Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Abstract

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10°C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to growth at temperatures between 50°C and 70°C at a frequency of less than 10-10. The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10-3 to 10-2 for single markers and about 10-7 for two unlinked markers. With the same procedures we were able to transfer the mesophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10-7, suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.