GENETIC-ANALYSIS OF FRUCTAN-HYPERPRODUCING STRAINS OF STREPTOCOCCUS-MUTANS

Fructan polymer, synthesized from sucrose by the extracellular fructos yltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries, It may serve as an extracellular storag e polysaccharide facilitating survival and acid production. It may als o have a role in adherence or accumulation of bacterial cells on the t ooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of incr eased production of fructan have been discovered. By using eight indep endent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Res triction fragment patterns of total cellular DNA were examined by usin g pulsed-field and conventional gel electrophoresis. Four genetic type s which appeared to correlate with the serotype of the organism and th e geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor di fferences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produc ed by these strains enhanced the virulence of these organisms in the o ral cavity, it was of interest to create mutants deficient in fructosi dase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperpro ducing strains as well as in S. mutans GS5, a strain which does not hy perproduce fructan. All of the fruA mutant strains were devoid of fruc tan hydrolase activity when levan was used as a substrate. However, th e fructan-hyperproducing strains retained the ability to hydrolyze inu lin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.