The study identifies two novel variants of the D-alanine-D-alanine ligase gene (ddl6 and ddl7) located within integron gene cassettes, which confer high-level resistance to D-cycloserine when expressed in E. coli and Bacillus subtilis. A single nucleotide polymorphism at position c.777 in ddl6 was found to significantly increase D-cycloserine resistance.