Abstract
In order to study virulence factors of the opportunistic oral pathogen Streptococcus mutans we have used Tn917 mutagenesis to generate mutants defective in specific phenotypic properties believed to be associated with virulence. This work describes the procedures required to use the temperature-sensitive transposon Tn917 delivery vector pTV1-OK, along with two techniques to recover inactivated genes. This vector has proven useful in generating transposon mutants of a poorly transformable S. mutans strain. We have successfully isolated mutants with diminished growth at pH 5.0, with defects in production of a mutacin, formerly known as BLIS or bacteriocin-like inhibitory substance and with nutritional requirements in minimal media including adenine, glutamate and arginine auxotrophy. The transposon has been used successfully in two strains of S. mutans, JH1005 and NG8, where transposition frequencies of 10−5 and 10−4 were observed, respectively. Rescue of inactivated genes was achieved using a recovery vector, pTV21Δ2TetM, that generates a replicative E. coli plasmid by reciprocal recombination with the mutant S. mutans chromosomal transposon site, and shot-gun cloning chromosomal DNA of mutants into pUC18, followed by selection of ampicillin and erythromycin resistant transformants in E. coli. The techniques described here can be adapted for generating mutants and recovering genes from a number of other Gram-positive and possibly Gram-negative bacteria.
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Cvitkovitch, D.G., Gutierrez, J.A., Crowley, P.J. et al. Tn917 transposon mutagenesis and marker rescue of interrupted genes of Streptococcus mutans. Methods Cell Sci 20, 1–12 (1998). https://doi.org/10.1023/A:1009711609646
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DOI: https://doi.org/10.1023/A:1009711609646