Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in Streptococcus gordonii

Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA Department of Oral Pathology, University of Sheffield, Sheffield S10 2TA, UK ABSTRACT Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme x -amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn 916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite 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 single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn 916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A ( abpA ), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn 916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity. * Author for correspondence: Frank A. Scannapieco. Tel: +1 716 829 2013. Fax: +1 716 829 3942. e-mail: Frank_Scannapieco@sdm.buffalo.edu Keywords: biofilms, dental plaque, salivary proteins, microbial adhesion/adherence, amylase-binding protein