FIG 2.

The rafX gene contributes to WTA biosynthesis in S. pneumoniae. (A and B) Representative analyses of TAs of S. pneumoniae strains by Western blotting and probing with a P-Cho-specific monoclonal antibody (TEPC-15). Bacteria were treated with a 0.5% sodium DOC solution to release TAs. Lanes 1 to 3 contained TAs from the WT, ΔrafX mutant, and complemented strains. CodY was used as a loading control. The positions of size markers (molecular masses in kilodaltons are shown) are indicated. (C) The amounts of TA in the cell wall (CW) fraction from the WT, ΔrafX mutant, and complemented (Compl) D39 strains were determined by sandwich ELISA. The amounts of TAs in the cell wall fraction (D), whole lysate (cells plus supernatant, E), supernatant (F) obtained from the WT, ΔrafX mutant, and complemented (Compl) R6 strains were determined by sandwich ELISA. TA concentrations (Con.) are expressed in arbitrary units, and values were obtained by multiplying the ratio of the OD value of a test sample to the background value obtained with normal C+Y medium by the dilution factor. (G) CRP binding assays. The inactivated WT, ΔrafX mutant, and complemented (Compl) R6 strains were used to absorb CRP, and the free CRP was determined after 4 h of absorption. Results are from three independent experiments (n = 3). Error bars indicate standard deviations. **, P < 0.01; ***, P < 0.001.