ISOLATION OF PLASMID PRLI FROM ARTHROBACTER-OXYDANS-317 AND DEMONSTRATION OF ITS ROLE IN STEROID 1(2)-DEHYDROGENATION

Althrobacter oxydans 317 capable of complete degradation of beta-sitos terol harbours two plasmids -pJLI and pRLI. Standard plasmid isolation methods can easily detect pJLI but not pRLI. The latter plasmid was i solated by a mild procedure using alpha,alpha'-dipyridyl as lytic agen t and separated from pJLI by agarose gel electrophoresis using the tec hnique of electro elution into throughs. It's molecular weight was fou nd to be about 3.5 kilo base (kb) pRLI was introduced into the plasmid cured A. oxydans 317 AL. Through genetic transformation using protopl asts of the recepient strain, a transformant strain A. oxydans 317 ALT bearing only pRLI plasmid was obtained. The transformant strain was u nable to grow on 4-androstene-3, 17-dione (AD) and 1,4-androstadiene-3 , 17-dione (ADD) as sole carbon source but a modest growth was observe d on 9 alpha-hydroxy-4-androslene-3, 17-dione (9 alpha-hydroxy AD) ind icating the ability for steriod 1(2)-dehydrogenation and a lack of the capacity for alpha,alpha-hydroxylation. The strain was able to form A DD from AD, and 1 (2)-dehydrogenation of 3-oxochol-4-en24-oic acid fro m beta-sitosterol. Direct estimation of steroid (1(2)-dehydrogenase ac tivity showed that the transformant strain and parent strain containin g pRLI had high enzyme activity where as strains lacking in this plasm id had negligible enzyme activity. It was concluded that pRLI is respo nsible for steroid 1 (2)-dehydrogenation in A. oxydans 317.