Unmarked gene modification in Streptococcus mutans by a cotransformation strategy with a thermosensitive plasmid

Unmarked gene modifications are desirable for various genetic analyses; however they difficult to construct without selectable markers. We describe here a new genetic have been difficult method for constructing unmarked mutants in Streptococcus mutans. The desired mutant allele is first constructed and introduced into the strain by cotransformation with pGhost4, a thermosensitive plasmid that replicates in several low G+C Gram positive bacteria. With this method, we have modified two different loci with high frequency by insertion or deletion in S. mutans. Because pGhost4 contains a broad host range thermosensitive replicon, this method can be applied to any transformable low G+C Gram positive bacteria, including oral streptococci.