Family of shuttle vectors for ruminal Bacteroides

Cheryl M. Wong, Athol V. Klieve, Brenton J. Hamdorf, Darren J. Schafer, Lambert Bräu, Shawn G M Seet, Keith Gregg*

*Corresponding author for this work

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 105 transformants/μg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11-0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0. 53% per generation.

Original languageEnglish
Pages (from-to)123-132
Number of pages10
JournalJournal of Molecular Microbiology and Biotechnology
Volume5
Issue number2
DOIs
Publication statusPublished - 2003
Externally publishedYes

Keywords

  • bacteria, ruminal
  • shuttle plasmid
  • transformation
  • genetic manipulation
  • fluoroacetate detoxification
  • Bacteroides

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