title: Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species.
abstract: Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 × 10-3for B. fragilis and 1.7 × 10-3for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kino
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan.
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Nakayama-Imaohji
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan; Department of Applied Bioscience, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Fujita
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan.
authors.forename: Ayano
authors.initials: A
authors.lastname: Tada
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Yoneda
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Murakami
authors.affiliation: Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Hashimoto
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Department of Applied Bioscience, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan.
authors.forename: Katsuichiro
authors.initials: K
authors.lastname: Okazaki
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1, Miki, Kagawa 761-0793, Japan. Electronic address: tomomi@med.kagawa-u.ac.jp.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kuwahara
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016667:Bacterial Capsules / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001439:Bacteroides / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010134:Fenclonine / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D005804:Genes, Lethal; D005818:Genetic Engineering; D005819:Genetic Markers; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D005819:Genetic Markers; D011135:Polysaccharides, Bacterial; D021122:Protein Subunits; D010652:Phenylalanine-tRNA Ligase; D010134:Fenclonine
keywords: Bacteroides; p-chloro-phenylalanine; pheS; tRNA