Markerless multiple-gene-deletion system for Streptococcus mutans

Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.