Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii.
The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS-PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.
Department of Oral Biology, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, 14214, USA.
Lina
L
Li
Jason M
JM
Tanzer
Frank A
FA
Scannapieco
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018441:Biofilms; D003001:Cloning, Molecular; D003773:Dental Plaque / Q000382:microbiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015183:Restriction Mapping; D012463:Saliva / Q000201:enzymology / Q000382:microbiology; D017422:Sequence Analysis, DNA; D013291:Streptococcus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D000681:Amylases