Fig 7.

Cps2E glycosyltransferase activities and CPS in S. pneumoniae mutants containing suppressor mutations in the absence of the original deletion mutations. (A) Glycosyltransferase assays were performed as described for Fig. 6A and in Materials and Methods, except that reactions were for 2 h and contained 0.125 μCi of UDP-[³H]Glc. For the Cps2E⁺ parent (JK903), membranes were assayed at 0.5 and 1 μg of total protein, which was in the linear range. All others were assayed at 2, 4, and 10 μg of total protein. Values are the means ± standard errors for the two or three membrane concentrations. (B) Cps2E and PspA proteins in membranes used for glycosyltransferase assays for panel A. Experiments were performed as described for Fig, 6B, loading 2 and 4 μg of total membrane protein in separate blots. Values are the means of the two blots, normalized to the Cps2E⁺ strain. (C) CPS immunoblot of strains. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antiserum to serotype 2 CPS, as described previously (56). PP, protoplast (membrane-containing) fraction; CW, cell wall fraction. Strains: Cps2E⁺, JK903; V196G, JK906; H258R, JK907; and Cps2E⁻, KA1522.