Evaluation of FruA/FruB in Fructan Catabolism by Streptococcus mutans

The ability of Streptococcus mutans to synthesize and degrade fructans has been shown to contribute to its cariogenicity. FruA is a secreted exo-beta-fructosidase and a proven virulence factor of S. mutans. Co-transcribed with fruA is fruB, a gene predicted to code for a fructosidase. However, non-polar fruA mutants of S. mutans UA159 and GS-5 lack all fructosidase activity, indicating that FruB does not play a direct role in fructan degradation, or that the fruB gene product is inactive in certain strains of S. mutans. Objectives: The objectives of this study was to determine whether FruB plays a role in fructan catabolism in other strains of S. mutans. Methods: In this study, PCR-ligation-mutagenesis was used to delete and replace fruA and fruB in various S. mutans strains. These mutants were analyzed for their capacity to utilize inulin, a fructose polymer, by fructanase assays and growth studies. Results: Mutants lacking fruA were generated in S. mutans laboratory strains [UA159, OMZ175, LM7, NG8] and low-passage clinical isolates [C32(5)1, C5(9)2, C2(5)3]. Similar to UA159, FruA-deficiency caused loss of fructanase activity and of the capacity to grow in tryptone-vitamin (TV) broth with inulin as the primary growth substrate (TVGI) in strains C32(5)1, C5(9)2, C2(5)3, LM7, and OMZ175. In contrast, S. mutans NG8 lacking FruA was able to grow albeit ~30% slower than the parental strain in TVGI. Construction and testing of fruB mutants is ongoing. Conclusion: These results confirm that FruA is the primary enzyme responsible for inulin hydrolysis in most S. mutans strains studied. In S. mutans NG8, however, a second fructosidase, possibly the product of the fruB gene, allows inulin hydrolysis. Studies are ongoing to evaluate fruB mutants of these S. mutans strains. This research was supported by a UFCD Student Summer Research Fellowship, NIH-NIDCR grant T32 DE007200, and NIH grant R01 DE12236.