Fig. 2: Glycosyl composition analysis of GAC released from cell wall by the chemical treatments and purified by size-exclusion and ion-exchange chromatography.
From: PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides
a Size-exclusion chromatography of GAC released from GAS cell wall by mild acid hydrolysis or b deamination with HONO. Upper and lower panels show the composition of the BioGel P150 fractions of the individual GAC preparations. Prior to the chromatography, the extracted GAC material was reduced chemically with sodium borohydride. Fractions were analyzed for phosphate content by malachite green assay following digestion with perchloric acid. Rha and GlcNAc contents were measured by GC-MS as TMS-methyl-glycosides. GlcNAcitol and 2,5-anhydromannitol contents were estimated by GC-MS as alditol acetates. The chromatographic profiles are representative of more than three separate experiments. c Ion-exchange chromatography of GAC released from the GAS cell wall by mild acid (upper panel) and HONO (lower panel). Fractions containing the GAC material from (a, b) were pooled, concentrated, desalted by spin column, loaded onto DEAE-Toyopearl and eluted with a NaCl gradient (0–0.5 M). Fractions were analyzed for total sugar content by anthrone assay and total phosphate content by malachite green assay following perchloric acid digestion. The experiments were performed at least three times and yielded the same results. Data from one representative experiment are shown. d Glycosyl composition analysis of the GAC material purified by ion-exchange chromatography. The GAC material released by either deamination with HONO (GACNA) or mild acid hydrolysis (GACMA) was analyzed as shown in (c). Fractions unbound (flow-through) and bound (eluted with a NaCl gradient) to the DEAE column were pooled, concentrated, desalted by spin column and analyzed by GC-MS to determine the Rha, GlcNAc, 2,5-anhydromannitol and GlcNAcitol concentrations as described above. Malachite green assay was used to assay the phosphate concentration. The concentrations of GlcNAc, 2,5-anhydromannitol, GlcNAcitol and phosphate are expressed as moles per 30 moles of Rha. Columns and error bars represent the mean and S.D., respectively (n = 4 biologically independent replicates). Source data are provided as a Source data file.
