Mariner‐based transposon mutagenesis for Bacteroides species. (20th May 2013)

• Record Type: Journal Article
• Authors: Ichimura, Minoru; Uchida, Keiko; Nakayama‐Imaohji, Haruyuki; Hirakawa, Hideki; Tada, Tomoyo; Morita, Hidetoshi; Yasutomo, Koji; Okazaki, Katsuichiro; Kuwahara, Tomomi
• Is Part Of: Journal of basic microbiology. Volume 54:issue 6(2014:Jun.)
• Page Start: 558
• Page End: 567
• Publication Date: 2013-05-20
• Subjects: Microbiology -- Periodicals; 579
• DOI: 10.1002/jobm.201200763
• Languages: English
• ISSNs: 0233-111X

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <sec id="jobm201200763-sec-0001" sec-type="section"> <p> <italic>Bacteroides</italic> is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of <italic>Bacteroides</italic> with epithelial function, the gut immune system and systemic metabolism in the host. The <italic>mariner</italic> family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the <italic>mariner</italic> family transposon is expected to identify key molecules for human‐<italic>Bacteroides</italic> symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (<italic>ermF</italic>/ITR), which harbors the erythromycin resistant marker (<italic>ermF</italic>) and the inverted repeat sequences (ITRs) recognized by <italic>Himar1</italic> transposase, to <italic>Bacteroides</italic> via electrotransformation. pMI07 successfully delivered <italic>ermF</italic>/ITR to the <italic>Bacteroides</italic> genomes and generated thousands of insertion mutants/μg of pMI07 in <italic>B. thetaiotaomicron</italic>, <italic>B. fragilis</italic>, <italic>B. ovatus</italic>, and also, although to a lesser extent, <italic>B. vulgatus</italic>. Analyses of the <italic>ermF</italic>/ITR insertion sites in <italic>B. thetaiotaomicron</italic> and <italic>B. vulgatus</italic> 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 <italic>Bacteroides</italic> species, which will enable identification of the genes responsible for their unique phenotypes.</p> </sec> </abstract>