A mariner transposon vector adapted for mutagenesis in oral streptococci.

Martin Nilsson,Niels Høiby,Michael Givskov,Natalia Christiansen,Svante Twetman,Tim Tolker‐Nielsen

doi:10.1002/mbo3.171

Martin Nilsson, Niels Høiby + Show 4 more

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https://doi.org/10.1002/mbo3.171

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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.

Highlights

Oral streptococci are Gram-positive, facultative anaerobes that prevail on tooth surfaces in complex multi-species biofilms called dental plaque
A mariner transposon vector was constructed with the aim to perform unbiased transposon mutagenesis in oral streptococci
In Streptococcus equi, 60% of transposon inserts had occurred within a 15 kb region of the genome (Slater et al 2003)

Summary

Introduction

Oral streptococci are Gram-positive, facultative anaerobes that prevail on tooth surfaces in complex multi-species biofilms called dental plaque. Due to environmental factors, the biofilm becomes cariogenic. There is a strong correlation between a prolonged acidic environment at the enamel and an increased population of Streptococcus mutans, which in the end leads to an accelerated progress of caries (Takahashi and Nyvad 2008). S. mutans has developed a variety of mechanisms to colonize the tooth surfaces and to outnumber other bacteria in a cariogenic biofilm (Lemos et al 2005; Ahn et al 2006). Oral streptococci may enter the blood stream, through dental procedures and during daily activities like eating and tooth brushing. They can potentially bind to for example laminin, fibrin, collagen, and platelets, and subsequently adhere to damaged heart valves and cause endocarditis (Beg et al 2002; Sato et al 2004)

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