Fig 2.

Cps2F rhamnosyltransferase activity. Cps2E-containing membranes (3 μg of total protein) were incubated with membranes containing the indicated glycosyltransferases (3 μg of total protein) or membrane controls (MC; 3 μg of total protein from vector-only E. coli strain RC124) and UDP-[¹⁴C]Glc only or UDP-[¹⁴C]Glc and dTDP-Rha. Reaction mixtures (75-μl total volume) were incubated for 1 h at 10°C and contained 0.008% NP-40, 10 mM MnCl₂, 1 mM DTT, 0.075 μCi of UDP-[¹⁴C]Glc (300 mCi/mmol), and, where indicated, 0.1 mM dTDP-Rha. Glycolipids were extracted, hydrolyzed, and separated by TLC as described in Materials and Methods. Lanes show enzymes in the reaction mixtures: E, Cps2E; T, Cps2T; MC, membrane control; F, Cps2F; G, Cps2G; and I, Cps2I. Arrows indicate the locations of the origin (O) and the products formed by Cps2E, Cps2T, and Cps2F. The Cps2E product migrates equivalently to a [¹⁴C]glucose standard.