Isolation of Salicylate-Degrading Plasmid from Pseudomonas putida

Pseudomonas putida로 부터 salicylate 분해 plasmid의 분리

The large plasmid (about 180 megadaltons) was isolated from the aquatic strain of Pseudomonas which was found to degrade salicylate. It was found that the plasmid could be isolated under gentle conditions in comparison with other methods. The yield of covalently closed circular DNA was enganced by heat treatment at $55^{\circ}C$ after denaturing the chromosomal DNA with alkaline sodium dodecyl sulfate (pH 12.45), and the plasmid DNA was selectively concentrated by utilizing 10% polyethylene glycol as final concentration. It was also found that the cured strains with mitomycin C did not show any growth on the medium containing salicylat6e, therefore, it was concluded that the plasmid might play and important role on the salicylate degradation.

자연계에서 분리한 Pseudomonas에서 크기가 매우 큰 plasmid (약 180 megadaltons:를 분리하였다. 이 plasmid는 기존의 plasmid 검출방법보다 더 온화한 조건에서 분리되었다. Alkalin., sodium dodecyl sulfate (pH 12. 45)를 사용하여 chromosomal DNA를 변성시킨 뒤 $55^{\circ}C$로 열처리 하여 covalently closed circular DNA 의 손실을 최대한으로 방지하였고, polyethylene glycol을 최종농도 10%로 첨가하여 plasmid DNA를 선택적으로 농축하였다. 한편, 이 plasmid는 mitomycin C를 처리하여 얻은 cured sitrains에서는 나타나지 않아 salicylate를 분해하는 새로운 plasmid로 확인되었다.