Construction of PA2146 Mutant Strain

PA2146 mutant strain construction was performed according to a previously described method with minor modifications (Luo et al., 2015 (link)). The primers used are listed in Table 2. Briefly, the upstream and downstream fragments of PA2146 were amplified with PA2146-up-F/R and PA2146-down-F/R, respectively. The amplified products were then fused by overlapping PCR, and the fused fragment was cloned into pLP12 (KnoGen Biotech, Guangzhou, China) using recombinant enzyme Exnase II (ClonExpress II, Vazyme) to generate recombinant plasmid pLPPA2146. The resulting plasmid was then transformed into E. coli DH5α, and the recombinant plasmids were confirmed by PCR using the primer pair pLP-U-F/R. Next, pLPPA2146 was transferred into E. coli β2163 and selected on LB agar containing 0.3 mM daptomycin (DAP) with 0.3% D-glucose. E. coli β2163 was then co-cultured with PAO1, and the insertion mutation was selected on LB agar containing 36 μg/mL tetracycline (TC) with 0.3% D-glucose and confirmed using the primer pair PA2146-up-F and PA2146-down-R. The PAO1ΔPA2146 strain was selected on LB agar with 0.4% L-arabinose. The PA2146 mutant was confirmed by sequencing using the primers PA2146-TF and PA2146-down-R.