FIG. 1.

Schematic overview of Cre-lox-based chromosomal deletion system for construction of B. coagulans DSM 1 ΔsigF. (A) Exchange of sigF for cat by double crossover. The lox66-cat-lox71 cassette flanked by the homologous regions for crossover is introduced into B. coagulans on a thermosensitive plasmid, pMH79, at the permissive temperature of 45°C and with selection for chloramphenicol resistance (7 μg·ml⁻¹). Double crossover recombination events are selected for by plating at 60°C, a nonpermissive temperature for the integration plasmid, while maintaining the antibiotic resistance pressure. (B) Removal of cat. The Cre-encoding plasmid is introduced on a thermosensitive plasmid (pMH66) carrying a tetracycline resistance gene for selection (1 μg·ml⁻¹). Cre recombines the lox66 and lox71 sites to a lox72 site that is no longer recognized by Cre. The Cre-encoding plasmid is cured by cultivation at 60°C and subsequent plating of a dilution series.