Bacterial strains and culture conditions
S. gordoniistrains Challis-S (wild type, a spontaneous streptomycin resistant mutant of strain Challis), and its AbpA-(strain ST) and AbpB-deficient (strain SE) mutants,[16,17], a Gtf-G-deficient mutant (CHΔgtf)  andS. mutans10449S, a spontaneous streptomycin resistant mutant of strain NCTC-10449, were recovered from frozen stocks on tryptic soy blood agar and incubated overnight in a candle jar. Streptococci were routinely cultured in tryptic soy broth (Difco, Detroit, MI. USA) containing 0.5% (w/v) yeast extract (TSBY) overnight at 37C.Escherichia coliwas grown in Luria-Bertani broth (LB) with constant shaking at 37°C and maintained on LB agar.
Construction of the AbpA expression system
The expression vector, pETBlue-1 Blunt (Novagen, Madison, WI. USA), was used to clone and express theabpAofS. gordonii. A 1.7 kbXbaI DNA fragment containingabpAwas purified from pCR2kb-7  and then inserted into pETBlue-1 Blunt vector, resulting in plasmid p101-1. The 1.6 kbEcoRI fragment from p101-1 was purified and digested with ApoI. The resultant 758 bpApoI fragment was eluted from 2% (w/v) agarose gel and cloned into the pETBlue-1 blunt vector, resulting in p102-4, theabpAexpression clone. All recombinant plasmids were introduced by chemical transformation into NovaBlue Singles™ Competent Cells (Novagen), and selected on LB agar supplemented with ampicillin (100 μg ml-). The orientations of cloned genes were confirmed by restriction enzyme analysis. Plasmids containing inserts of correct size and orientation were purified using Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA), and the fidelity of the cloned region verified by sequencing.
Expression of AbpA in E. coli
TheabpAplasmid p102-4 was transformed into Turner™(DE3)pLacI Competent Cells (Novagen) which were grown to 1.0 OD600in 250 ml LB supplemented with ampicillin (50 μg ml-1) and 1% glucose. The cells were induced by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG) for 2 h at 37°C with constant shaking. Cells were harvested by centrifugation at 4000 × g for 15 min at 4°C, and the cell pellet (~2.3 g wet weight) was stored frozen at -20°C. The inducedE. colicell pellet was suspended in 50 ml of EasyLyse™ Bacterial Protein extraction solution (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5% Triton X-100, 0.1 mM MgCl2and 20 μl EasyLyse™ enzyme mix) (Epicentre, Madison, WI, USA). The insoluble cellular debris was removed by centrifugation (10,000 × g) at 4°C for 10 min and the soluble supernatant transferred to a clean tube. The total supernatant protein concentrations were estimated using BCA reagent (Bio-Rad, Hercules, CA, USA), and the supernatant containing the rAbpA was stored at -20°C.
Purification of rAbpA
rAbpA was concentrated by anion exchange Macro-prep DEAE methacrylate (Biorad) in a 5 ml column equilibrated with 10 mM Tris buffer, pH 7.3. Crude supernatant (10 ml) containing 2 mg protein ml-1of theE. coliextract that included rAbpA was loaded onto the column and eluted by a 0 to 1 M linear NaCl gradient. Twenty-5 ml fractions were collected and monitored at 280 nm. All fractions were further analyzed by 12% SDS-PAGE and stained with either 0.1% (w/v) Coomassie brilliant blue R-250 or SYPRO red (Cambrex, Rockland, MI, USA). N-terminal amino acid sequencing was performed to verify the amino acid sequence of rAbpA (data not shown). Purified proteins were lyophilized and stored for further studies.
Western immunoblotting or amylase ligand binding assay
Western blotting  was performed by electrotransfer of proteins from SDS-PAGE gels to Immobilon-P membranes (Millipore, Bedford, MA, USA). For the amylase ligand-binding assay, after blocking with 5% (w/v) non-fat milk in TBST, membranes were incubated with a solution of purified amylase (Sigma, St. Louis, MO, USA; Cat # A-1031. A stock solution of 10 mg/ml was made in deionized water and stored at -20°C.) in TBST for 30 min. After washing, the blots were incubated with either polyclonal rabbit anti-AbpA  or polyclonal anti-human α-amylase (Sigma), washed, and then incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (Promega). Finally, the blots were developed using the ProtoBlot Western Blot AP system (Promega).
Matrix assisted laser desorption-ionization-time-of-flight analysis
The pure, lyophilized rAbpA was dissolved in water. Approximately 1 p-gram of the protein was mixed with sinapinic acid solution (10 mg ml-1in 60% acetonitrile, 0.1% tri-fluoroacetic acid), spotted onto the sample analysis plate, and dried. The protein was then analyzed by matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Bremen, Germany). Mass spectra were acquired with an accelerating voltage of 19 kV using linear mode. A molecular mass of 18121.024 Da was observed for rAbpA by MALI-TOF (data not shown). The instrument was externally calibrated with carbonic anhydrase and bovine serum albumin.
For mass fingerprinting, about 100 μg of the pure lyophilized protein was resuspended in 20 μl of 0.25 mg ml-1sequence grade trypsin (Promega) in water and incubated overnight at 37°C. Mass spectra were obtained using reflection mode. The monoisotopic masses obtained for the peptides were in the range of 919.523 to 1985.091. The molecular masses of the digested peptides obtained by MALDI-TOF showed coverage of 47% and were matched with the peptide masses obtained using Peptidemass software (data not shown). The instrument was calibrated externally with ACTH and angiotensin.
Inhibition of binding of (125I)α-amylase by rAbpA toS. gordonii
These experiments were performed essentially as previously described (Scannapieco, et al. 1989). Briefly, freshly grownS. gordoniicells were suspended in PBS containing 0.01% (w/v) lipid-free bovine serum albumin (BSA) to a final concentration of 3 × 109cells ml-1. Experiments were performed in polypropylene micro-centrifuge tubes pre-coated with 0.1% (w/v) BSA to reduce nonspecific binding of ligand with the tube wall. Reaction mixtures of 0.1 ml, containing 1.1 pmole of (125I) α-amylase in PBS, and various amounts of rAbpA were incubated at room temperature for 30 min. To this mixture bacteria suspended in PBS were added to final volume of 0.5 ml, mixed gently, incubated for 30 min, and the reaction terminated by centrifugation at room temperature followed by three 1 ml washes with PBS. The amount of radioactivity, reflecting amylase bound to the bacteria, was measured with a gamma counter (Model 5500; Beckman City, CA, USA).
Precipitation of amylase-interactive proteins fromS. gordoniiculture supernatants
Supernatants were collected from overnight cultures by centrifugation at 5000 × g for 10 min at room temperature and passed through a 0.2 micron filter. Precipitation of proteins was induced by the addition of purified salivary amylase (50 μg ml-1) to the supernatant (Liet al., 2002). After incubation at room temperature for 2 h, the precipitate containing amylase and streptococcal proteins was recovered by centrifugation at 5000 × g for 10 min, resuspended in sample buffer (0.06 M Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 0.05% (v/v) 2-β-mercaptoethanol and 0.00125% bromophenol blue, and boiled for 3 min. Subsequently, the precipitate components were resolved on 12% SDS-PAGE, stained with SYPRO RED in 7.5% (v/v) acetic acid, and photographed under UV light.
Glucosyltranferase activity assay
Gtf activity was evaluated by both qualitative and quantitative assays. For qualitative assays, activity was determined in polyacrylamide gels as previously described . Briefly, cell free supernatants were run on SDS-PAGE followed by overnight incubation of gel in 3% sucrose. The glucan bands synthesized by the GTF were visualized by staining with periodic acid and pararosanaline.
The component sucrase and transferase activities of Gtf-B were differentially quantified as previously described [33,34]. Briefly, 0.6 μg of soluble Gtf-B (kindly provided by Dr. Ann Vacca-Smith, University of Rochester, NY, USA) in 2 μl of 0.2 M sodium phosphate buffer (pH 6.0), was incubated in the presence or absence of various amounts of soluble rAbpA (2 μl) and/or amylase (2 μl in buffer) at room temperature for 5 min. To this, 50 μl of buffer and 40 μl of 1 M sucrose in water were added to a final volume of 100 μl and the reaction mixture was incubated overnight at 37°C. The amounts of free glucose and fructose in the reaction mixture were measured by an enzymatic-redox reaction train using hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphoglucose isomerase, and NADP+(F-Kit, R-Biopharm, Mannheim, Germany). The amount of resulting fructose in the reaction mixture represents sucrase activity; the difference between the amounts of free glucose and free fructose in the reaction mixture corresponds to the amount of glucosyl residues transferred to glucan, and thus the transferase activity . Means (± S.E.) were used to describe the enzyme activities.
Interaction of rAbpA and amylase with Gtf-B of S. mutans
ELISA was used to verify the interaction of rAbpA and amylase with Gtf-B. Two-fold dilutions of Gtf-B or rAbpA were coated onto wells of ELISA plates. The plates were washed and blocked as per manufacturers instructions (Kirkegaard and Perry, Gaithersburg, MD). To each well, 1 μg of purified amylase, Gtf-B or rAbpA was added and incubated at room temperature for 2 hr. After washing, the samples were probed with polyclonal anti-Gtf-B (kindly provided by Dr. Ann Vacca-Smith), anti-AbpA (25), or anti-amylase antibodies, as appropriate. The plates were then incubated, washed, and probed with the secondary antibody provided in the ELISA kit. The plates were developed using the kit substrate according to the manufacturer's instructions. Following color development, which was proportional to the amount of target protein interacting with the protein bound to the plate, the OD of the solution was read at 630 nm in a plate reader.
Biofilm experiments were performed in round-bottomed polystyrene microtiter plates (Nalge Nunc International, Rochester, New York, USA). Plates containing 200 μl of biofilm media (BM)  in the presence or absence of 1% sucrose and/or 25% filter sterilized saliva per well were inoculated with either WT or mutant strains ofS. gordonii(5 × 107CFU per well) from an overnight culture grown in TSBY. After 20 h of incubation at 37°C, resultant bacterial growth in each well was quantified by measuring the absorbance at 595 nm. The wells were then washed with PBS and the biofilm that formed on the walls of the wells was fixed for 5 min by adding 200 μl of methanol at room temperature. Following air drying, 100 μl of 0.1% crystal violet (CV) solution was added to each well. After 5 min, the wells were rinsed three times with 200 μl of distilled water and air-dried. To each well, 200 ml of 95% ethanol was added and placed in a rocker for 15 min. The CV eluted from the biofilm was then quantified by measuring the absorbance at 630 nm using an ELISA plate reader.
Statistical analyses were conducted using SPSS software, version 11. Analyses of the enzyme activity were done using the two-sided t-test. Differences were considered significant when a P value of 0.05 was obtained.