title: A mariner transposon vector adapted for mutagenesis in oral streptococci.
abstract: This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen.
authors.affiliation: Costerton Biofilm Center, Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Nilsson
authors.forename: Natalia
authors.initials: N
authors.lastname: Christiansen
authors.forename: Niels
authors.initials: N
authors.lastname: Høiby
authors.forename: Svante
authors.initials: S
authors.lastname: Twetman
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.forename: Tim
authors.initials: T
authors.lastname: Tolker-Nielsen
mesh_terms: D004261:DNA Replication / Q000528:radiation effects; D004251:DNA Transposable Elements; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D013295:Streptococcus mutans / Q000235:genetics*; D013696:Temperature
chemical_list: D004251:DNA Transposable Elements
keywords: Biofilm; Streptococcus mutans; extracellular matrix; glycosyltransferases; mariner; mutagenesis; transposon.
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