Open AccessEfficient Electrotransformation of Bacteroides fragilis
Author(s) -
Minoru Ichimura,
Haruyuki NakayamaImaohji,
Shin Wakimoto,
Hidetoshi Morita,
Tetsuya Hayashi,
Tomomi Kuwahara
Publication year - 2010
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.02420-09
Subject(s) - bacteroides fragilis , electroporation , plasmid , transformation (genetics) , biology , microbiology and biotechnology , shuttle vector , escherichia coli , incubation , pbr322 , dna , gene , genetics , recombinant dna , vector (molecular biology) , biochemistry , antibiotics
This study describes refined electroporation parameters for efficient transformation ofBacteroides fragilis by plasmids prepared from laboratory strains ofEscherichia coli . Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the fourE. coli -Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable forB. fragilis transformation, and it generated 2- to 900-fold more transformants (about 104 transformants per μg pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested,B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion ofB. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained inB. fragilis strain NCTC9343 only when a homologously prepared (i.e.,in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of aB. fragilis gene usingin vivo -methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation ofBacteroides species.