High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis

A high osmolarity electroporation method has been developed for the efficient transformation of Bacillus subtilis and B. licheniformis. The presence of high concentrations of the osmoticums, sorbitol and mannitol, in the electroporation, growth and recovery media resulted in an approximately 5000-fold increase in the transformation efficiency of B. subtilis, with a maximum value of 1.4×106 transformants per μg DNA. The transformation efficiency increased with electric field strength in the range of 12–23 kV/cm using the high osmolarity method. This enhanced transformation efficiency correlated with improved cell survival rate, indicating that the high osmolarity media have some protective action on the electrically injured cells. The application of the optimised high osmolarity method to transformation of B. licheniformis resulted in an increase in transformation efficiency by approximately 400-folds, with a maximum value of 1.8×104 transformants per μg DNA. As with B. subtilis, the highest transformation efficiency of B. licheniformis was achieved at a very high field strength (21 kV/cm). These results indicate that a combination of high osmolarity and high field strength may be a useful approach to improving the transformation efficiency of some gram-positive bacterial species which are able to grow in a high osmolarity medium.