helical oligomeric structure that winds around the cell (for recent review and references see Ref. 6Lutkenhaus J. Annu. Rev. Biochem... 2007; 76: 539-562Google Scholar). Our previous study of a mutant lacking phosphatidylethanolamine (PE) and containing highly elevated levels of phosphatidylglycerol (PG) and CL demonstrated a strong inhibition of cell division and aggregation of MinD and FtsZ/FtsA proteins at domains enriched in CL (7Mileykovskaya E. Fishov I. Fu X. Corbin B.D. Margolin W. Dowhan W. J. Biol. Chem... 2003; 278: 22193-22198Google Scholar, 8Mileykovskaya E. Sun Q. Margolin W. Dowhan W. J. Bacteriol... 1998; 180: 4252-4257Google Scholar). To further investigate the role of lipids in the process of cell division, we chose an E. coli mutant with an opposite extreme in phospholipid composition to PE-lacking mutants, namely a ΔpgsA mutant (pgsA encodes phosphatidylglycerol phosphate synthase, which catalyzes the committed step to PG and CL synthesis (9Raetz C.R. Dowhan W. J. Biol. Chem... 1990; 265: 1235-1238Google Scholar)). This mutant is devoid of PG and CL (contribute ∼20 mole % of phospholipids in wild type) and contains higher levels of PE (∼90 mole % versus 80 mole % in wild type) (10Kikuchi S. Shibuya I. Matsumoto K. J. Bacteriol... 2000; 182: 371-376Google Scholar, 11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). Interestingly, the ΔpgsA null mutant accumulates elevated amounts of the phospholipid precursors, phosphatidic acid (PA) (∼4 mole %) and CDP-diacylglycerol (∼3 mole %), which are also anionic lipids that were proposed to fulfill the structural and functional roles of PG and CL (10Kikuchi S. Shibuya I. Matsumoto K. J. Bacteriol... 2000; 182: 371-376Google Scholar, 11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). These results suggest that a minimum of 5–10% anionic lipid is required to support viability. Another minor anionic phospholipid, N-acylphosphatidylethanolamine was suggested to be present in wild-type E. coli (12Helmy F.M. Mueller T.E. Juracka A. J. Planar Chromatogr.. 2002; 15: 23-27Google Scholar), which, if proven true, might be also elevated in this mutant. Finally, if anionic lipids are essential for cell division, then we would expect these normally minor lipids to segregate into similar anionic lipid domains, as does CL. In this report we identified N-acyl-PE in E. coli and, along with PA, its enrichment in polar/septal membrane domains of the ΔpgsA mutant UE54 (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar) lacking PG and CL. Thus E. coli has a mechanism for preferential segregation of anionic phospholipids to the polar/septal regions where several amphitropic proteins, which show preference for interaction with anionic phospholipids in vitro, are functionally located. Strain Construction—E. coli K-12 strains MG1655 (www.genome.wisc.edu/sequencing/k12.htm), UE53 (MG1655 lpp-2 Δara714 rcsF::mini-Tn10 cam), and UE54 (MG1655 lpp-2 Δara714 rcsF::mini-Tn10 cam pgsA::FRT-kan-FRT) have been previously described (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). To construct a UE54 strain expressing a green fluorescent protein (GFP) derivative of MinD (UEM541), a single-copy fusion Ptrc90-GFP-minD-minE (inserted in chromosome at the attB site), which is closely linked to an Apr marker, from strain WM1264 (13Corbin B.D. Yu X.C. Margolin W. EMBO J.. 2002; 21: 1998-2008Google Scholar) was transferred to UE54 by phage P1 transduction followed by selection for ampicillin resistance. For construction of a UE54 strain producing a GFP derivative of FtsZ (UEM542) a single-copy fusion Ptrc208-ftsZ-GFP (inserted in the chromosome at the attL site and closely linked to an Apr marker) from strain EC448 (14Weiss D.S. Chen J.C. Ghigo J.M. Boyd D. Beckwith J. J. Bacteriol... 1999; 181: 508-520Google Scholar) was transferred to UE54 by phage P1 transduction followed by selection for ampicillin resistance. To obtain a minicell-forming derivative of UE54 (UEM543) fadR::Tn10 linked to minCDE::kan from strain WM1192 (13Corbin B.D. Yu X.C. Margolin W. EMBO J.. 2002; 21: 1998-2008Google Scholar) was transferred to UE54 by phage P1 transduction. Transductants were selected for tetracycline resistance and screened for the Δmin phenotype. Growth of Cells—All cells were grown at 30 °C in LB medium. For mass spectrometry (MS) analysis strains were grown in LB medium to an A600 of 1.0, harvested by centrifugation at 500 × g for 15 min at 4 °C, washed once with phosphate-buffered saline (PBS) (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4) and then resuspended in the same buffer. Pellets were frozen at -80 °C until lipids were extracted. Isolation of Minicells—Minicells were isolated according to a previous study (5Koppelman C.M. Den Blaauwen T. Duursma M.C. Heeren R.M. Nanninga N. J. Bacteriol... 2001; 183: 6144-6147Google Scholar) with modifications. Briefly, cells were grown to an optical density of A450 of 0.2 in LB medium with 0.1% glucose, harvested, and resuspended in 100 ml of BSG buffer (1.5 m NaCl, 20 mm KH2PO4, 50 mm Na2HPO4 (pH 7.7), 0.1% (w/v) gelatin). Large cells were isolated by centrifugation at 500 × g for 10 min, and the pellet was resuspended in 10 ml of 50 mm KH2PO4/K2HPO4 (pH 7.2), 5 mm MgSO4 (buffer A) and stored at -80 °C for lipid analysis. The supernatant was centrifuged at 20,000 × g for 20 min. The resulting pellet was resuspended in 5 ml of BSG, loaded onto a sucrose gradient generated from layers of 3-ml 20%, 12-ml 10%, and 18-ml 3% (w/v) sucrose solutions in BSG, and then centrifuged at 2500 × g for 10 min in a swinging bucket rotor. The minicell fraction in the 3% sucrose layer was collected. The sucrose gradient centrifugation step was repeated up to six times. The purity was checked by 4′,6′-diamidino-2-phenylindole (DAPI, Molecular Probes, Inc) staining and fluorescence microscopy (see below) to verify an enrichment of minicells over large cells. The final minicell preparation was washed in buffer A, pelleted by centrifuge at 2500 × g for 10 min, resuspended in 1–2 ml of the same buffer, and frozen at -80 °C. Liposome Preparation—CL from beef heart and 1-palmitoyl 2-oleoylphosphatidic acid were purchased from Avanti Polar Lipids (Alabaster, AL). Liposomes were prepared by water bath sonication for 1–2 h at 0 °C in a buffer containing 25 mm Tris-HCl, pH 7.5, and 50 mm KCl at a lipid concentration of 2–4 mg/ml, and were stored at -80 °C. TLC of Lipid Extracts—The cell pellet was resuspended in 0.1 ml of 0.5 m NaCl in 0.1 n HCl, and lipids were extracted by chloroform/methanol and examined by TLC separation according to a previous study (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). Microscopy Study—To observe FtsZ-GFP localization or GFP-MinD movement in UE541 or UE542, overnight cultures were grown in LB medium with 10 μm chloramphenicol or kanamycin to an A600 of 1.6 and diluted 1:50 in the same growth medium followed by growth to A600 of 0.6 at 30 °C in the presence of 10 μm isopropyl β-d-1-thiogalactopyranoside. This condition was chosen because it resulted in levels of FtsZ-GFP or GFP-MinD high enough to be detected by the camera but low enough to have practically no impact on MinD oscillation and cell division in wild-type cells. Both types of cells were immobilized on microscope slides in 1% agarose as described before (7Mileykovskaya E. Fishov I. Fu X. Corbin B.D. Margolin W. Dowhan W. J. Biol. Chem... 2003; 278: 22193-22198Google Scholar). Fluorescence images and differential interference contrast (DIC) were observed with a 100× oil immersion objective on an Olympus BX60 microscope fitted with a GFP filter cube, and captured with a light-sensitive Photometrics CoolSnap FX cooled charge-coupled device camera driven by QED image capturing software and saved as Adobe Photoshop TIF files. Cell lengths were measured with Object Image software (Norbert Vischer). Time-lapse images of GFP-MinD were taken every 6–8 s, each with 2- to 4-s exposures using 2 × 2 binning. For microscopic examination of minicells and nucleoids of living cells, samples were stained with DAPI at a final concentration of 1 μg/ml and viewed with a standard DAPI filter. The membrane of UE54 cells was stained with lipophilic FM4-64 (Molecular Probes, Inc.) at a final concentration of 4 μg/ml as described before (15Ghosh A.S. Young K.D. J. Bacteriol... 2005; 187: 1913-1922Google Scholar). For staining of anionic lipids in UE54 cells, NAO was added directly to the culture (final concentration 400 nm) in the growth medium and incubated for 1 h at room temperature. Liposomes (1 μm of lipids) were stained with 2 μm NAO in buffer containing 25 mm Tris-HCl (pH 7.5) and 50 mm KCl, washed by centrifugation, and resuspended in the same buffer. The stained cells or liposomes were immobilized on a cover glass with 1% agarose and viewed for green and red fluorescence images (2Mileykovskaya E. Dowhan W. J. Bacteriol... 2000; 182: 1172-1175Google Scholar) as described above. Extraction of Lipids From Cells for MS—Lipids were extracted from UE54 and MG1655 whole cells, UEM543 large cells separated from minicells, and purified minicells with a chloroform/methanol/aqueous Bligh and Dyer extraction system (16Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol... 1959; 37: 911-917Google Scholar). Whole cell extracts were prepared as follows. Cell pellets resulting from the growth of MG1655 or UE54 culture in 50 ml of LB as described above were resuspended in 4 ml of PBS. Chloroform (5 ml) and methanol (10 ml) were added to generate a single-phase neutral extract (1:2:0.8, chloroform:methanol:PBS) (16Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol... 1959; 37: 911-917Google Scholar). Cell debris was removed from the extract by centrifugation. The supernatant was transferred to a fresh tube and converted to a two-phase neutral extract (2:2:1.8, chloroform:methanol:PBS) by the addition of 5 ml of chloroform and 5 ml of PBS. Phases were resolved by centrifugation, and the upper phase was discarded. The lower phase was washed with 15 ml of pre-equilibrated neutral lower phase and centrifuged a second time to resolve the phases. The lower phase was dried under nitrogen, and the dried lipid films were stored at -20 °C until analysis. Lipid extracts were prepared from large cells and minicells as follows. Fifty microliters of the large cells or 150 μl of minicells were used for each extraction. The aqueous volume was increased to 0.4 ml by the addition of PBS. Chloroform (0.5 ml) and methanol (1.0 ml) were added to generate a single-phase neutral extract (1:2:0.8 chloroform:methanol:PBS). Protein separation from the samples, conversion of the single-phase extract into two-phase system, and obtaining of the dry lipid films from organic phase were performed as described above and previously (17Schaaf G. Betts L. Garrett T.A. Raetz C.R. Bankaitis V.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun... 2006; 62: 1156-1160Google Scholar). For quantification of lipid species using MS synthetic lipid standards were co-extracted with the cell pellets. For quantification of N-acyl-PE, 391 pmol of synthetic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-nonadecanoyl (55:2 N-acyl-PE) standard (Avanti Polar Lipids, Alabaster, AL) was added to the resuspended cell pellets prior to addition of chloroform and methanol. For quantification of the ratio of PA to PE, 1.9 nmol of synthetic 12:0, 13:0 PA, and 1.82 nmol of 12:0, 13:0 PE (Avanti Polar Lipids, Alabaster, Al) were added to the initial extraction mixture. For quantification of the ratio of N-acyl-PE to PE, 0.98 nmol of synthetic 55:2 N-acyl-PE was used in place of the PA standard. DEAE Fractionation of Total Lipid Extracts—Total lipid extracts derived from UE54 and MG1655 whole cells were fractionated on DE-52 cellulose (Whatman) as described previously (18Kanjilal-Kolar S. Basu S.S. Kanipes M.I. Guan Z. Garrett T.A. Raetz C.R. J. Biol. Chem... 2006; 281: 12865-12878Google Scholar). Mass Spectrometry—For analysis of total lipid composition dried lipid films were dissolved in 100 μl of chloroform/methanol (2:1) and infused into a quadrupole time-of-flight tandem mass spectrometer (QStar® XL, Applied Biosystems) at 6 μl/min. The mass spectra were obtained by scanning from 40–2000 atomic mass units in the negative-ion, multichannel acquisition mode with the ESI source operating at the following settings: nebulizer gas, 20.0 p.s.i.; curtain gas, 20.0 p.s.i.; ion spray voltage, -4200 V; declustering potential, -55 V; focusing potential, -265 V; and declustering potential 2, -15 V. Spectra were acquired for 1 min at 1 scan per second and analyzed using Analyst QS 1.1 software (Applied Biosystems). Collision-induced decomposition MS (MS/MS) was performed using collision energy of -40.0 V and N2 as the collision gas. Spectra were acquired for up to 5 min at 1 scan per second. Quantification of lipids was accomplished using reversed-phase liquid chromatography MS (LC-MS). For the quantification of N-acyl-PE in whole cells, the dried lipid film was resuspended in 300 μl of CHCl3, 200 μl was transferred to a Target DP vial containing a Target Polyspring Inset (National Scientific), dried under nitrogen, and finally, resuspended in 100 μl of mobile phase A containing 10% CHCl3. For quantification of the ratio of PA to PE the dried lipid film was resuspended in 0.8 ml of CHCl3, 200 μl was transferred to a Target DP vial containing a Target Polyspring Inset dried under nitrogen, and then resuspended in 100 μl of mobile phase solvent A containing 10% CHCl3. Reversed-phase LC-MS was performed as described previously (19Garrett T.A. Guan Z. Raetz C.R. Methods Enzymol.. 2007; 432: 117-143Google Scholar) by using a Shimadzu LC system (comprising a solvent degasser, two LC-10A pumps and a SCL-10A system controller) coupled to a QSTAR XL quadrupole time-of-flight tandem mass spectrometer (Applied Biosystems/MDS Sciex). A Zorbax SB-C8 reversed-phase column (Agilent, Palo Alto, CA) was used for all LC-MS analyses. For each LC-MS analysis 30 μl of sample (prepared as described above) was injected on to the reversed-phase column. LC was operated at a flow rate of 200 μl/min with a linear gradient as follows: 100% mobile phase A (methanol:acetonitrile:aqueous 1 mm ammonium acetate, 60:20:20) was held for 2 min and then linearly increased to 100% mobile phase B (100% ethanol containing 1 mm ammonium acetate) over 14 min and held at 100% mobile phase B for 4 min. The column was re-equilibrated to 100% mobile phase A for 2 min prior to the next injection. The post-column split diverted ∼10% of the LC flow to the ESI source of the mass spectrometer. All MS data were analyzed using Analyst QS software (Applied Biosystems/MDS Sciex). Individual lipid species were quantified by taking the ratio of the peak area of the extracted ion current from the LC-MS chromatogram to the peak area of the extracted ion current for the appropriate synthetic internal standard (19Garrett T.A. Guan Z. Raetz C.R. Methods Enzymol.. 2007; 432: 117-143Google Scholar). Lack of PG and CL Decreases the Length of the UE54 Mutant Cell—TLC analysis of total lipid extracts from the ΔpgsA strain (UE54) according to a previous study (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar) showed no differences in its phospholipid composition compared with other ΔpgsA strains reported before (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). As with the other ΔpgsA strains, UE54 is viable only if it carries two additional gene mutations. An lpp mutation is necessary to prevent accumulation of the nascent major outer membrane lipoprotein, which requires PG for its modification (10Kikuchi S. Shibuya I. Matsumoto K. J. Bacteriol... 2000; 182: 371-376Google Scholar, 20Matsumoto K. Mol. Microbiol... 2001; 39: 1427-1433Google Scholar, 21Suzuki M. Hara H. Matsumoto K. J. Bacteriol... 2002; 184: 5418-5425Google Scholar). The Rcs phosphorelay system is constitutively activated in ΔpgsA mutants resulting in the cell lyses at 37–42 °C, which is prevented by a ΔrcsF mutation (22Nagahama H. Sakamoto Y. Matsumoto K. Hara H. J. Gen. Appl. Microbiol... 2006; 52: 91-98Google Scholar). We compared the growth rates of UE54 lacking PG and CL and its two parental pgsA+ strains, MG1655 and UE53 (lpp-2 ΔrcsF), with wild-type phospholipid composition. To exclude additional factors contributing to cell growth and division at high temperatures all strains were grown at 30 °C. In agreement with previous findings for the ΔpgsA mutant (10Kikuchi S. Shibuya I. Matsumoto K. J. Bacteriol... 2000; 182: 371-376Google Scholar), the doubling times of UE54 were ∼90 min versus 60 min for the two parental strains (data not shown). During the exponential phase of growth a similar average cell length was found for MG1655 and UE53 (3.66 ± 0.1 μm), whereas the UE54 cells were ∼20% shorter (2.93 ± 0.06 μm) (Fig. 1A). Cell width was the same (1.25 ± 0.05 μm) for all three strains. FtsZ-GFP Localization and GFP-MinD Oscillation—FtsZ-GFP (green fluorescent protein), produced from a single copy gene fusion, was introduced into UE54 to generate UEM542. Fig. 1B shows the normal mid-cell localization of FtsZ-GFP in the majority of UEM542 cells. FtsZ rings were observed in ∼77% of cells (206 cells analyzed). We also examined the dynamic localization of GFP-MinD in the UEM541 cells expressing a single-copy gene fusion of GFP-MinD along with MinE (see "Experimental Procedures"). In these cells, GFP-MinD oscillated from pole to pole with cycle times between 60 and 80 s (Fig. 1C) similar to that of UE53 with wild-type phospholipid composition (data not shown). Staining of Anionic Lipid Membrane Domains with NAO—To study possible formation of anionic lipid membrane domains in ΔpgsA cells, we used a visualization technique developed previously for staining CL domains in bacteria with the fluorescent dye NAO (2Mileykovskaya E. Dowhan W. J. Bacteriol... 2000; 182: 1172-1175Google Scholar) and successfully used by other groups (3Kawai F. Shoda M. Harashima R. Sadaie Y. Hara H. Matsumoto K. J. Bacteriol... 2004; 186: 1475-1483Google Scholar, 4Bernal P. Munoz-Rojas J. Hurtado A. Ramos J.L. Segura A. Environ. Microbiol... 2007; 9: 1135-1145Google Scholar, 23Romantsov T. Helbig S. Culham D.E. Gill C. Stalker L. Wood J.M. Mol. Microbiol... 2007; 64: 1455-1465Google Scholar). Association of NAO with CL results in the appearance of a red emission maximum in the dye fluorescence spectrum, and CL membrane domains emitting both green and red fluorescence were previously demonstrated in E. coli and Bacillus subtilis with wild-type phospholipid composition (2Mileykovskaya E. Dowhan W. J. Bacteriol... 2000; 182: 1172-1175Google Scholar, 3Kawai F. Shoda M. Harashima R. Sadaie Y. Hara H. Matsumoto K. J. Bacteriol... 2004; 186: 1475-1483Google Scholar, 23Romantsov T. Helbig S. Culham D.E. Gill C. Stalker L. Wood J.M. Mol. Microbiol... 2007; 64: 1455-1465Google Scholar, 24Mileykovskaya E. Mol. Microbiol... 2007; 64: 1419-1422Google Scholar). In contrast to NAO bound to CL, NAO bound to other anionic phospholipids does not exhibit specific red emission. Only green fluorescence of the membrane in Δcls mutants of B. subtilis (3Kawai F. Shoda M. Harashima R. Sadaie Y. Hara H. Matsumoto K. J. Bacteriol... 2004; 186: 1475-1483Google Scholar) and E. coli (2Mileykovskaya E. Dowhan W. J. Bacteriol... 2000; 182: 1172-1175Google Scholar, 23Romantsov T. Helbig S. Culham D.E. Gill C. Stalker L. Wood J.M. Mol. Microbiol... 2007; 64: 1455-1465Google Scholar) or the PG-containing membrane domain of Streptococcus pyogenes (25Rosch J.W. Hsu F.F. Caparon M.G. J. Bacteriol... 2007; 189: 801-806Google Scholar) was observed. In vitro experiments with PA containing liposomes demonstrated this phospholipid also binds NAO, but only green fluorescence of the liposome was observed under the microscope (Fig. 2A). Staining of UE54 mutant cells with NAO revealed green fluorescence membrane domains in the regions of the cell poles and at the cell center (Fig. 2, B and C). Some cells in the population showed only cell pole domain fluorescence while other cells exhibited both cell pole and mid-cell or only mid-cell domain positioning of NAO, which may be a result of cells at different stages of the cell cycle. Staining of cells with a nonspecific lipophilic fluorescent dye, FM4-64, demonstrated a uniform distribution along the membrane (Fig. 2D). Comparison of green fluorescence domain positioning in UE54 and UE53 cells (Fig. 2E) suggests that in contrast to UE53 the polar staining in UE54 cells containing a mid-cell domain appears less frequently than staining of the central domain. This might be an indication of preferential targeting of anionic phospholipids to division site in UE54 cells. However, we cannot completely exclude that the domains might not be equally visible due to different orientations of the UE54 cells in the field. These results are consistent with the targeting of anionic lipids to the cell poles and division sites of UE54 cells. To reveal the nature of anionic lipids concentrated at cell poles/septal sites, we analyzed the phospholipid composition of minicells (as described below), which are produced due to abnormal cell division occurring at the cell poles. MS of Lipid Extracts from UE54 and MG1655 Cells—Initially we compared lipid composition of UE54 to MG1655. Analyses were performed by negative-ion electrospray ionization time-of-flight MS (ESI-MS). As expected the lipid composition of the two strains differed drastically. MG1655 showed predominant ions corresponding to PG (Fig. 3A). The ions at m/z 691.44, 719.45, 733.48, 747.48, 761.50, and 773.51 represent [M-H]- ions of PG with 30:1, 32:1, 33:cyclopropane (cp), 34:1, 35:cp, 36:2 carbon acyl chains, respectively. Ions corresponding to [M-H]- ions of PE with 32:1 and 34:1 acyl chains were present but at lower abundance (m/z 688.48 and 716.49, respectively). Although PE represents roughly 80% of the total phospholipids in wild-type cells (9Raetz C.R. Dowhan W. J. Biol. Chem... 1990; 265: 1235-1238Google Scholar), it ionizes much less efficiently than PG in the negative-ion mode, and, therefore its true abundance is not reflected by its ionization (17Schaaf G. Betts L. Garrett T.A. Raetz C.R. Bankaitis V.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun... 2006; 62: 1156-1160Google Scholar). To confirm the presence of PE, CL, and PA in MG1655, the total lipid extract was fractionated on DEAE-cellulose (18Kanjilal-Kolar S. Basu S.S. Kanipes M.I. Guan Z. Garrett T.A. Raetz C.R. J. Biol. Chem... 2006; 281: 12865-12878Google Scholar), which separates the phospholipids according to charge. ESI-MS of the appropriate fractions revealed ions corresponding to PE, CL, and PA as expected (data not shown). In contrast, the mass spectrum of the lipid extract derived from UE54 cells contained predominately ions corresponding to PE (Fig. 3B) consistent with the inability of these cells to make PG and CL (11Shiba Y. Yokoyama Y. Aono Y. Kiuchi T. Kusaka J. Matsumoto K. Hara H. J. Bacteriol... 2004; 186: 6526-6535Google Scholar). The ions at m/z 688.47, 702.48, 716.50, and 742.51 represent [M-H]- ions of PE with 32:1, 33:cp, 34:1, and 36:2 acyl chains, respectively. Also present were ions corresponding to PA, and chloride adducts of diacylglycerol. The ions at m/z 601.44, 629.47, and 655.48 represent [M+Cl]- ions of diacylglycerol with 32:1, 34:1, and 36:2 acyl chains, respectively. The ions at m/z 645.43 and 673.47 represent [M-H]- ions of PA with 32:1 and 34:1 acyl chains, respectively. Mass spectrometry of the UE54 lipid extract following fractionation on DEAE-cellulose also reveals additional species of PA (see below) and the presence of ions corresponding to CDP-diacylglycerol (data not shown), both precursors to PG and CL. All lipids were identified by exact mass and MS/MS, verifying by more rigorous means the pre
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