Unmarked gene deletion mutagenesis of gtfB and gtfC in Streptococcus mutans using a targeted hit-and-run strategy with a thermosensitive plasmid.
Unmarked gene deletion of the Streptococcus mutans gtfB-gtfC locus was achieved using a thermosensitive plasmid. DNA fragments flanking the locus were amplified by polymerase chain reaction and jointly ligated into pG+host5, which was transformed into S. mutans at 37 degrees C to facilitate integration. A transformant was then grown at 28 degrees C for 60 generations without antibiotics to facilitate excision. Antibiotic sensitive clones appeared at a frequency of about 99% and were analyzed for deletions of gtfB, gtfC and a part of mbrA by the lack of insoluble glucan synthesis, sensitivity to bacitracin, and polymerase chain reaction. Targeted gene deletions occurred at a frequency of 2.5%.
Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, IL 60612, USA.
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D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001414:Bacitracin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005784:Gene Amplification; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005936:Glucans / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011135:Polysaccharides, Bacterial / Q000235:genetics; D013295:Streptococcus mutans / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial / Q000235:genetics
D000900:Anti-Bacterial Agents; D005936:Glucans; D011135:Polysaccharides, Bacterial; D001414:Bacitracin; C036655:1,3-alpha-D-glucan synthase; D005964:Glucosyltransferases; C110463:glucosyltransferase C