Mariner‐based transposon mutagenesis for Bacteroides species

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.

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

“…Transposon mutagenesis is currently available only for a small number of intestinal bacteria, including bifidobacteria (1,2,7,(24)(25)(26), mainly because of their low transformation efficiency. Hence, high efficiency is essential for transposon mutagenesis of these species.…”

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

“…The purified plasmid was subsequently introduced into B . fragilis by electroporation as previously described [ 41 , 42 ], employing conditions of 2.5 kV, 25 μF, and 200 Ω. Em-resistant colonies, in which the target plasmid had integrated into the chromosome through a single genetic crossover, were selected and grown in non-selective GAM broth. Dilutions of the overnight culture were spread onto GAM agar plates to provide an appropriate colony number; the plates were then replica plated onto GAM and GAM/Em plates to screen for Em-sensitive clones that resolved the diploid through a second homologous recombination.…”

The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis

“…The transposon insertion sites in the Tn4351-based mutant library of B. thetaiotaomicron VPI-5482 were identified by nested arbitrarily primed PCR (AP-PCR), as described previously [52]. Overnight cultures of the mutants were mixed with 20 µL of 50 mM NaOH and boiled for 10 min.…”