Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici , and Its Use for Development of a Host-Vector System in Propionibacteria

Abstract

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G؉C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 ؋ 10 6 CFU/g of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10 4 to 10 7 CFU/g of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.Propionibacteria, which have a wide range of probiotic activity, are used in making dairy foods, such as cheese, for the production of vitamin B 12 , tetrapyrrole compounds, and propionic acid (8,15,21), in bread baking, as starters for ensilage, and in some pharmaceutical preparations (35). To elucidate the biosynthetic pathways of vitamin B 12 and siroheme in Propionibacterium, we previously identified several genes coding for the enzymes involved in production of tetrapyrrole derivatives (hemYHBXRL) (11,12) and vitamin B 12 (cobA, cbiO) (30).Development of genetic manipulation in propionibacteria has progressed slowly due to a lack of detailed information on the genetics of the bacteria and a lack of an appropriate plasmid that can serve as a possible transformation vector. A number of plasmids from Propionibacterium acidipropionici, P. freudenreichii, and P. jensenii, ranging in size from 4.4 to more than 119 MDa, have been described (19,24). However, neither analysis of a plasmid DNA sequence nor construction of a vector for propionibacteria has been reported. To establish a versatile vector system to facilitate genetic analysis and to allow the transfer of a gene of interest, we investigated the development of a host-vector system in propionibacteria.We succeeded in determining the co...

Genetics and molecular biology of propionibacteria

“…The nucleotide sequence of pLME106 from P. jensenii DF1 was determined by Stierli [26,42]. This sequence is 100% identical to the nucleotide sequence of plasmid pRGO1 from P. acidipropionici E214 [36], determined by Kiatpapan et al [17,18]. The size of plasmid pLME106/pRGO1 is 6868 bp, the G+C content is 65%.…”

“…It was suggested that pLME106/pRGO1 is a composite plasmid, which must have gained ppnA and orf5 from another organism [29]. ORF3, encoding a putative protein of unknown function, was not described by Kiatpapan et al [17,18] either.…”

“…The region that comprises the earlier described identical order of enzyme restriction sites conserved between plasmids pRGO1 and pRGO2 contains the two ORFs responsible for the theta replication of pRGO1/pLME106 as well as ORF5 and ORF6 (respectively ORF8 and ORF9 in pLME106), whose functions are unknown, but which have been assumed to play a role in replication of plasmid pRGO1 [17]. 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.…”

“…In order to find small plasmids suitable for vector constructions, Kiatpapan et al [17] investigated 50 Propionibacterium strains from three different culture collections for the presence of plasmids but they did not report their screening results.…”

“…Due to their low G+C content ORF4 and ORF5 were not considered by Kiatpapan et al [17]. ORF5 encodes a putative protein of unknown function and resides downstream of ppnA.…”