ABSTRACTFive genes (cps2E,cps2T,cps2F,cps2G, andcps2I) are predicted to encode the glycosyltransferases responsible for synthesis of theStreptococcus pneumoniaeserotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinantS. pneumoniaeenzymes Cps2L, Cps2M, Cps2N, and Cps2O. Inin vitroassays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose.cps2T,cps2F, andcps2Ideletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2Gmutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2Imutants contained different secondary suppressor mutations incps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2Tmutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.