Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans

Abstract

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.

Bacterial Extracellular Polysaccharides in Biofilm Formation and Function

“…Upon exposure to sucrose, levan accumulates on dental plaques and is catabolized to acid when the environmental sugar is depleted. Prolonged exposure promotes dental carries (165). While levan is not required for S. mutans biofilm formation, supplementation of sucrose to biofilms in vitro yields thicker structures, which contain more levan and are more resistant to shear stress (92,166,167).…”

Regulation of Expression of the Fructan Hydrolase Gene of Streptococcus mutans GS-5 by Induction and Carbon Catabolite Repression

“…More recently, it has been found that although many strains of S. mutans produce only FruA, some strains of S. mutans can produce two fructan-hydrolyzing activities. The major one is FruA, and the second activity is an inulinase with no detectable activity on levans (33). The environmental factors specifically regulating fruA expression and the molecular basis for the apparent differential expression of fructan hydrolase activity remain to be elucidated.…”