Development of antibiotics by monitoring the inhibition of the ATP-binding to DnaA, the initiator protein of chromosomal DNA replication in bacteria

金黄色葡萄球菌引起人类机会性疾病。在这项研究中，我们研究了金黄色葡萄球菌DnaA蛋白，复制启动子的生化性质。先前对大肠杆菌DnaA蛋白的研究表明，DnaA蛋白的ATP结合形式是活性的，而ADP结合形式是无活性的。纯化的金黄色葡萄球菌DnaA蛋白对ATP和ADP也表现出高亲和力。我们发现，磷脂酰甘油，一种酸性磷脂，刺激ADP从ADP-DnaA复合物的释放，导致DnaA蛋白的活化。赖氨酰磷脂酰甘油是一种碱性磷脂，可抑制磷脂酰甘油的作用。因此，磷脂酰甘油可以负调节DnaA蛋白的再活化。我们在这里提出了一个新的模式，通过增加细胞质膜中碱性磷脂的含量来调节DNA复制的起始，我们分离出了金黄色葡萄球菌的突变体，它们的DNA复制是温度敏感的。通过互补分析，我们确定了polC，dnaE，和dnaC基因是必不可少的DNA复制的细菌。我们还利用家蚕建立了传染病动物模型。该系统将为防治传染病的药物提供一个有用的筛选系统。

Staphylococcus aureus causes opportunistic diseases for humans. In this study, we examined biochemical nature of Staphylococcus aureus DnaA protein, the replication initiator. Previous studies with Escherichia coli DnaA protein demonstrated that the ATP-binding form of DnaA protein is active, whereas the ADP-binding form is inactive. Purified Staphylococcus aureus DnaA proteins also showed high affinity for ATP and ADP. We showed that phosphatidylglycerol, an acidic phospholipids, stimulated the release of ADP from the ADP-DnaA complex, resulting in the activation of DnaA protein. Lysylphosphatidylglycerol, a basic phospholipid, was shown to inhibit the action of phosphatidylglycerol. Thus, phosphatidylglycerol may negatively regulate re-activation of DnaA protein. We propose here a new model of the regulation of initiation of DNA replication by increase in the content of basic phospholipids in cytoplasmic membranes.We isolated mutants of Staphylococcus aureus whose DNA replication were temperature-sensitive. By complementation analysis, we identified polC, dnaE, and dnaC genes which are essential for DNA replication in the bacteria. We also established an animal model of infectious diseases by using silkworms. The system will provide a useful screening system for medicines against infectious diseases.