Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

Abstract

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 g of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (>10 8 colonies per g 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.The genus Propionibacterium can be divided into two groups, a group containing the classical (or dairy) propionibacteria and a group containing the cutaneous propionibacteria (6). Members of the first group, especially Propionibacterium freudenreichii, play an essential role in the manufacture of Swiss and related types of cheeses (12). Other industrial applications are found in the production of propionic acid and vitamin B 12 (5, 28). Of growing interest, but less well documented, are the probiotic properties ascribed to some propionibacterial strains (16,21).Strain improvement and, in general, study of this economically important group of bacteria would be greatly facilitated by the availability of a system for genetic modification. This report describes isolation and characterization of a 3.6-kb plasmid and successful use of this plasmid in the construction of a set of Escherichia coli-Propionibacterium shuttle vectors. Reproducible transformation of P. freudenreichii strains with these shuttle vectors was achieved by means of electroporation. MATERIALS AND METHODSBacterial strains and plasmids. Propionibacterium strains were obtained from the Belgian Coordinated Collections of Microorganisms/LMG (Ghent, Belgium), from the American Type Culture Collection (Rockville, Md.), and from the Deutsche Sammlung von Mikroorganismen (Braunschweig, Germany). P. freudenreichii subsp. freudenreichii VTB1 was obtained from DSM Food Specialties' industrial collection. For amplification of newly constructed shuttle vector DNA E. coli DH5␣ was used. pBluescript SKIIϩ was obtained from Stratagene (La Jolla, Calif.).Media and growth conditions. E. coli DH5␣ was cultivated at 37°C in L medium (24) supplemented with 50 g of ampicillin per ml if necessary. Propionibacteria were cultivated anaerobically at 30°C in MRS (7) or SLB medium (8) supplemented with an appropriate antibiotic when plasmid isolation was to be performed or in SLB medium when electroporation was to be performed.Isolatio...

Genetics and molecular biology of propionibacteria

“…Plasmids pRGO5 and pRGO7, which were described to be homologous in some parts to pRGO1 and pRGO2 (based on hybridisation experiments), might contain these kind of rep genes, too. On the other hand, p545 and pRGO1 did not show any positive signals in hybridisation experiments [16] at high stringency conditions, but still revealed 35.3% identity in a 249 amino acid overlap of ORF1.…”

“…Recently, two reports on an efficient electrotransformation system for Propionibacterium have been published [16,17]. Kiatpapan et al [17] describe the development of an E. coli -Propionibacterium shuttle vector pPK705, based on part of the pRG01 plasmid, containing the replication region of this plasmid, and the E. coli cloning vector pUC18.…”

“…Attempts to transform Propionibacterium with plasmids originating from other Gram-positive bacteria, including plasmids known to have a broad host-range, failed [16,17,24]. One of the barriers that was expected to play an important role is the existence of restriction/modification systems in Propionibacterium.…”

“…Jore et al [16], (van Luijk, unpublished) examined 75 Propionibacterium strains for the presence of plasmids. Only six strains harboured small plasmids, 3.6-10 kb in size.…”

“…The size of plasmid pLME106/pRGO1 is 6868 bp, the G+C content is 65%. Jore et al [16] determined the nucleotide sequence of p545 from P. freudenreichii LMG 16545. Its size is 3555 bp and its G+C content 63%.…”