Objectives: The ability of Streptococcus mutans to cause the formation of dental caries is predicated, in large part, by its ability to adapt to acidic conditions in dental plaque. In order to dissect the number of genes and gene products involved with acid-adaptation in S. mutans, it is of interest to develop means to rapidly create mutations in individual genes. Insertional inactivation of genes often involves inserting an antibiotic resistance cassette into a restriction site in the gene of interest, which can be problematic if the restriction site must be engineered. In this study, we explored the possibility of using a transposable element containing an erythromycin marker, which could be selected in oral streptococci. Methods: Our approach was to clone the erythromycin-resistance cassette from pTS19E into plasmid pMOD-2 (Epicentre, Madison WI), which contains a transposable element bearing a multiple cloning site. The resulting plasmid contained a modified transposon, referred to as EZ::TnErm. Samples of plasmid DNA, pGEM-7, containing a clone of the S. mutans endonuclease III gene, were mixed with EZ::TnErm and transposition was initiated by addition of enzymes contained in the EZ::TN in-vitro transposition system (Epicentre). Reaction products were subsequently used to transform Escherichia coli to erythromycin-resistance. Plasmid DNA samples, isolated from E. coli transformants, were screened for insertions in endonuclease III by restriction digestion. Candidate plasmids were sequenced to determine the precise insertion point of EZ::TnErm. Results: Of 60 plasmids screened, 4 contained an insertion in endonuclease III, while the remainder carried insertions of the Erm^R-marker elsewhere in the plasmid. Conclusion: Our results indicated that the EZ::TN system can be utilized to insertionally inactivate genes from S. mutans and is a viable alternative to traditional methods.

This work was supported in part by the Rochester Short-term Training-Health Professional Schools (T35-DE07189), the Training Program in Oral Infectious Diseases (T32 DE07165) and DE10174.

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