Direct measurement of genetic recombination between bacilus subtilis auxotrophs through protoplast fusion

Abstract

Both cultural conditions and analytical methods were tried to vary in order to obtain a high frequency in genetic recombination through protoplast fusion. Three types of auxotrophic mutants of Bacillus subtilis, BD 170 (trpC 2, thr-5), NA 64 (met, ade), and RM 125 (leuA 8, arg-15), were used as test organisms. Cells grown in nutrient broth were subjected to protoplasting. Transferring cells at the late log phase always gave better results. It was also found that growing the cells under hypertonical conditions was better for the subsequent protoplast regeneration. Adding various osmotic stabilizers such as sucrose (0.5 M), NaCl (0.5 M), sorbitol (0.5 M) or sodium succinate (0.33 M) to the nutrient broth separately, increased the regeneration frequency by 10-fold. For the regeneration medium additions of 1.5\% agar and 3\% PVP to the basal medium (DPA medium without horse serum and agar) could improve the regeneration rate. The use of casein hydrolysate as nitrogen source in the regeneration medium was found to be indispensable. The replacement of casein hydrolysate with other nitrogen sources such as ammonium bisulphate and yeast nitrogen base decreased regeneration efficiency drastically; only 1/80 to 1/120 of that obtained on casein hydrolysate containing medium. The naked protoplasts could regenerate better when they were plated on the regeneration agar premixed in soft agar (0.5\%) to make a thin layer on the top of the solid agar medium instead of being plated out directly. The regeneration efficiency increased by 50-fold through the thin layer method. Applying the optimal conditions obtained, fusion between protoplasts originated from the three auxotrophs of B. subtilis was performed. The results were evaluated by measuring recombination frequencies determined in two ways; direct selection and single colony analysis which requires to pick about 1,000 individual colonies and transfer to selective media. The two analytical methods gave the same result, 2 to ...