Construction of the RA0C_1534 deletion mutant in the streptomycin-resistant strain ATCCs based on the suicide plasmid pORS

The process of knockout based on pORS was done as described in previous study with a little modification [24]. Briefly, the 780-bp upstream sequence of RA0C_1534 was amplified using the primers RA0C_1534 upP1 (containing a NcoI site) and RA0C_1534 upP2. The 796-bp downstream sequence of RA0C_1534 was amplified with the primers RA0C_1534 downP1 and RA0C_1534 downP2 (containing a XhoI site). The two PCR products were ligated using the overlap PCR method. The fused fragment was purified and digested with NcoI and XhoI. Then, the fragment was cloned into pORS, which had been digested with the same enzymes, to generate pORS::RA0C_1534 up-down. The plasmid pORS::RA0C_1534 up-down was introduced into streptomycin-resistant strain ATCCs by conjugation according to a previously described method [12, 24].

After the first recombination, the positive clone was isolated on the blood plate with Cfx (1 μg/mL) and tested by PCR using the primers CfxP1 and CfxP2 (Table 2). The correct clone was inoculated in GCB liquid medium for overnight. About 10⁵ bacterial cells were spread on blood plates with 100 μg/mL streptomycin to screen the mutants which had lost the plasmid after a second recombination event. Mutation was identified by PCR using the primers RA0C_1534 CompP1 and RA0C_1534 CompP2 (Table 2).