Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

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Abstract
A 3.6-kb endogenous plasmid was isolated from aPropionibacterium 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 anEscherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli andP. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacteriumtransformant dramatically increased the efficiency of transformation (≥108 colonies per μg 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.