Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector

Abstract

The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis.

Keywords: Bacteroides fragilis; Mariner transposon; Mutant library; Rescue cloning; Semi random PCR; Transposon mutagenesis.

Fig. 1

Transposon mutagenesis of B. fragilis 638R. A) The mariner transposon vector pSAM-Bt. IRL, Inverted repeat left; ermG-provides erythromycin resistance in B. fragilis; rp4 oriT/oriR6K-conditional origin of replication facilitates plasmid replication only in E. coli; Amp-ampicillin resistance gene for E. coli. Plasmid pYV07 was created by cloning Km-kanamycin resistance gene at the XbaI site of pSAM-Bt. B) Transposon mutants are generated by mating E. coli S-17 λ pir-pYV07 with B. fragilis 638R. C) When pYV07 enters B. fragilis 638R, the mariner transposase, whose expression is driven by B. thetaiotaomicron promoter (BT1331), inserts the transposon DNA (ie. IRL, ermG, Km and IRR) into the genome. The transposon mutants become resistant to erythromycin. D) Transposon disrupted gene identification by SRP-PCR and E) Transposon disrupted gene identification by rescue cloning. Mutant genomic DNA can be cut with BglII and cloned into BglII-digested pUC19. Sequencing with SAMseq3 and SAMseqR primer yields transposon junction DNA.

Fig. 2

Schematic representation of B. fragilis 638R transposon mutants. B. fragilis 638R has 4,416 genes in its genome. B. fragilis transposon mutants are generated using pYV07 and identified by SRP-PCR. The mutant name and the transposon insertion position (locus tag number) are indicated in brackets.