Transformation of Lactiplantibacillus plantarum and Apilactobacillus kunkeei is influenced by recipient cell growth temperature, vector replicon, and DNA methylation.

The effects of recipient cell growth temperature, vector choice, and DNA methylation on transformation efficiency were explored for Lactiplantibacillus plantarum strain B38 and Apilactobacillus kunkeei strains YH15 and 3L. All three parameters significantly affected transformation efficiency. L. plantarum B38 and A. kunkeei YH15 transformed at higher efficiencies with the pTW8 vector than with the pTRKH2 vector; conversely, A. kunkeei 3L transformed at higher efficiency with pTRKH2. Mean transformation efficiencies as high as 7.8 × 10 colony forming units (CFU) μg were obtained with pTW8 for B38, as high as 1.2 × 10 CFU μg with pTW8 for YH15, and as high as 3.4 × 10 CFU μg with pTRKH2 for 3L. With respect to methylation, B38 and YH15 transformed at higher efficiencies with DNA that lacked dam methylation, while 3L transformed at higher efficiency with DNA that was dam methylated. Methylation at Escherichia coli dcm sites did not affect the ability of pTRKH2 or pTW8 to transform these strains. Recipient cell growth at 21 °C rather than at 37 °C significantly increased transformation efficiencies when using each strain's preferred vector and methylation state; pTW8 without dam methylation for B38 and YH15 and pTRKH2 with dam methylation for 3L.