Molecular organization and expression of the gtfA gene of Streptococcus mutans LM7

The Streptococcus mutans LM7 gene gtfA was cloned in Escherichia coli along with flanking regions of the chromosome as a fragment representing 10.3 kilobases (kb) of streptococcal DNA. Restriction endonuclease mapping revealed that the cloned DNA consisted of four EcoRI fragments with gtfA sucrase activity localized to one fragment, EcoRI-B (2.4 kb). Subsequent analysis with E. coli minicells indicated that three polypeptides were encoded on the 10.3-kb insert (55 [GtfA], 45, and 35 kilodaltons). Neither the 45- nor 35-kilodalton polypeptide exhibited any detectable sucrase activity. The approximate positions and directions of transcription of the two larger proteins were determined from minicell protein profiles displaying truncated versions of these polypeptides. The restriction endonuclease data for the cloned gtfA gene were used to develop a strategy for insertional inactivation of this locus in vivo. An internal HincII fragment of the gtfA gene was removed and replaced with a DNA fragment containing a tetracycline resistance determinant. This new recombinant plasmid was linearized and then transformed into S. mutans GS5 and S. mutans V403 where it was incapable of replication. It was predicted that Tcr colonies would result from double-crossover recombinational events involving homologous regions flanking the gtfA gene. This was verified by Southern DNA hybridization analyses. The inactivation of the gtfA gene in both S. mutans GS5 and S. mutans V403 resulted in a decrease of water-soluble exopolysaccharide but no detectable changes in the amounts of water-insoluble polymers.