Cloning and expression in Escherichia coli of the extreme thermophile

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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