INSERTIONAL INACTIVATION OF THE STREPTOCOCCUS-MUTANS DEXA (DEXTRANASE) GENE RESULTS IN ALTERED ADHERENCE AND DEXTRAN CATABOLISM

Streptococcus mutans is able to synthesize extracellular glucans from sucrose which contribute to adherence of these bacteria. Extracellular dextranase can partially degrade the glucans, and may therefore affect virulence of S. mutans. In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random Sau3Al-digested fragments of chromosomal DNA from S. mutans into the BamHI site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia coli but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into S. mutans LT11, allowing insertional inactivation through homologous recombination. Two transformants were identified which did not possess dextranase activity. Integration of a single copy of the plasmid into the chromosome of these transformants was confirmed by Southern hybridization analysis. Chromosomal DNA fragments flanking the plasmid were recovered using a marker rescue technique, and sequenced. Comparison with known sequences using the BLASTX program showed 56% homology at the amino acid level between the sequenced gene fragment and dextranase from Streptococcus sobrinus, strongly suggesting that the S. mutans dextranase gene (dexA) had been inactivated. The colony morphology of the dextranase mutants when grown on Todd-Hewitt agar containing sucrose was altered compared to the parent strain, with an apparent build-up of extracellular polymer. The mutants were also more adherent to a smooth surf;ace than LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation. In contrast to LT11, neither the dexA mutants nor a mutant in the dexB gene, which encodes a dextran glucosidase, were able to ferment dextran to produce acid, supporting an earlier hypothesis that both enzymes are required for metabolism of dextran. From the results obtained by inactivating the dexA gene, a role for dextranase is suggested in controlling the amount and nature of extracellular glucans, in adherence of S. mutans, and in the utilization of glucans as a carbohydrate source.