Protoplast fusion in Bacillus Megaterium QM B1551

Bibliography: pages [60]-71.This thesis represents work done to determine the optimal conditions for protoplast formation and regeneration of Bacillus megaterium QM B1551 and- to study the possibility that PEG-induced protoplast fusion might be used to establish linkage between markers on the chromosome. To determine the conditions which would increase protoplasting efficiency, lysozyme concentration, exposure time to lysozyme, temperature of protoplasting, pH of hypertonic buffer used for protoplasting, growth stage at which protoplasting occurs and shaking of the protoplasting suspension were varied. Conditions for regeneration were tested by varying regeneration media, lysozyme concentration, and temperature of regeneration. The results indicated that 100% protoplast formation could be obtained when mid-logarithmic (A₆₆₀ = 0.5 - 0.6) cells were exposed to 250 ug/ml final concentration of lysozyme for 30 minutes at 32°C, in a hypertonic media at pH 6.5 while shaking gently. Efficient regeneration was found when protoplasts were made with 250 ug/ml lysozyme and plated on 10% sucrose nutrient agar, then incubated at 30°C. Several different fusions involving two, three and four factor crosses were attempted and all possible types of recombinants could be detected but recombination frequencies varied from fusion to fusion and between the various loci used. Non-complementing diploids were also detected. They were isolated, analyzed and found to behave like those reported in B. subtilis. Although fusion cannot be used for mapping, it was successfully demonstrated that the method could be used to transfer plasmids among Bacilli at frequencies from 3.8 x 10⁻⁶ to 2.3 x 10⁻⁵.M.S. (Master of Science