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Applied and Environmental Microbiology, February 2001, p. 499-503, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.499-503.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

J. P. M. Jore,^1,* N. van Luijk,¹ R. G. M. Luiten,² M. J. van der Werf,¹ and P. H. Pouwels¹

TNO Nutrition and Food Research, 3700 AJ Zeist,¹ and DSM Anti-Infectives, 2600 MA Delft,² The Netherlands

Received 9 August 2000/Accepted 7 November 2000

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 µg of DNA, use of vector DNA reisolated from a Propionibacterium transformant 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.

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^* Corresponding author. Mailing address: TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, The Netherlands. Phone: 31 30 69 444 68. Fax: 31 30 69 444 66. E-mail: jore{at}voeding.tno.nl.

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Applied and Environmental Microbiology, February 2001, p. 499-503, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.499-503.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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