Lysylphosphatidylglycerol (LPG) is a basic phospholipid in which l-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The Km values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 μM and 6·9 μM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef]
CastumaC. E., CrookeE., KornbergA.1993; Fluid membranes with acidic domains activate DnaA, the initiator protein of replication in Escherichia coli. J Biol Chem 268:24665–24668
HoutsmullerU. M. T., van DeenenL. L. M.1965; On the amino acid esters of phosphatidyl glycerol from bacteria. Biochim Biophys Acta 106:564–576[CrossRef]
IchihashiN., KurokawaK., MatsuoM., KaitoC., SekimizuK.2003; 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. J Biol Chem 278:28778–28786[CrossRef]
KatayamaT., KubotaT., KurokawaK., CrookeE., SekimizuK.1998; The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase. Cell 94:61–71[CrossRef]
KenwardM. A., BrownM. R., FryerJ. J.1979; The influence of calcium or manganese on the resistance to EDTA, polymyxin B or cold shock, and the composition of Pseudomonas aeruginosa grown in glucose- or magnesium-depleted batch cultures. J Appl Bacteriol 47:489–503[CrossRef]
KristianS. A., DurrM., Van StrijpJ. A., NeumeisterB., PeschelA.2003; MprF-mediated lysinylation of phospholipids in Staphylococcus aureus leads to protection against oxygen-independent neutrophil killing. Infect Immun 71:546–549[CrossRef]
KurokawaK., NishidaS., EmotoA., SekimizuK., KatayamaT.1999; Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli. EMBO J 18:6642–6652[CrossRef]
LennarzW. J., NesbittJ. A.3rd, ReissJ.1966; The participation of sRNA in the enzymatic synthesis of O-l-lysyl phosphatidylglycerol in Staphylococcus aureus. Proc Natl Acad Sci U S A 55:934–941[CrossRef]
LennarzW. J., BonsenP. P., van DeenenL. L.1967; Substrate specificity of O-l-lysylphosphatidylglycerol synthetase.Enzymatic studies on the structure of O-l-lysylphosphatidylglycerol. . Biochemistry 6:2307–2312[CrossRef]
MiyazakiC., KurodaM., OhtaA., ShibuyaI.1985; Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis. Proc Natl Acad Sci U S A 82:7530–7534[CrossRef]
MizushimaT., IshikawaY., ObanaE., HaseM., KubotaT., KatayamaT., KunitakeT., WatanabeE., SekimizuK.1996; Influence of cluster formation of acidic phospholipids on decrease in the affinity for ATP of DnaA protein. J Biol Chem 271:3633–3638[CrossRef]
NesbittJ. A.3rd, LennarzW. J.1968; Participation of aminoacyl transfer ribonucleic acid in aminoacyl phosphatidylglycerol synthesis. I. Specificity of lysyl phosphatidylglycerol synthetase. J Biol Chem 243:3088–3095
NishidaS., FujimitsuK., SekimizuK., OhmuraT., UedaT., KatayamaT.2002; A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidence from a mutant DnaA protein defective in regulatory ATP hydrolysisin vitro and in vivo. J Biol Chem 277:14986–14995[CrossRef]
PeschelA., JackR. W., OttoM. & 9 other authors; 2001; Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine. J Exp Med 193:1067–1076[CrossRef]
SekimizuK., KornbergA.1988; Cardiolipin activation of DnaA protein, the initiation protein of replication in Escherichia coli. J Biol Chem 263:7131–7135
SekimizuK., BramhillD., KornbergA.1987; ATP activates DnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell 50:259–265[CrossRef]
StaubitzP., PeschelA.2002; MprF-mediated lysinylation of phospholipids in Bacillus subtilis – protection against bacteriocins in terrestrial habitats?. Microbiology 148:3331–3332
TomuraA., IshikawaT., SagaraY., MikiT., SekimizuK.1993; Requirement of phosphatidylglycerol for flagellation of Escherichia coli. FEBS Lett 329:287–290[CrossRef]
XiaW., DowhanW.1995; In vivo evidence for the involvement of anionic phospholipids in initiation of DNA replication inEscherichia coli. Proc Natl Acad Sci U S A 92:783–787[CrossRef]