Isolation of plasmid pRLI from Arthrobacter oxydans 317 and demonstration of its role in steroid 1 (2)-dehydrogenation

Arthrobacter oxydans 317 capable of complete degradation of β‐sitosterol harbours two plasmids ‐pJLI and pRLI. Standard plasmid isolation methods can easily detect pJLI but not pRLI. The latter plasmid was isolated by a mild procedure using α,α′‐dipyridyl as lytic agent and separated from pJLI by agarose gel electrophoresis using the technique of electro elution into throughs. It's molecular weight was found to be about 3.5 kilo base (kb) pRLI was introduced into the plasmid cured A. oxydans 317 AL. Through genetic transformation using protoplasts of the recepient strain, a transformant strain A. oxydans 317 ALT bearing only pRLI plasmid was obtained. The transformant strain was unable 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 observed on 9α‐hydroxy‐4‐androstene‐3, 17‐dione (9α‐hydroxy AD) indicating the ability for steriod 1 (2)‐dehydrogenation and a lack of the capacity for α,α‐hydroxylation. The strain was able to form ADD from AD, and 1 (2)‐dehydrogenation of 3‐oxochol‐4‐en24‐oic acid from β‐sitosterol. Direct estimation of steroid (1 (2)‐dehydrogenase activity showed that the transformant strain and parent strain containing pRLI had high enzyme activity where as strains lacking in this plasmid had negligible enzyme activity. It was concluded that pRLI is responsible for steroid 1 (2)‐dehydrogenation in A. oxydans 317.