Open AccessA mariner transposon vector adapted for mutagenesis in oral streptococci
Author(s) -
Nilsson Martin,
Christiansen Natalia,
Høiby Niels,
Twetman Svante,
Givskov Michael,
TolkerNielsen Tim
Publication year - 2014
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.171
Subject(s) - transposable element , transposon mutagenesis , biology , sleeping beauty transposon system , genetics , transposase , p element , gene , mutant , tn3 transposon
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 pMN 100, contains the temperature‐sensitive origin of replication repATs ‐ pWV 01, 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 pMN 100 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.