Pseudomonas fluorescens에 있는 하나의 Plasmid가 말론산 대사에 관련된 유전자를 가지고 있다는 증거

Evience that a Plasmid Endoces Genes for Metabolism of Malonte in Pseudomonas fluorescens

말론산을 탄소원으로 하여 성장하는 Pseudomonas fluorescens에서 60 kb 정도의 plasmid를 발견하였다. 이 plasmid를 curing한 Pseudomonas는 malonate 배지에서 자라지 못하였고 plasmid도 소실되었다. 또한 이 plasmid를 transformation과 conjugation으로 각각 E. coli와 cured P. fluorescens로 이동시킨 결과 이 plasmid를 받은 transformed E. coli와 conjugant P. fluorescens는 malonate를 탄소원으로 하여 성장하였고 malonate 대사에 관련된 효소인 malonate decarboxylase와 acetyl-CoA synthetase의 활성이 측정되었다. Western blotting을 통하여 tansformed E. coli에서 P. fluorescens와 동일한 acetyl-CoA synthetase가 malonate에 의해 induction됨을 확인하였다.

Pseudomonas fluorescens which is able to utilize malonate as a sole carbon source was found to contain a novel 60 kb plasmid, which encodes the genes for the proteins to assimilate malonate, including malonate decarboxylase and acetyl-CoA synthetase. The evidence is as follows: The Pseudomonas cured with mitomycin C was unable to grow on malonate-medium as well as it lost plasmid. The plasmid isolated from the Pseudomonas could be introduced into E. coli strain JM103 and DH1 by transformation. The transformed E. coli was able to grow on malonate-medium and could transmit its plasmid back to the cured P. fluorescens by conjugation. The existence of the plasmid in the transformed E. coli was confirmed by hybridization with a labeled probe prepared from 12 kb segment of the plasmid. Dot hybridization showed that the copy number of the plasmid in the transformed E. coli is at least 13 times higher than in the wild type P. fluorescens. The two key enzymes, malonate decarboxylase and acetyl-CoA synthetase, were inducible by malonate in the transformed E. coli.