Abstract
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).
Highlights
Chromosomal DNA replication in bacteria is regulated at the initiation step [1, 2], where the activity and quantity of the initiator DnaA protein is critically controlled [3,4,5]
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
S. aureus DnaA protein has a high affinity for ATP and ADP with Kd values of 1 and 5 nM, respectively (Fig. 1)
Summary
Reagents—[␣-32P]ATP (110 TBq/mmol), [␣-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. ATP (ADP) Binding of DnaA Protein—The standard reaction (50 l) contained 1 M [␣-32P]ATP (44,000 cpm/pmol) or [3H]ADP (18,700 cpm/ pmol), purified S. aureus DnaA protein (50 –250 ng), 40 mM HEPESKOH (pH 7.6), 100 mM potassium glutamate, 40 M magnesium acetate, 0.5 mM EDTA, 1 mM DTT, 0.05 mg/ml bovine serum albumin, and 10% sucrose. Purified S. aureus DnaA protein (5 pmol) was mixed with various concentrations of [␣-32P]ATP (1.5–50 nM, 15 Ci/mmol) or [3H]ADP (1.8 –100 nM, 32 Ci/ mmol) in 100ϫ volume of the standard reaction (5 ml) for ATP binding or 10ϫ volume (500 l) for ADP binding (Fig. 1). Exposure of ATP-DnaA to Lipids—For S. aureus DnaA protein, the [␣-32P]ATP-DnaA complex (0.2– 0.5 pmol) was formed as described above in 50 l of standard reaction buffer and magnesium acetate was added to 10 mM.
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