title: A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes.
abstract: 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.
authors.affiliation:  Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.affiliation:  Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
authors.forename: Y-G
authors.initials: YG
authors.lastname: Kim
authors.affiliation:  Yakult Central Institute for Microbiological Research, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsuji
authors.affiliation:  Yakult Central Institute for Microbiological Research, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kiwaki
authors.affiliation:  Yakult Central Institute for Microbiological Research, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nomoto
authors.affiliation:  Yakult Central Institute for Microbiological Research, Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Tanaka
authors.affiliation:  Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Okada
authors.affiliation:  Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Danbara
mesh_terms: 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
chemical_list: D004251:DNA Transposable Elements