FIGURE 4.
Site-directed mutagenesis identified amino acid residues crucial for enzymatic activity. A, in vivo activity of the mutant capsule polymerases (as indicated) was tested with Nm strain WUE171 as recipient. Mutant genes were introduced by homologous recombination and capsule production was quantified in a whole cell ELISA. Values are given as means of three independent experiments in relationship to the wild-type recipient (100%). The capsule-deficient strain WUE 2661-Δ CPS served as a negative control. B, display of recombinant purified wild-type and mutant forms of SiaDW-135 in a Coomassie Blue-stained 10% SDS-PAGE. C, activity of recombinant purified wild-type and mutant capsule polymerases (proteins as shown in B) was determined in a radioactive incorporation assay with CPS(h) as acceptor. Of note, combination of the inactive mutants E307A and S972A in one reaction mixture restored activity to 70% of wild-type.