FIG 4.

ORF4 is critical for stability and copy number control of SNJ1-based plasmids. (a) Relative copy numbers of SNJ1-based plasmids pYC-SHS (with orf4) and pYC-SHS-4M (without orf4) in CJ7 cells with or without MMC treatment. “WT” represents wild-type pYC-SHS plasmid, “Δorf4” represents pYC-SHS-4M (start codon mutation of orf4), and “Δorf4+orf4” represents pFJ6-1-656 complemented pYC-SHS-4M. These plasmids were transformed to CJ7 strain and cultured to late exponential phase (24 h) in 18% MGM+Mev and treated with MMC (1 μg ml⁻¹) for 30 min, while cultures not treated with MMC were set as controls. Cells were collected by centrifugation and washed twice in the same volume of 18% MGM to remove MMC. Cell pellets were resuspended in 18% MGM+Mev and cultured for 24 h. Samples were taken for qPCR analysis using the primer pairs vector-F/vector-R, orf14-F/orf14-R, and radA-F/radA-R to determine the plasmid copy numbers relative to the chromosome. Three independent experiments were performed, and the error bars indicate the standard deviations. (b) Plasmid stability of pYC-SHS and pYC-SHS-4M during passage. Portions (100 μl) of stationary-phase cultures of CJ7/pYC-SHS, CJ7/pYC-SHS-4M, and CJ7/pYC-SHS-4M+pFJ6-1-656 were inoculated into 5 ml of Halo-2 medium every day. Samples were taken and measured by qPCR using the primer pairs vector-F/vector-R, orf14-F/orf14-R, and radA-F/radA-R for 5 days. Three independent experiments were performed, and error bars indicate the standard deviations.