FIGURE 3.

tarM disruption leads to altered WTA that lacks the α-GlcNAc residues. A, PAGE analysis of WTA is shown. The indicated WTA samples (100 nmol of phosphate/lane) were resolved in polyacrylamide gels and visualized by Alcian blue silver staining. The PAGE migration behaviors of WTA from wild type (lane 1) and tarM-complemented mutant strain (lane 3) were almost the same; the tarM mutant WTA migrated much faster (lane 2) and exhibited weaker staining with Alcian blue-silver solution. B, purified WTA spotted on a nitrocellulose membrane was incubated with GlcNAc binding biotinylated WGA and visualized with streptavidin-HRP. In contrast to wild-type WTA, mutant WTA could not be stained with WGA, indicating the loss of GlcNAc from the K6 WTA backbone. C, ¹H NMR spectrum of WTA from RN4220 derivatives is shown. The two characteristic signals for GlcNAc at δ 5.07 and 2.08 (anomeric proton of α-GlcN and methyl group of NAc, respectively) were found and are indicated in the spectra of wild type and the tarM complement (K6/tarM) WTA but were missing in the spectrum of mutant K6 WTA, indicating that disruption of tarM in the mutant K6 leads to the loss of the α-GlcNAc from WTA and that α-GlcNAc modification could be restored by complementation with a plasmid encoding tarM.