A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes.
OBJECTIVE
Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes.


RESULTS
We optimized the conditions for transformation using a plasmid pUCYIT356-1-Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome.


CONCLUSIONS
Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition.


CONCLUSIONS
The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.
Ito
M
M
 Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
Kim
Y-G
YG
 Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
Tsuji
H
H
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
Kiwaki
M
M
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
Nomoto
K
K
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
Tanaka
R
R
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
Okada
N
N
 Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
Danbara
H
H
 Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
D004251:DNA Transposable Elements; D016680:Genome, Bacterial; D006801:Humans; D007780:Lacticaseibacillus casei / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D019936:Probiotics; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements