Transposon mutagenesis of the anaerobic commensal,  Bacteroides fragilis, using the EZ::TN5 transposome.
Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.
GLAVAHCS, Los Angeles, CA, USA; UCLA School of Medicine, Los Angeles, CA 90073, USA. veereshagy@gmail.com
Yaligara
Y
Veeranagouda
Fasahath
F
Husain
Hannah M
HM
Wexler
D001426:Bacterial Proteins / Q000235:genetics; D001441:Bacteroides fragilis / Q000235:genetics*; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D008780:Methyltransferases / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin; D019895:Transposases / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008780:Methyltransferases; C437393:ErmF protein, Bacteroides; D019895:Transposases