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 salivary enzyme alpha-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 Tn916 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 Tn916 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 Tn916. 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.
Rogers
Jeffrey D
JD
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Haase
Elaine M
EM
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Brown
Alan E
AE
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Douglas
Charles W I
CWI
Department of Oral Pathology, University of Sheffield, Sheffield S10 2TA, UK.
Gwynn
Justin P
JP
Department of Oral Pathology, University of Sheffield, Sheffield S10 2TA, UK.
Scannapieco
Frank A
FA
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA.
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008040:Genetic Linkage; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012463:Saliva / Q000201:enzymology; D017422:Sequence Analysis, DNA; D013291:Streptococcus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014169:Transformation, Bacterial; D014357:Trypsin / Q000378:metabolism
C112818:AbpA protein, Streptococcus gordonii; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D000681:Amylases; D014357:Trypsin