Improvement of transformation efficiency through in vitro methylation and SacII site mutation of plasmid vector in Bifidobacterium longum MG1

Abstract

The different cleavage patterns of pYBamy59 plasmid isolated from E. coli DH5alpha and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for low transformation efficiency was related to the restriction modification (R-M) system. To confirm correlation between R-M system and transformation efficiency, in vitro methylation and site directed mutagenesis was performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of sequence recognized by SacII endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacII digestion. Site directed mutagenesis which removed SacII sites from pYBamy59 or in vitro methylation of pYBamy59 showed eight to fifteen-fold increases in the transformation efficiency over intact pYBamy59. Modification of SacII related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve transformation efficiency in this strain. The result showed that R-M system is a factor to limit introduction of exogenous DNA and in vitro modification was a convenient method to overcome barrier of R-M system for transformation.