2.2.2. Construction of the rif-orf5 Gene Complementation Mutant Δrif-orf5::orf5

First, the rif-orf5 gene complementation vector pSET152-orf5 was constructed. The targeted gene rif-orf5 was amplified using the genomic DNA of A. mediterranei S699 as a template. The purified PCR fragment was digested with NdeI and XbaI, and cloned into the downstream of the rifKp promoter in pSET152 through Gibson assembly [20]. Similarly, the assembled product was transformed into DH5α-competent cells, and positive clones were verified by restriction enzyme digestion and sequencing (Figure S4). The gene complementation vector pSET152-orf5 was transformed into the rif-orf5 gene knock-out mutant Δrif-orf5 by electroporation. Apramycin-sensitive (AprS) colonies were selected and confirmed to be the rif-orf5 gene complementation mutant Δrif-orf5::orf5 by PCR amplification (Figure S5).

Primers used in this study are shown in Table S1.