Article Figures & Data
Figures
- Fig. 1.
ELISA-based detection of levan production with UPC-10. Biofilms of A. naeslundii WVY45 (column 1) or FTF1 (column 2) or of S. salivarius 57.1 (column 3) were formed in microtiter wells in the presence of sucrose. Levan production by the bacteria was detected with a mouse monoclonal antibody against β2,6-linked fructans as detailed in Materials and Methods. The values presented are the means of three separate experiments performed in triplicate, and the error bars show the standard deviations. The negative controls were cells grown without sucrose.
- Fig. 2.
(A) Coomassie blue-stained, histidine-tagged FTF. The recombinant, histidine-tagged FTF protein was purified from E. coli after induction with 1 mM IPTG using the denaturing protocol recommended by Qiagen. The purified protein (1 μg) was electrophoresed in an SDS–10% PAGE gel, and the gel was stained using Coomassie blue. Molecular weight standards were from LTI. (B) Western blot analysis with a rabbit anti-FTF antisera. Antisera was raised to the purified histidine-tagged FTF protein (1.4 mg of total protein) in rabbits at Lampire Biologicals. Following separation of the supernatant proteins or cell extracts of A. naeslundii WVU45 and FTF1 and E. coli FTFLB4 (3), which carries and expressed the ftf gene on a multicopy plasmid, the proteins were transferred to PVDF membranes. The blots were probed with the anti-FTF antisera at a dilution of 1:100 and subsequently probed with a goat anti-rabbit IgG peroxidase-conjugated antibody at a dilution of 1:1,500. Supernatant proteins from A. naeslundii WVU45 (lane 1) or A. naeslundii FTF1 (lane 2), whole-cell lysates fromE. coli FTFLB4 (3) (lane 3), and whole-cell lysates of A. naeslundii WVU45 (lane 4) and FTF1 (lane 5) were tested. Std., prestained molecular weight standards from LTI. The asterisk indicates the position of the 70-kDa FTF protein in A. naeslundii WVU45 supernatant and in E. coli FTFLB4 (3).
- Fig. 3.
Detection of levan production by proteins following SDS-PAGE. Protein samples from culture supernatants of A. naeslundii WVU45 (lane 1) and FTF1 (lane 3) or from whole-cell extracts of WVU45 (lane 2) and FTF1 (lane 4) were separated in a 10% polyacrylamide gel and subsequently transferred to PVDF membranes. The filters were incubated overnight at 37°C in 50 mM sucrose. The membranes were briefly rinsed in TBS and incubated with monoclonal antibody UPC-10 as detailed in Materials and Methods. After multiple washes, the membranes were incubated with a goat anti-mouse, peroxidaase-conjugated antibody, and the immune reactivity with levans was disclosed. Std., prestained protein molecular weight standards.
- Fig. 4.
Levanase-, inulinase-, and sucrase-specific activities in supernatant and whole-cell lysates of A. naeslundiistrains WVU45 and Lev1. Cells were grown to mid-exponential phase in ADM containing sucrose (columns 1 and 2), glucose (columns 3 and 4), or fructose (columns 5 and 6), and the levanase activity was measured in the culture fluid (supernatant) or whole-cell lysates (cells) of the wild-type strain WVU45 (columns 1, 3, and 5) or the LevJ-deficient strain (columns 2, 4, and 6). The results represent the mean of at least three separate experiments performed in a minimum of triplicate. Error bars show the standard deviation. In all cases of measurements of supernatant activities, the mutant and wild-type strains are statistically different (P < 0.05) by ttest, as is the case for cell-associated levanase activity in sucrose- and glucose-grown cells (columns 1 to 4) and for the cell-associated sucrase activity in all carbohydrates tested.
