Efficient transformation system for Propionibacterium freudenreichii based on a novel vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely, Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii, Whereas electroporation of P, freudenreichii with vector DNA isolated from an E, coli transformant yielded 10 to 30 colonies per mug of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (greater than or equal to 10(8) colonies per mug of DNA), It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.