1887

Abstract

Summary: 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 In order to isolate mutants unable to produce dextranase, a DNA library was constructed by inserting random 3AI-digested fragments of chromosomal DNA from into the HI site of the streptococcal integration vector pVA891, which is able to replicate in but does not possess a streptococcal origin of replication. The resultant plasmids were introduced into 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 , strongly suggesting that the dextranase gene () 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 LT11 but there was no apparent difference in sucrose-dependent cell-cell aggregation. In contrast to LT11, neither the mutants nor a mutant in the 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 gene, a role for dextranase is suggested in controlling the amount and nature of extracellular glucans, in adherence of , and in the utilization of glucans as a carbohydrate source.

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1995-11-01
2021-10-17
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