title: Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.
abstract: Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.
authors.affiliation: Department of Microbiology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ito
authors.affiliation: Department of Microbiology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo, Japan.
authors.forename: Yun-Gi
authors.initials: YG
authors.lastname: Kim
authors.affiliation: Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsuji
authors.affiliation: Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kiwaki
authors.affiliation: Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nomoto
authors.affiliation: Department of Microbiology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Danbara
authors.affiliation: Department of Microbiology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001435:Bacteriophages / Q000502:physiology; D002473:Cell Wall / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005798:Genes, Bacterial; D007109:Immunity / Q000235:genetics*; D007780:Lacticaseibacillus casei / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D008088:Listeriosis / Q000382:microbiology / Q000473:pathology; D008769:Methylnitronitrosoguanidine; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D019936:Probiotics / Q000378:metabolism*; D016415:Sequence Alignment; D013154:Spleen / Q000378:metabolism / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D016207:Cytokines; D004251:DNA Transposable Elements; D004187:Disaccharides; D010457:Peptidoglycan; D008769:Methylnitronitrosoguanidine; D001217:Aspartate-Ammonia Ligase