Fig 6.

Cps2E glycosyltransferase activities in S. pneumoniae mutants containing original deletion mutations and cps2E suppressor mutations. (A) Membranes were used to measure incorporation of [³H]Glc from UDP-[³H]Glc to an organically soluble product. Results are the means ± standard errors for two independent membrane preparations assayed in separate experiments (with two replicates per sample). Samples were assayed using 0.2 and 0.5 μg of total membrane protein, which was in the linear range for the parent Cps2E activity. Activities are expressed as CPM/μg of total membrane protein. (B) Cps2E protein in membranes used for glycosyltransferase assays of panel A. Samples containing 5 μg of total membrane protein were separated by SDS-12% PAGE, transferred to a nitrocellulose membrane, and probed with polyclonal antiserum to Cps2E (25). Relative protein levels were determined by densitometry and normalized to that of the Cps2E⁺ parent (D39). The values below the blot represent the means from three independent membrane preparations, two of which were used for the glycosyltransferase assays in panel A. A representative blot is shown. All samples were run on the same gel/blot; a replicate sample of M338I was spliced out of the image. Strains: Cps2E⁻, KA1522; Cps2E⁺, D39; H258R, DJ916; A162S, DJ921; D167Y, BX547; L200F, BX552; V196G, BX605; M338I, DJ918; and R307S, DJ919.