Mariner-based transposon mutagenesis for Bacteroides species

Minoru Ichimura, Keiko Uchida, Haruyuki Nakayama-Imaohji, Hideki Hirakawa, Tomoyo Tada, Hidetoshi Morita, Koji Yasutomo, Katsuichiro Okazaki, Tomomi Kuwahara

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.

Original languageEnglish
Pages (from-to)558-567
Number of pages10
JournalJournal of basic microbiology
Volume54
Issue number6
DOIs
Publication statusPublished - Jun 2014

Keywords

  • Bacteroides
  • Electrotransformation
  • Mariner
  • Transposon
  • Transposon mutagenesis

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology

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  • Cite this

    Ichimura, M., Uchida, K., Nakayama-Imaohji, H., Hirakawa, H., Tada, T., Morita, H., Yasutomo, K., Okazaki, K., & Kuwahara, T. (2014). Mariner-based transposon mutagenesis for Bacteroides species. Journal of basic microbiology, 54(6), 558-567. https://doi.org/10.1002/jobm.201200763