Insertional inactivation of the Streptococcus mutans dexA (dextranase) gene results in altered adherence and dextran catabolism

Abstract

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 Sau3ACdigested fragments of chromosomal DNA from 5. mutans into the BamHl site of the streptococcal integration vector pVA891, which is able to replicate in Escherichia COIi 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 conf irmed 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 surface than LTII 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.

Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

“…Dextranase-deficient mutants of S. mutans (DexA Ϫ ) are more adherent to a smooth surface than the parent strain, but no difference in sucrose-dependent cell-cell aggregation has been observed (24). Endodextranase activity evidently provides primer or branch points for glucosyltransferases and thus contributes to the complexity of the glucan structure (25,44).…”

“…The dextranase of S. mutans breaks down glucans to isomaltooligosaccharides, which are then transported into the cell via the products of the multiple sugar metabolism (msm) operon. In the cell, the oligosaccharides are further degraded to glucose by the products of the dexB gene, a dextran glucosidase (24). S. sobrinus and S. salivarius do not have such a mechanism and are unable to utilize dextrans or isomaltosaccharides as the sole carbon source (44,112).…”

“…Oral streptococci are predominant producers of the dextranases (24,46,55,220). Strains of S. mutans constitutively produce both endo-and exodextranases (162,171,220), whereas S. sobrinus synthesizes only endodextranase (10,222).…”

Sequence Analysis of the Streptococcus mutans Ingbritt dexA Gene Encoding Extracellular Dextranase

“…Experiments using a dextranase-deficient mutant obtained by insertional inactivation of the dextranase gene have shown that dextranase might play a role in controlling the amount and the nature of extracellular glucans, in adherence of S. mutans, and in the utilization of glucans as a carbohydrate source (5). In addition, a dextranase-negative mutant, which was obtained by chemical mutagenesis, was less cariogenic to experimental rats than the parent strain (37).…”

“…The molecular mass of the recombinant dextranase produced by E. coli cells is not clear because the complete dex gene of S. sobrinus UAB66 has not been cloned yet. Colby et al (5) reported that intact dextranase shows anomalous migration on SDS-PAGE and the exact size of dextranase can only be determined from the deduced amino acid sequence. As compared with the molecular masses from the deduced amino acid sequences, DexA (94.5 kDa) from S. mutans Ingbritt is smaller than that (143.3 kDa) from S. sobrinus UAB66 .…”