FIG 3.

Glucose utilization through the pentose phosphate pathway is essential in the smooth phase variants of str. R20291. (A) Gene and reaction essentiality results for glycolysis and the pentose phosphate pathway across both the rough and smooth phase variant context-specific models. Components were deemed essential if models failed to generate <1% of optimal biomass flux. (B and C) Colony morphologies resulting from smooth and rough variants of C. difficile str. R20291 grown on either BHI or BDM ± glucose (2 mg/ml) after 48 h of growth (phase contrast 20/40, ×4 magnification). Defined medium colonies were then subcultured onto BHI medium for an additional 24 h as indicated. Increased colony perimeter was found to be the defining characteristic of the rough colony morphology. This feature was quantified for multiple colonies under each permutation of colony variant and growth medium (n ≥ 4). (D) Colony perimeter for smooth and rough progenitor colony variants grown on BHIS (P value < 0.001). (E and F) Smooth (E) or rough (F) colony variant perimeter during subculture onto each of the BDM carbon source medium formulations (P values < 0.05). Significant differences determined by Wilcoxon rank sum test with Benjamini-Hochberg correction when necessary.