IDENTIFICATION AND ANALYSIS OF A GENE (ABPA) ENCODING A MAJOR AMYLASE-BINDING PROTEIN IN STREPTOCOCCUS-GORDONII

Oral streptococci such as Streptococcus gordonii bind the abundant sal ivary enzyme alpha-amylase. This interaction may be important in denta l plague formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two mos t common plaque-mediated diseases. The conjugative transposon Tn916 wa s used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant de ficient in amylase-binding (Challis Tn1) was identified. While wild-ty pe strains of S. gordonii released both 20 kDa and 82 kDa amylase-bind ing proteins into culture supernatants, Challis Tn1 expressed the 82 k Da but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of 5 gordonii Challis by hydroxyapa tite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP(A)TDAAT(R)NND. A novel strategy, based on the sin gle-specific-primer polymerase chain reaction technique, enabled the g ene inactivated by Tn916 to be cloned. Analysis of the resultant nucle otide sequence revealed an open reading frame of 585 bp, designated am ylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), i mmediately downstream from the insertion site of Tn916. This protein p ossessed a potential signal peptide followed by a region having identi ty with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the bindi ng of amylase to 5 gordonii. Knowledge of the nature of amylase-bindin g proteins may provide a better understanding of the role of these pro teins in the colonization of 5 gordonii in the oral cavity.