Figure 4

Biochemical analysis of the mp65Δ mutant. (A) Localization of β-glucan after glutaraldehyde fixation in the mp65Δ mutant, determined by Immunoelectron microscopy (IEM). This method of preparation avoids the use of osmium tetroxide and uranyl acetate and permits good cell preservation of the wild type (wt: Panel 1), mp65Δ mutant (hom: Panel 2) and revertant (rev: Panel 3) strains following post embedding labeling with the mAb 1E12 and followed by gold-labeled secondary antibody. The magnification bar corresponds to 0.5 μm. For more details, see the Methods section. (B) Expression of β-glucan in the mp65Δ mutant, as determined by flow cytometry. The β-glucan content is expressed in arbitrary units (A.U.) and was calculated as the ratio of the labeled samples on the mean fluorescence channel (mfc) of the corresponding negative controls. Each column represents the mean of 3 experiments, with the bars representing standard deviations (Mann-Whitney U test was used for statistical assessment). (C) Quantitative analysis of the cell wall sugar content by HPIC. The determination of the three principal cell wall polysaccharides (chitin, glucan and mannan) was performed, after extraction with acid hydrolysis, using HPIC with a Dionex Bio-LC system. The results are the mean of 3 independent experiments. The bars indicate standard deviations.