Genomic DNA Extraction and PCR Confirmation of Isogenic Mutants

Genomic DNA was extracted from wild-type SM101 and its derivative strains using the MasterPure Gram-positive bacterial DNA purification kit (Epicentre, Paris, France). All previously constructed [28 (link)] plasmids used in this study were isolated from C. perfringens type A strain 13, the cloning host strain, using a QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.
The PCR reactions for confirming the isogenic mutants and complemented strains were conducted using 2× DreamTaq Green PCR Master Mix (ThermoFisher Scientific) and appropriate primers listed in Table 2. The following PCR amplification condition was used for this experiment: (1) 95 °C for 5 min; (2) 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, and (3) a final extension for 5 min at 72 °C.
In addition, LongAmp Taq polymerase (New England Biolabs, Ipswich, MA, USA) was used for confirmation of CPR1953KO-1954COM, CPR1954KO-1953COM, CPR1953KO-1954COM-H410A, and CPR1954KO-1953COM-H430A constructs. The following PCR parameters were used: (1) 95 °C for 5 min; (2) 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 65 °C for 50 s per Kb, and (3) a final extension for 10 min at 65 °C. Finally, an aliquot of each PCR reaction (20 μL) was electrophoresed on a 2% agarose gel and then visualized after staining with ethidium bromide.