Construction of a counterselection-based in-frame deletion system for genetic studies of Streptococcus mutans

Genetic studies of Streptococcus mutans have benefited greatly from the numerous techniques that have been successfully adapted for use in this organism. One notable exception is the lack of a negative selection system that can be employed for the easy isolation of markerless in-frame deletions. In this study, we report the development of a galK/galactose-based negative selection system in S. mutans for this purpose. This system consists of a recipient strain (IFD140) that contains a deletion in the galKTE operon and a suicide vector (pIFD-Sm) that carries the S. mutans galK open reading frame fused to the constitutive lactate dehydrogenase (ldh) promoter. Using this system we created a markerless in-frame deletion in the beta-galactosidase (lacG) gene within the S. mutans lactose operon. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of the galactose-resistant colonies and resulted in in-frame deletions in 50% of the screened isolates. Based on the ratio of galactose-resistant cells to total cells, we determined that plasmid excision occurred at a frequency of approximately 1/3000 cells. Furthermore, the simplicity of this system should make it adaptable for use in numerous other gram-positive and gram-negative organisms.