title: Construction of a counterselection-based in-frame deletion system for genetic studies of Streptococcus mutans.
abstract: 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.
authors.affiliation: Department of Oral Biology, UCLA School of Dentistry, Los Angeles, CA 90095-1668, USA. jwiz138@ucla.edu
authors.forename: J
authors.initials: J
authors.lastname: Merritt
authors.forename: P
authors.initials: P
authors.lastname: Tsang
authors.forename: L
authors.initials: L
authors.lastname: Zheng
authors.forename: W
authors.initials: W
authors.lastname: Shi
authors.forename: F
authors.initials: F
authors.lastname: Qi
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D005686:Galactokinase / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D005686:Galactokinase; D001616:beta-Galactosidase; D005690:Galactose