Characterization of an amylase-binding component of Streptococcus gordonii G9B

Abstract

The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited '25I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of '25I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 1251-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall. * Corresponding author. with proteolytic enzymes abolishes amylase binding to S. gordonii, implicating a surface protein as the amylasebinding component (11, 12, 39). However, amylase binding could also be abrogated by treating the bacteria with periodate, suggesting a role for streptococcal carbohydrate in this interaction (39). Recently, several amylase-binding components have been identified on S. gordonii NCTC 7868 (Challis) (12). Little is known, however, about the chemical composition of these molecules or their distribution on the streptococcal surface. Thus, the goal of the present study was to characterize and localize the amylase-binding component(s) on S. gordonii G9B. MATERIALS AND METHODS Bacterial strains and culture conditions. The following amylase-binding strains were studied: S. gordonii G9B (37), Challis (NCTC 7868), Blackburn (NCTC 10231), and FAS4 (39) and S. crista CR311 (22). The non-amylase-binding strain S. sanguis ATCC 10556 was also used in control

Structure of Human Salivary α-Amylase at 1.6 Å Resolution: Implications for its Role in the Oral Cavity

“…* However, studies directed towards this are in progress. Recently, the streptococcal amylase-binding component has been characterized (Scannapieco, Haraszthy & Levine, 1992). The complex between this protein and salivary amylase should provide a more detailed picture of the bacterial binding and its effect on salivary amylase conformation and structure.…”

Effect of starch and amylase on the expression of amylase‐binding protein A in Streptococcus gordonii

“…Amylase-binding streptococci are numerous in the supra-gingival plaque of host species that express functional salivary a-amylase (Scannapieco et al, 1992(Scannapieco et al, , 1993(Scannapieco et al, , 1994. A representative amylase-binding species is Streptococcus gordonii, a commensal bacterium typically associated with oral health (Kolenbrander, 2000).…”

“…A representative amylase-binding species is Streptococcus gordonii, a commensal bacterium typically associated with oral health (Kolenbrander, 2000). Streptococcus gordonii binds salivary amylase to its surface with high efficiency and specificity via the surface expressed 20-kDa amylase-binding protein A (AbpA) (Scannapieco et al, 1992). AbpA is attached to the nascent cell wall at sites of cell division, and later, as the cell matures, is shed into the environment (Scannapieco et al, 1992).…”

“…Streptococcus gordonii binds salivary amylase to its surface with high efficiency and specificity via the surface expressed 20-kDa amylase-binding protein A (AbpA) (Scannapieco et al, 1992). AbpA is attached to the nascent cell wall at sites of cell division, and later, as the cell matures, is shed into the environment (Scannapieco et al, 1992). Though AbpA is the major protein responsible for amylase binding to bacterial cell surface, it is transiently associated with the cell wall and abundantly present in culture supernatants (Rogers et al, 1998).…”

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

“…This interaction is thought to be important for streptococcal colonization of the oral cavity [7^9]. Preliminary studies that identi¢ed a 20-kDa protein produced by S. gordonii as the major amylase receptor [10,11] were later con¢rmed using a transposon-mediated mutation selection strategy [12]. Stable isogenic mutants de¢cient in the 20-kDa protein adhere less well to amylase-coated hydroxyapatite and are de¢cient in bio¢lm formation in a saliva-coated £ow cell system when compared to their wild-type parental strains [9].…”