Protoplast Fusion Techniques in the Study of Antibiotic-Producing Streptomycetes
Steel, Douglas J.
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Streptomyces coelicolor and S. achromogenes subsp. rubradiris were investigated in order to discover conditions under which protoplast formation and fusion would take place with a high frequency. Variations were made in the protocol to increase the yield and viability of protoplasts, their fusion, and regeneration into colonies. Optimal growth conditions included growth on TYG and TYG-R media at 28 C or higher. Seed cultures were most viable at two days of incubation, and gave rise to a protoplast formation and fusion efficiency of over 90%. A new method of disposing of mycelial contaminants was developed using a modified plastic centrifugation tube packed with glass wool, and using suction filtration. Both intra- and interspecific fusions were conducted, and recombinant isolates were then tested for phenotype, mapped, and grown into stable cultures. Bacterial contamination posed a major barrier, and three methods were tested to minimize it, utilizing growth pattern differences between Streptomycetes and the contaminants. They were found to be most sensitive to the antibiotics cephalothin, novobiocin and bacitracin. The antibiotic activities of isolates were measured at one-day intervals, and the most active fractions were detected by thin-layer chromatography. These fractions were compared to known Rf values. Little correspondence was found, suggesting that novel antibiotics, or higher levels of antibiotics previously present at low levels were being displayed.