New integrative method to generate Bacillus subtilis recombinant strains free of selection markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactThetaamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P-lysA promoter with that of the P-blaP beta-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the Mal cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the Mal cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.