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 have been difficult to construct without selectable markers. We describe here a new genetic 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.
Biswas
Indranil
I
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion 57069, USA. ibiswas@usd.edu
Drake
Laura
L
Johnson
Sean
S
Thielen
Damon
D
D006358:Hot Temperature; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D013295:Streptococcus mutans / Q000235:genetics*; D014170:Transformation, Genetic