Short Report

LOF and GOF mutations in Streptomyces spp. by sodium azide

Keshav Bhattarai(1), Kiran Babu Tiwari(1, 2, 3*) and Vishwanath Prasad Agrawal(1)
1Department of Biochemistry, Universal Science College, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal
3Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal
*Address of correspondence: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal, email:gp120cdnashuffling@gmail.com

Role of sodium azide as chemical mutagen has been studied in plants, fruit flies, rats etc. Several studies on loss of function (LOF) mutation and gain of function (GOF) mutation in different organisms could be seen, however GOF mutation is not yet reported in Streptomyces spp.Therefore, we attempted to explore on the beneficial mutations it can bring about while working with the actinomycetes isolates from Khumbu, Everest region.

A Streptomyces strain was isolated from a soil sample and purified at the Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). The bacteria were then grown on plates containing 12.5, 25, 37.5 or 50ppm sodium azide. The sub-cultured colonies from 12.5ppm (S1), 25ppm (S2) and 37.5ppm (S3) containing plates were asporogenic and more yellowish, brittle, irregular and smaller in size compared to the wild strain (S0). Growth was not observed on plate containing 50ppm sodium azide indicating the lethal dose of sodium azide on the organism.

Treatment of Streptomyces with 12.5ppm of sodium azide enabled bacteria to utilize nitrate, whereas alternate carbon sources (sucrose, mannitol and salicin) were metabolized by those bacteria treated with 37.5ppm of the chemical. Thus the modified biochemistry of the treated Streptomyces strain could be due to the mutagenic effect of the Sodium azide.

To explore possible mutations in the treated Streptomyces strain, a RAPD primer (sequence CTGGCGTGAC; GC 70%; mp 34ºC) was used to investigate the DNA polymorphisms. Electrophoresis result in 1% agarose gel showed five bands in S0 while two bands were missing in mutant S3. The other mutants showed similar band patterns as of wild type. It was concluded that point mutations on the primer-binding site resulted in the loss of bands for S3. Furthermore, similar band patterns in other mutants with S0 may be due to point mutations between primer binding flanking ends, which went undetected. Hence, additional primer evaluation is required to identify potential differences in the S1 and S2 strains.

Further, studies on antibacterial activity of the mutants (viz. S1, S2 and S3) and wild strain (S0) were done against Staphylococcus aureus, Escherichia coli, Bacillus subtillis, B. thuringiensis, Pseudomonas aeruginosa, Klebsella pneumoniae and Proteus vulgaris. Marked increase in the antibacterial acivity as GOF mutation was observed against B. subtillis, a food poisoning bacterium.

In conclusion, these preliminary findings opened the possibilities of strain improvement with the use of chemical mutagen, which may have wider applications in the modern chemotherapy researches.