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Cloning and expression in Escherichia coli of the extreme thermophile

极端嗜热菌的克隆及其在大肠杆菌中的表达

基本信息

批准号：
04660077
负责人：
MATSUZAKI Hiroshi
金额：
$ 1.34万
依托单位：
Saitama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

To achieve the functional expression and regulation mechanism of acidic phospholipids cardiolipin (CL) and the precursor product, phosphatidylglycerol (PG), we have strated to clone the structure genes for the cardiolipin synthesis of the extreme thermofile Thermus thermophilus and to contribute to the large scale production of phospholipids.T.themophilus HB8 chromosomal DNA fragments were packaged in a P1 phage with pNS582tet14Ad (Dupont) or in a plasmid pWSK129. With these plasmids, several mutants in Escherichia coli, the pgsA3 mutants, defective phosphatidylglycerophosphate (PGP) synthase and deficient in acidic phospholipids, or the cls : kan null mutants, defective in CLsynthase and deficient in cardiolipin and the temperature sensitive pssA1 mutants defective in phosphatidylserine synthase were transformed. The transformants were selected with available antibiotics and further selected the plasmid harboring strains responsible for complementation of pgsA3 or cls : kan mutation. … More pgsA3 mutants cannot form colonies on an NBY agar plate, and nonmotile. Nine strains which were capable of growth on a NBY agar plate and 5 motile strains were isolated. However, none transformants which indicated the increased level of the acidic phospholipids, CL and PG were isolated. To isolate the candidates clones efficintly from the above gene library of Thermus thermophilus our constructed strains were verified. CL contents in membrane increased significantly in the stationary phase. Activity of the cardiolipin synthase encoded by cls also increased about 10-fold in the stationary phase. A null cls mutant (cls : kan) lost viability to 10^<-4> of the wild-type cells during prolonged incubation for 5 days. The results suggested that CL was required for the growth of stationary phase. pgsA3 mutants defective PGP synthase and thus deficient in acidic phospholipids, indicated the conditional leathality on a NBY agar plate. The pgsA3 mutants were also found to be defective in flagellar formation and completely nonmotile. This mutation was found to repress the transcription of the flagellar master operon, flhD-flhC.These properties were used to select the transformants harboring and expressing the structure gene for cardiolipin synthesis of Thermus thermophilus.Indicated data in this investigation are useful to clone and characterize the structure genes for the cardiolipin synthesis of extreme thermophiles or other microorganisms. Less

为了解酸性磷脂心磷脂（CL）及其前体产物磷脂酰甘油（PG）的功能表达和调控机制，我们已经克隆了嗜热栖热菌（Thermus thermophilus）合成心磷脂的结构基因，并为大规模生产磷脂做出了贡献。或在质粒pWSK 129中。用这些质粒转化大肠杆菌中的几种突变体，pgsA 3突变体，缺陷型磷脂酰甘油磷酸（PGP）合酶和缺陷型酸性磷脂，或cls：kan无效突变体，缺陷型CL合酶和缺陷型心磷脂，以及温度敏感型pssA 1突变体，缺陷型磷脂酰丝氨酸合酶。用可用的抗生素选择转化体，并进一步选择携带负责pgsA 3或cls：kan突变互补的质粒的菌株。 ...更多信息 pgsA 3突变体不能在NBY琼脂平板上形成菌落，并且不运动。在NBY琼脂平板上分离到9株能生长的菌株和5株能运动的菌株。然而，没有分离到表明酸性磷脂、CL和PG水平增加的转化体。为了从上述嗜热栖热菌基因文库中有效地分离候选克隆，对所构建的菌株进行了验证。在稳定期，细胞膜CL含量显著增加。由cls编码的心磷脂合酶的活性在稳定期也增加了约10倍。无效cls突变体（cls：kan）<-4>在延长的孵育5天期间丧失了对10%野生型细胞的活力。结果表明，CL是固定相生长所必需的。pgsA 3突变体缺陷PGP合酶，因此缺乏酸性磷脂，表明在NBY琼脂平板上的条件性致死。pgsA 3突变体也被发现是有缺陷的鞭毛形成和完全nonmotile。利用该突变抑制鞭毛主操纵子flhD-flhC的转录，筛选了携带和表达嗜热栖热菌心磷脂合成结构基因的转化子，为极端嗜热菌或其他微生物心磷脂合成结构基因的克隆和鉴定提供了依据。少