Inhibitory Effects of Basic or Neutral Phospholipid on Acidic Phospholipid-mediated Dissociation of Adenine Nucleotide Bound to DnaA Protein, the Initiator of Chromosomal DNA Replication
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m212202200
ISSN1083-351X
AutoresNorikazu Ichihashi, Kenji Kurokawa, Miki Matsuo, Chikara Kaito, Kazuhisa Sekimizu,
Tópico(s)DNA Repair Mechanisms
ResumoDnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s). DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s). Chromosomal DNA replication in bacteria is regulated at the initiation step (1Cooper S. Helmstetter C.E. J. Mol. Biol. 1968; 31: 519-540Crossref PubMed Scopus (696) Google Scholar, 2Donachie W.D. Nature. 1968; 219: 1077-1079Crossref PubMed Scopus (434) Google Scholar), where the activity and quantity of the initiator DnaA protein is critically controlled (3Kornberg A. Baker T. DNA Replication. 2nd ed. W. H. Freeman and Co., New York1992Google Scholar, 4Boye E. Lobner-Olesen A. Skarstad K. EMBO Rep. 2000; 1: 479-483Crossref PubMed Scopus (130) Google Scholar, 5Katayama T. Mol. Microbiol. 2001; 41: 9-17Crossref PubMed Scopus (66) Google Scholar). DNA replication in Escherichia coli is initiated by the binding of DnaA protein to the DnaA boxes in the oriC, the origin of chromosomal DNA replication, followed by melting of the duplex at the three AT-rich 13-mers that locate close to the DnaA boxes. Loading of DnaB helicase in complex with DnaC protein leads to priming by DnaG primase, and DNA is synthesized by the DNA polymerase III holoenzyme (3Kornberg A. Baker T. DNA Replication. 2nd ed. W. H. Freeman and Co., New York1992Google Scholar, 6Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (512) Google Scholar). Based on biochemical and molecular genetic research in E. coli, DnaA protein activity is proposed to be regulated by its binding of adenine nucleotides. DnaA protein has a high affinity for ATP and ADP, and the ATP binding form of DnaA protein (ATP-DnaA) initiates DNA replication in vitro, whereas the ADP binding form (ADP-DnaA) does not (7Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (346) Google Scholar). In E. coli cells, the majority of DnaA protein is present as ADP-DnaA, and ATP-DnaA increases at the time of initiation (8Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar). The ratio of ATP-DnaA to ADP-DnaA is regulated by at least four elements, including de novo synthesis of DnaA protein accompanied by formation of ATP-DnaA, an intrinsic ATP hydrolysis activity that generates ADP-DnaA from ATP-DnaA, a stimulation factor for the ATP hydrolysis named RIDA 1The abbreviations used are: RIDA, regulatory inactivation of DnaA; PG, phosphatidylglycerol; CL, cardiolipin; LPG, lysylphosphatidylglycerol; PE, phosphatidylethanolamine; TLC, thin layer chromatography; DTT, dithiothreitol; LB, Luria Bertani.1The abbreviations used are: RIDA, regulatory inactivation of DnaA; PG, phosphatidylglycerol; CL, cardiolipin; LPG, lysylphosphatidylglycerol; PE, phosphatidylethanolamine; TLC, thin layer chromatography; DTT, dithiothreitol; LB, Luria Bertani. (for regulatory inactivation of DnaA), and an exchange reaction of bound adenine nucleotide. RIDA requires Hda protein and a β-clamp of the DNA polymerase III holoenzyme as essential components and stimulates DnaA ATPase coupling with progressive DNA replication by the DNA polymerase III holoenzyme (8Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 10Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (215) Google Scholar). Thus, RIDA suppresses the initiation of DNA replication (5Katayama T. Mol. Microbiol. 2001; 41: 9-17Crossref PubMed Scopus (66) Google Scholar). Acidic phospholipid is a well characterized factor that facilitates the exchange reaction of ADP to ATP bound to DnaA protein (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar, 12Yung B.Y. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7202-7205Crossref PubMed Scopus (118) Google Scholar, 13Crooke E. Castuma C.E. Kornberg A. J. Biol. Chem. 1992; 267: 16779-16782Abstract Full Text PDF PubMed Google Scholar, 14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar). Acidic phospholipids in a fluid membrane facilitate dissociation of ADP bound to DnaA protein in vitro, and the resultant free form of DnaA protein is reactivated through binding to ATP, which is present at high concentrations under physiological conditions. In the conditional pgsA deletion mutant that is defective for the synthesis of acidic phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL), initiation of replication at oriC does not occur (15Xia W. Dowhan W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 783-787Crossref PubMed Scopus (121) Google Scholar). Moreover, the growth defect of the pgsA mutant is restored by expression of DnaA proteins possessing certain mutations (16Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). E. coli DnaA protein is located at the cell membrane (17Sekimizu K. Yung B.Y. Kornberg A. J. Biol. Chem. 1988; 263: 7136-7140Abstract Full Text PDF PubMed Google Scholar, 18Newman G. Crooke E. J. Bacteriol. 2000; 182: 2604-2610Crossref PubMed Scopus (41) Google Scholar). In addition, there is direct evidence of an exchange reaction of ADP to ATP bound to DnaA protein in the cells (9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar). These findings strongly suggest that reactivation of DnaA protein by acidic phospholipids is indispensable for the initiation of DNA replication at oriC. DnaA protein of other bacteria was purified (19Fukuoka T. Moriya S. Yoshikawa H. Ogasawara N. J. Biochem. (Tokyo). 1990; 107: 732-739Crossref PubMed Scopus (58) Google Scholar, 20Majka J. Messer W. Schrempf H. Zakrzewska-Czerwinska J. J. Bacteriol. 1997; 179: 2426-2432Crossref PubMed Google Scholar, 21Schaper S. Nardmann J. Luder G. Lurz R. Speck C. Messer W. J. Mol. Biol. 2000; 299: 655-665Crossref PubMed Scopus (26) Google Scholar, 22Zawilak A. Cebrat S. Mackiewicz P. Krol-Hulewicz A. Jakimowicz D. Messer W. Gosciniak G. Zakrzewska-Czerwinska J. Nucleic Acids Res. 2001; 29: 2251-2259Crossref PubMed Scopus (46) Google Scholar), and its interaction with acidic phospholipids has been investigated (23Yamamoto K. Rajagopalan M. Madiraju M. J. Biochem. (Tokyo). 2002; 131: 219-224Crossref PubMed Scopus (5) Google Scholar). How the reactivation of DnaA protein by phospholipids is coordinated with cell cycle progression remains uncertain. All previous studies of the interaction of E. coli DnaA protein with acidic phospholipids used chemically synthesized lipids or those isolated from eukaryotic cells. Little attention has been paid to alterations of lipid composition caused by physiological changes in the growth condition. In the present study, we focused on a Gram-positive bacterium, Staphylococcus aureus, in which lipid composition is altered from the exponential phase to the stationary growth phase (24Houtsmuller U.M.T. van Deenen L.L.M. Biochim. Biophys. Acta. 1965; 106: 564-576Crossref PubMed Scopus (58) Google Scholar). The amino acid sequence of domains 3 and 4 of S. aureus DnaA protein has 49% identity with that of E. coli DnaA protein (25Katayama H. Mizushima T. Miki T. Sekimizu K. Biol. Pharm. Bull. 1997; 20: 820-822Crossref PubMed Scopus (5) Google Scholar). This region is responsible for ATP binding, ATP hydrolysis, membrane interaction, and DNA binding properties of the E. coli DnaA protein (26Skarstad K. Boye E. Biochim. Biophys. Acta. 1994; 1217: 111-130Crossref PubMed Scopus (168) Google Scholar). Therefore, the properties of DnaA protein for ATP binding and interaction with acidic phospholipids are expected to be conserved between the two bacterial species. Here we describe that total lipid isolated from exponentially growing S. aureus cells releases ATP from ATP-DnaA, whereas total lipid isolated from stationary phase cells does not. Moreover, in the stationary phase, the activities of the acidic phospholipids, PG and CL, are inhibited by a basic phospholipid, lysylphosphatidylglycerol (LPG). Reagents—[α-32P]ATP (110 TBq/mmol), [α-32P]dCTP (110 TBq/mmol), and [32P]orthophosphate (370 MBq/ml) were purchased from Amersham Biosciences. [2,8-3H]ADP (1.48 TBq/mmol) was purchased from PerkinElmer Life Sciences. Phosphatidylethanolamine (PE) (egg yolk) was purchased from Avanti Polar Lipid Inc. PG (egg yolk) was purchased from Avanti Polar Lipid Inc. or from Doosan Serdary Research Laboratories. CL (bovine heart) was purchased from Sigma. Bacteria and Plasmids—S. aureus strain RN4220 (27Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (821) Google Scholar) was kindly provided by Dr. Kenichi Hiramatsu (Juntendo University). S. aureus strain CK1001 was constructed as described below. S. aureus strain Cowan I (28Steele Jr., G. Ankerst J. Sjogren H.O. Vang J. Lannerstad O. Int. J. Cancer. 1975; 15: 180-189Crossref PubMed Scopus (8) Google Scholar) and E. coli strain KA450 (ΔoriC1071::Tn10, dnaA17(Am), rnhA199(Am)) (9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar) were from our laboratory stock. Shuttle vectors between E. coli and S. aureus, pHY300PLK and pND50 (29Inoue R. Kaito C. Tanabe M. Kamura K. Akimitsu N. Sekimizu K. Mol. Genet. Genomics. 2001; 266: 564-571Crossref PubMed Scopus (59) Google Scholar), were from Takara (Japan) and Dr. Matsuhisa Inoue (Kitasato University), respectively. S. aureus DnaA-overproducing plasmid was constructed as follows. The coding region of the S. aureus dnaA gene (1.4 kbp) (25Katayama H. Mizushima T. Miki T. Sekimizu K. Biol. Pharm. Bull. 1997; 20: 820-822Crossref PubMed Scopus (5) Google Scholar) was amplified by polymerase chain reaction (PCR) using primers 5′-gggaattccatatgtcggaaaaaaagaaatttggg-3′ and 5′-ccggaattcttatacatttcttatttctttttc-3′ and chromosomal DNA extracted from S. aureus Cowan I as template. EcoRI and NdeI restriction sites or the EcoRI site were added to the 5′ or 3′ ends of the coding region, respectively. The EcoRI fragment of the PCR product was subcloned into the EcoRI site of M13mp19. The NdeI-EcoRI fragment of the resultant plasmid containing the S. aureus dnaA coding region was replaced with the E. coli dnaA coding region of E. coli DnaA overproducer pMZ001, which contains an arabinose promoter (30Mizushima T. Nishida S. Kurokawa K. Katayama T. Miki T. Sekimizu K. EMBO J. 1997; 16: 3724-3730Crossref PubMed Scopus (60) Google Scholar). The resultant plasmid, named pSAdnaA002, was used for overproduction of S. aureus DnaA protein. pHYmprF was obtained by insertion of the DNA fragment, which was amplified by PCR using primers 5′-tgaaacgagtatttgccacttga-3′ and 5′-tccaagcgcttcaggcataa-3′ and chromosomal DNA extracted from S. aureus RN4220 as template, into the SmaI site of pHY300PLK in the same orientation as the tetracycline resistance gene. Bacterial Culture Conditions—E. coli and S. aureus cells were grown in Luria Bertani (LB) medium (1% tryptone (Difco), 0.5% yeast extract (Difco), and 1% NaCl). Thymine, up to 50 μg/ml, ampicillin, up to 50 μg/ml, tetracycline, up to 5 μg/ml, or chroramphenicol, up to 12.5 μg/ml, was added if required. For S. aureus lipid preparations, a 0.5-ml aliquot of overnight culture was diluted into 100 ml of LB medium in a 225-ml round tube (Falcon), capped tightly, and incubated at 37 °C. Cells were harvested at the exponential phase (OD660 of 0.3) or at the stationary phase (20 h incubation with an OD660 of ∼2.5). Purification of S. aureus DnaA Protein—E. coli strain KA450 harboring pSAdnaA002 was grown in 3 liters of LB medium containing thymine and ampicillin at 30 °C. When the OD660 value reached 0.5, 60 ml of 50% l-(+)-arabinose was added (final 1%), and the culture was further incubated for 3 h. Cells were harvested by centrifugation at 4 °C, resuspended in buffer C′ (50 mm HEPES-KOH (pH 7.6), 1 mm EDTA, 2 mm dithiothreitol (DTT), 20% glycerol) containing 0.25 m KCl (1 ml/1 g of cell paste), frozen in liquid nitrogen, and stored at –80 °C. After thawing the frozen cell suspension on ice, lysozyme and spermidine were added up to 0.3 mg/ml and 20 mm, respectively. The suspension was mixed by inversion, placed on ice for 30 min, frozen in liquid nitrogen, and thawed on ice. After brief sonication, the extract was collected by centrifugation in a Beckman 80Ti rotor at 35,000 rpm for 20 min at 4 °C. Ammonium sulfate was slowly added to the supernatant up to 0.2 g/ml with stirring. After additional stirring for 3 h, precipitates were collected by centrifugation in an 80Ti rotor at 30,000 rpm for 20 min at 4 °C, dissolved in buffer C′ containing 10 mm magnesium acetate, and dialyzed for 12 h against 1.6 liters of buffer C′ containing 10 mm magnesium acetate with replacement of the buffer once. After centrifugation in an 80Ti rotor at 30,000 rpm for 20 min, the supernatant was applied to a MonoS HR5/5 column (Amersham Biosciences fast protein liquid chromatography) equilibrated with buffer C′ containing 10 mm magnesium acetate. DnaA protein was eluted with a linear gradient of 0 to 1 m KCl in buffer C′ containing 10 mm magnesium acetate. The DnaA protein fraction that was active for ATP binding was used as purified S. aureus DnaA fraction. This fraction had a single band on SDS-polyacrylamide gel (10.5%) electrophoresis stained with Coomassie Brilliant Blue R250. Protein concentration was determined by the Lowry method (31Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. Purification of E. coli DnaA Protein—E. coli DnaA protein was purified as described previously (32Kubota T. Katayama T. Ito Y. Mizushima T. Sekimizu K. Biochem. Biophys. Res. Commun. 1997; 232: 130-135Crossref PubMed Scopus (38) Google Scholar), except for using an E. coli DnaA overproducer, pBAD-dnaA, 2T. Ogawa, unpublished data. that was kindly provided from Dr. T. Ogawa (Nagoya University, Nagoya, Japan). Isolation of S. aureus Lipid—Exponentially growing cells and stationary phase cells were prepared as described above. Total lipid was extracted by a modified Bligh-Dyer method as described previously (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar), dried with a rotary evaporator, and suspended in water by sonication (Branson Sonifier 450). The S. aureus phospholipids were purified from total lipid obtained during either the exponentially growing phase or the stationary phase as indicated. LPG was purified by thin layer chromatography (TLC) developed with chloroform/methanol/acetic acid (65:25:10, v/v/v) using a silica gel plate (Silicagel 60, type PK6F, Whatman). PG and CL were further subjected to a second TLC with chloroform, methanol, and 29% ammonium (65:35:5, v/v/v). Re-extraction of the lipid from the thin layer plate was performed as follows. One of the TLC plates was sprayed with 100 mg/ml CuSO4 containing 8% phosphoric acid and heated to detect lipids (33Entezami A.A. Venables B.J. Daugherty K.E. J. Chromatogr. A. 1987; 387: 323-331Crossref PubMed Scopus (14) Google Scholar). The region containing PG, CL, and LPG was scraped off the other thin layer plates, and lipids were extracted by stirring for 30 min at 4 °C in chloroform/methanol (2:1, v/v). Methanol and distilled water were added to the samples up to a chloroform/methanol/water ratio of 1:1:0.9 (v/v/v) followed by vigorous mixing. After centrifugation at 3000 rpm for 3 min, the lower phase was collected, dried with a rotary evaporator, dissolved in a small amount of chloroform/methanol (2:1, v/v), and stored at –20 °C. All procedures, except for evaporation, were performed at 4 °C. Each purified phospholipid fraction had a single spot by TLC (Silicagel 60, Merck) with three different solvent systems; chloroform/methanol/water (65:25:4, v/v/v), chloroform/methanol/29% ammonium (65:35:5, v/v/v), and chloroform/methanol/acetic acid (65:25:10, v/v/v). Quantification of Phospholipids—The amount of phosphorus was determined (34Chen J.P.S. Toribara T.Y. Warner H. Anal. Chem. 1956; 28: 1756-1758Crossref Scopus (5824) Google Scholar), and the concentration of phospholipids was calculated assuming an average molecular weight of 750 for total lipid, 750 for PG, 850 for LPG, 1400 for CL, and 750 for PE. ATP (ADP) Binding of DnaA Protein—The standard reaction (50 μl) contained 1 μm [α-32P]ATP (44,000 cpm/pmol) or [3H]ADP (18,700 cpm/pmol), purified S. aureus DnaA protein (50–250 ng), 40 mm HEPES-KOH (pH 7.6), 100 mm potassium glutamate, 40 μm magnesium acetate, 0.5 mm EDTA, 1 mm DTT, 0.05 mg/ml bovine serum albumin, and 10% sucrose. DTT was added just before use. Specific radioactivities of [α-32P]ATP or [3H]ADP were kept constant (Fig. 1). Purified S. aureus DnaA protein (5 pmol) was mixed with various concentrations of [α-32P]ATP (1.5–50 nm, 15 Ci/mmol) or [3H]ADP (1.8–100 nm, 32 Ci/mmol) in 100× volume of the standard reaction (5 ml) for ATP binding or 10× volume (500 μl) for ADP binding (Fig. 1). After incubation on ice for 15 min, the solution was filtered through a nitrocellulose membrane (Millipore HA 0.45 μm, 24 mm diameter) presoaked in the wash buffer (40 mm HEPES-KOH (pH 7.6), 152 mm KCl, 10 mm magnesium acetate, 0.2 mm EDTA, 1 mm DTT, 20% (v/v) glycerol). The filter was washed with 20 ml of ice-cold wash buffer and dried under an infrared lamp. Radioactivity retained on the filter was measured in a liquid scintillation counter. ATP binding assay for E. coli DnaA protein was performed as described previously (7Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (346) Google Scholar). Exposure of ATP-DnaA to Lipids—For S. aureus DnaA protein, the [α-32P]ATP-DnaA complex (0.2–0.5 pmol) was formed as described above in 50 μl of standard reaction buffer and magnesium acetate was added to 10 mm. Lipid suspension and excess cold ATP (final 1 mm) were added, and the mixture was incubated at 4 °C for 14 min or as indicated. Reactions were also performed at 37 °C for indicated time periods (Fig. 4). Samples were filtered as described above and radioactivity retained on the membrane was measured. When basic or zwitterionic lipids were mixed with PG, each lipid was mixed in organic solvent, dried, and suspended in 250 μl of water (PG, 1.2 mg/ml). Lipid suspensions were sufficiently sonicated before use. An ATP releasing assay for E. coli DnaA protein was performed under conditions for E. coli DnaA protein, as described previously (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar). ATP binding and ATP releasing assays for both S. aureus DnaA protein and E. coli DnaA protein were performed under conditions for S. aureus DnaA protein, and releasing reactions were performed on ice or at 37 °C for various time periods (Fig. 4). Construction of S. aureus mprF Mutant—The S. aureus mprF mutant was constructed by integrational disruption through a single-crossover event (35Lindsay J.A. Foster S.J. Microbiology. 2001; 147: 1259-1266Crossref PubMed Scopus (70) Google Scholar) as follows. An 804-bp DNA fragment corresponding to the internal region of the mprF gene was amplified by PCR using 5′-gcaatcacattgtatcgggag-3′ and 5′-cgggatccggtacaaaatagtacgcaa-3′ primers and chromosomal DNA extracted from RN4220 as template, then cloned into the SmaI site of the plasmid, pCK20. pCK20, containing both the E. coli pUC19-derived origin and the chloramphenicol-resistant gene, was constructed by self-ligation of the AflII-AvaII fragment of pND50 (29Inoue R. Kaito C. Tanabe M. Kamura K. Akimitsu N. Sekimizu K. Mol. Genet. Genomics. 2001; 266: 564-571Crossref PubMed Scopus (59) Google Scholar) to delete the S. aureus pUB110-derived origin. The resultant plasmid (pCK20-mprF) was electroporated into S. aureus RN4220, and homologous recombinants were selected on LB-agar plates containing 12.5 μg/ml chloramphenicol and named CK1001. Southern blot hybridization was performed to ensure a single integration of pCK20-mprF into the chromosomal mprF locus. Flow Cytometric Analysis—Flow cytometry analysis of S. aureus cells was performed as described previously (36Skarstad K. Bernander R. Boye E. Methods Enzymol. 1995; 262: 604-613Crossref PubMed Scopus (40) Google Scholar) with slight modifications. The exponentially growing cells at 37 °C with an OD660 of 0.2 were treated with 100 μg/ml rifampicin and 10 μg/ml cephalexin for 4 h. The samples were harvested by centrifugation, washed, and resuspended in 0.1 ml of wash buffer. Fixing solution (1 ml) was added to the suspensions and mixed by vortex. The fixed cells were treated with 800 μg/ml RNase A at 50 °C for 2 h and sonicated for 30 s to disrupt conjugation of the cells. After washing with TE buffer, samples were stained with a combination of ethidium bromide and mithramycin. Cells were analyzed using a Becton Dickinson FACSCalibur. Effects of Total Lipid Isolated from S. aureus Cells on the Dissociation of ATP-DnaA—S. aureus DnaA protein was overexpressed in E. coli and purified to near homogeneity. Purified protein had a single band of ∼50 kDa judged by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue R-250 staining. S. aureus DnaA protein has a high affinity for ATP and ADP with K d values of 1 and 5 nm, respectively (Fig. 1). One molecule of DnaA protein bound to 0.1–0.3 molecules of ATP (Fig. 1), depending on the different preparations. Lipids from S. aureus were isolated during the exponential phase and stationary growth phase to compare their ability to facilitate the release of ATP from ATP-DnaA. Total lipid isolated from exponentially growing cells facilitated the release of ATP from DnaA protein (Fig. 2). The activity was dependent on the lipid dose and exposure time (Fig. 2). On the other hand, total lipid from stationary phase cells did not have this activity (Fig. 2). Unlike S. aureus, there was little difference in the action of the E. coli lipids between the two growth phases as described before (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar). PG Facilitates the Dissociation of ATP-DnaA—To determine the mechanism underlying the alterable interaction of DnaA protein with lipids, we examined which lipid was responsible for facilitating the dissociation of ATP-DnaA. Total lipid isolated from exponentially growing cells was fractionated by two-dimensional TLC and separated into eight fractions (Fig. 3A). In S. aureus cells, PG, CL, and LPG are the major phospholipids that form ∼98% of the total phospholipids (37White D. Frerman F. J. Bacteriol. 1967; 94: 1854-1867Crossref PubMed Google Scholar). Staining with Dittmer-Lester reagents revealed that fractions 4, 5, and 7 contained lipid phosphates (data not shown). Staining with ninhydrin reagent revealed that fraction 7 also contained amino groups (data not shown), indicating that fraction 7 contained LPG. The R F value of the lipid in fraction 5 on one-dimensional TLC was much the same as that of PG isolated from egg yolk (data not shown). Mass spectrometric analyses indicated that the molecular mass of the most abundant lipid in fraction 5 was 736, which corresponded to PG. Lipid in fraction 4 was determined to be CL by comparing the R F value from one-dimensional TLC with CL from bovine heart. Mass spectrometric analyses supported this conclusion (data not shown). Lipid in each fraction was isolated from the thin layer silica gel plate, suspended in water, and subjected to assay for the release of ATP from ATP-DnaA. Fraction 5 (PG) had the most potent activity (Fig. 3C). Fraction 8, which contained lipids from the entire region except for fractions 1–7, had weak, but significant activity (Fig. 3C). It remains uncertain which lipid(s) is responsible for the activity in fraction 8. There was no activity in fraction 4 (CL), possibly caused by the small amount of CL in the exponentially growing phase. CL purified from stationary phase cells had releasing activity as shown below (Fig. 5). PG purified from Fraction 5 facilitated the release of ATP bound to DnaA protein in a dose-dependent manner (data not shown) and the recovery of the activity from total lipid in fraction 5 was ∼45%. PG also facilitated the dissociation of ADP-DnaA (data not shown). These results suggest that PG was mainly responsible for facilitating the release of the ATP bound to DnaA protein in the total lipid of exponentially growing S. aureus cells, and confirmed the importance of PG in bacterial cell membranes for interaction with DnaA protein. There is a possibility that the PG-induced release of the nucleotide from S. aureus DnaA protein was the result of irreversible denaturation of the protein. Thus, we examined the ATP binding activity of S. aureus DnaA protein in the presence of PG. DnaA protein (4.2 pmol) was incubated on ice for 15 min in 5 μl of reaction buffer with PG (egg yolk; 3.1 μg) and 0.5–10 μm [α-32P]ATP (44,000 cpm/pmol), and bound nucleotides were then determined. The results indicated that DnaA protein binds to ATP in the presence of PG with a higher K d value (1.5 μm; data not shown) than in the absence of PG (1 nm; Fig. 1). The stoichiometry of bound ATP to DnaA protein in the presence of PG was 0.1, which was equivalent to that without PG. The results suggest that PG-induced dissociation of the nucleotide from S. aureus DnaA protein is not the result of irreversible denaturation of the protein, but rather of a decreased in affinity for the nucleotides. Effects of Temperature on the Dissociation of ATP-DnaA— Dissociation of the nucleotide form of E. coli DnaA protein by acidic phospholipid membrane required a temperature above the phase transition point (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar, 14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar). On the other hand, the nucleotide dissociation from S. aureus DnaA protein proceeded on ice (Fig. 2). To examine whether the difference in these results was the result of a difference in the assay conditions, dissociation of ATP from S. aureus DnaA was compared with that from E. coli DnaA under the same assay conditions. When the dissociation of E. coli DnaA-ATP was performed under the conditions for S. aureus DnaA used in Fig. 2, dissociation of ATP from E. coli DnaA protein by PG did not occur on ice, but was observed at 37 °C (Fig. 4, C and D), consistent with the results under the conditions for E. coli DnaA protein (11Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar, 14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar). On the other hand, dissociation of ATP from S. aureus DnaA protein by PG took shorter time periods at 37 °C than on ice (Fig. 4, A and B). Therefore, facilitation of the
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