The Bacillus subtilis L-42 mutant strain, which displays limited growth and inability to cope with hyperosmotic shock in a defined medium with a K+ concentration of < 1 mmol/L, was isolated by non-specific transposon insertional mutagenesis followed by an enrichment selection in media with K+ concentration < 0.5 mmol/L. The growth rate (as the main physiological characteristic) was determined to test the viability of the isolated mutant in media with various concentrations of K+, different values of osmolarity and pH. The mutant revealed a significant decrease in growth rate when cultivated in media with K+ concentration < 1 mmol/L and at hyperosmolarity. Localization of the insertional mutation was provided, based on genetic characteristics of the used transposon. Only 1 insertion of recombinant transposon was found in the mutant chromosome, localized into the yxkO gene (a putative ribokinase with unknown biological function).