Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria

P. Kiatpapan, Y. Hashimoto, H. Nakamura, Y. Z. Piao, H. Ono, Mitsuo Yamashita, Y. Murooka

Research output: Contribution to journalArticle

40 Citations (Scopus)

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 x 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg 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.

Original languageEnglish
Pages (from-to)4688-4695
Number of pages8
JournalApplied and Environmental Microbiology
Volume66
Issue number11
DOIs
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Propionibacterium acidipropionici
Propionibacterium
Propionibacterium freudenreichii subsp. shermanii
plasmid
genetic vectors
plasmids
amino acid sequences
Propionibacterium freudenreichii subsp. freudenreichii
translation (genetics)
open reading frames
amino acid
DNA
Propionibacterium freudenreichii
hygromycin B
electroporation
beta-fructofuranosidase
Gram-positive bacteria
sequence homology
bacterium
gene

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria. / Kiatpapan, P.; Hashimoto, Y.; Nakamura, H.; Piao, Y. Z.; Ono, H.; Yamashita, Mitsuo; Murooka, Y.

In: Applied and Environmental Microbiology, Vol. 66, No. 11, 2000, p. 4688-4695.

Research output: Contribution to journalArticle

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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 x 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg 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.",
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AU - Kiatpapan, P.

AU - Hashimoto, Y.

AU - Nakamura, H.

AU - Piao, Y. Z.

AU - Ono, H.

AU - Yamashita, Mitsuo

AU - Murooka, Y.

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