Research Description

Molecular Characterization of Fructans Catabolism by Streptococcus mutans

Streptococcus mutans is generally considered the primary etiological agent of human dental caries. The ability of this organism to produce and utilize fructose polymers has been shown to directly contribute to caries formation. In S. mutans UA159 and several other clinical and laboratory strains studied, fructanase (fructan hydrolase, FruA), a β-exo-fructosidase, appears to be the sole enzyme involved in utilization of β(2,1)- and β(2,6)-linked extracellular fructose polymers. Deficiency of FruA causes loss of the ability to utilize both β(2,1)-linked inulins and β(2,6)-linked levans. Different from UA159 and other tested strains, however, FruA-altered mutants of strain NG8 still possess the capacity to grow on inulin as the sole carbohydrate source, albeit at a significantly slower growth rate, suggesting the presence of either a newly truncated fructanase protein in NG8 or the prescence of an entirely new fructanase protein. Co-transcribed with fruA, is a gene, fruB, that is predicted to code for a fructosidase, although the exact role of FruB has not yet been established. Objectives: The objectives of this project are to use recombinant proteins to examine the interactions and biochemical characteristics of the fructosidases in S. mutans NG8 and to investigate the role of FruB in fructan catabolism in S. mutans. Methods: In this study, sequencing analysis will be used to identify the exact mutant genomic sequence of NG8 mutant TW268, a derivative of NG8 mutant that has the fruA and fruB genes inactivated by deletion and replacement with an antibiotic resistance marker. TW268 was generated using transposon insertion. The growth of this NG8 mutant will be compared to that of the S. mutans TW237, a FruA/B-deficient mutant of UA159. Mutants will be grown under Bioscreen Assay on a tryptone-vitamin (TV) base medium with inulin as the sole carbohydrate source. Growth analysis of the mutant S. mutans strains will determine whether or not fructanase activity is pesent this data along with sequencing results will reveal whether or not a new fructanase or simply a truncated protein is responsible for mutant growth. Biochemical characterization of the inulinase of strains NG8 will be conducted using recombinant protein expressed in E. coli and in the supernatant fractions of the fruAB mutant strain. Outcome: A variety of molecular and biochemical techniques will elucidate the structure and characteristics of the mutated FruA inulinase enzyme from S. mutans TW268. These enzymes play important roles in human and plant diseases, and are of considerable economic significance because of their applications in the food industry.