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

金黄色葡萄球菌会给人类带来机会性疾病。在这项研究中，我们检测了复制启动子金黄色葡萄球菌DNAA蛋白的生化性质。以前对大肠杆菌Dna A蛋白的研究表明，Dna A蛋白的ATP结合型是活性的，而ADP结合型是不活性的。纯化的金黄色葡萄球菌DNAA蛋白也表现出对ATP和ADP的高亲和力。我们发现，磷脂酰甘油，一种酸性磷脂，刺激ADP-DNAA复合体释放ADP，导致DNAA蛋白激活。赖氨酸磷脂酰甘油是一种碱性磷脂，它能抑制磷脂酰甘油的作用。因此，磷脂酰甘油可能负向调节DNAA蛋白的重新激活。我们提出了一种通过增加细胞膜中碱性磷脂含量来调控DNA复制启动的新模型。我们分离了DNA复制对温度敏感的金黄色葡萄球菌突变株。通过互补分析，我们确定了在细菌中DNA复制所必需的PolC、dna E和dna C基因。我们还利用家蚕建立了传染病动物模型。该系统将为治疗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.