Antibiotic Production, Activity Spectra and Plasmid Analysis of Streptomyces species Isolated from Soil
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Abstract
Antibiotic producing Streptomyces strains were isolated by spread-plating aliquots of ten-fold serially diluted pre-treated soil samples on a chemically defined Streptomyces medium, ISPI, using the crowded-plate technique. Cultures on the crowded plate were bioautographed against Staphylococcus aureus (ATCC 25932).Nine of the bioactive strains (UY1 – UY9), which showed appreciable antagonism and clear inhibition zones were selected and grown as pure cultures in ISP1 medium. Antibiotic production and activity of the bioactive Streptomyces strains were carried out in submerged fermentation in the production medium, ISP2. Cells were harvested by centrifugation, processed into antibiotic-filtrates and sensitivity test on the test organisms by the modified agar-well diffusion technique, indicated high potency. Broth cultures of strains in ISP1 were subjected to plasmid analysis. Rapid screening for plasmid revealed bands of plasmid DNA. Plasmid DNA extracted by a miniprep method and plasmid profiling conducted on 1.0 % agarose gel electrophoresis, revealed probable identical single linear copies of high molecular weight plasmid DNA bands for the strains, aligning somewhat above the 10,000 base-pairs (bp), high molecular weight DNA biomarker. The role of plasmid in antibiotic production and activity was studied by plasmid curing with sub-inhibitory concentrations of acridine-orange and tested for activity and profile on agarose gel electrophoresis, which indicated the loss of antibiotic production and activity, disappearance and non-alignment of any plasmid bands on the gel electrophoresis. Transformation of the competent cured strains demonstrated antibiotics production and activity as well as the re-appearance of plasmid bands on agarose -gel electrophoresis like the native strains. The comparison between the native, cured and transformed Streptomycesisolates, showed that there was significant (p< 0.05) difference in antibiotic production and activity amongst these three forms of the Streptomyces strains. These possibly suggest and confirmed that the antibiotic production and activity of the isolated Streptomyces strains are plasmid –mediated.
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