Cloning and expression in Escherichia coli of the extreme thermophile

为了研究酸性磷脂cardiolipin （CL）及其前体产物磷脂酰甘油（PG）的功能表达和调控机制，我们克隆了嗜热嗜热菌（Thermus thermophilus）合成cardiolipin的结构基因，为大规模生产磷脂提供了基础。将嗜热嗜热菌HB8染色体DNA片段与pNS582tet14Ad （Dupont）一起包装在P1噬菌体中，或将其包装在质粒pWSK129中。利用这些质粒转化了大肠杆菌中的pgsA3突变体、PGP合成酶缺陷突变体和酸性磷脂缺陷突变体、cl1 - kan零突变体、clp合成酶缺陷突变体和心磷脂缺陷突变体以及温度敏感突变体pssA1。用现有的抗生素选择转化子，并进一步选择携带负责pgsA3或cls: kan突变的菌株的质粒。更多的pgsA3突变体不能在NBY琼脂平板上形成菌落，且无运动能力。分离得到9株能在NBY琼脂平板上生长的菌株和5株可运动的菌株。然而，没有分离到酸性磷脂、CL和PG水平升高的转化子。为了从上述嗜热热菌基因库中有效分离候选克隆，我们对所构建的菌株进行了验证。膜中CL含量在固定期显著增加。由cls编码的心磷脂合成酶的活性在固定期也增加了约10倍。null cls突变体（cls: kan）在延长5天的孵育期间失去了10^<-4>的野生型细胞的生存能力。结果表明，固定相的生长需要CL。pgsA3突变体PGP合成酶缺陷，酸性磷脂缺乏，在NBY琼脂平板上显示有条件死亡。pgsA3突变体在鞭毛形成方面也存在缺陷，并且完全没有运动能力。发现该突变抑制鞭毛主操纵子flhD-flhC的转录。利用这些特性来选择含有和表达嗜热热菌心磷脂合成结构基因的转化子。本研究结果对极端嗜热菌或其他微生物的心磷脂合成结构基因的克隆和表征具有重要意义。少

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