Unmarked gene modification in

Unmarked gene modifications are desirable for various genetic analyses; however, they have been difficult to construct without selectable markers. We describe here a new genetic method for constructing unmarked mutants in . 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 . 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.