The <i>mar1Δ</i> mutant has an aberrant cell surface.

<p>(A) The <i>mar1Δ</i> strain has a capsule defect. Cells were incubated in capsule-inducing conditions (CO₂-independent tissue culture (TC) medium, 37°C) with shaking for 72 hours. Capsule was assessed by India ink counterstaining, followed by imaging. (B) The <i>mar1Δ</i> strain sheds capsule comparable to WT. Shed capsule polysaccharide was measured by blotting of culture supernatant, using an anti-GXM antibody to probe for capsule as described previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007126#ppat.1007126.ref030" target="_blank">30</a>]. (C) The <i>mar1Δ</i> mutant displays increased staining for exposed chitin and chitooligomers in tissue culture medium. Cells were incubated for 16–18 hours at 30°C in rich medium (YPD) or 37°C in tissue culture medium (TC), followed by staining with FITC-conjugated wheat germ agglutinin (WGA) for exposed chitin/chitooligomers, or calcofluor white (CFW) for total chitin. Stained cells were imaged by fluorescent microscopy with the appropriate filters. (D) Average fluorescence of at least 100 individual cells was measured using ImageJ/Fiji software. ****, p < 0.0001 as determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) The <i>mar1Δ</i> mutant displays increased chitosan staining. Cells were incubated for 16–18 hours at 30°C (YPD) or 37°C (TC), followed by staining with eosin Y (EY) for chitosan. Stained cells were imaged by fluorescent microscopy. (F) The <i>mar1Δ</i> cell wall does not have increased total chitin or chitosan. Cells were incubated for 16–18 hours at 37°C in TC medium, followed by cell wall isolation. Chitin and chitosan levels were quantified using a modified 3-methyl-2-benzothiazolinone hydrazine hydrochloride (MBTH) colorimetric assay as described previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007126#ppat.1007126.ref016" target="_blank">16</a>]. Data represent means of 3 independent cell wall isolations (n = 3 for each strain). Ns, not significant as determined by two-way ANOVA with Sidak’s multiple comparisons test.