Skip to main content
. 2019 Oct 28;17(10):e3000379. doi: 10.1371/journal.pbio.3000379

Fig 2. Reversible selection of distinct colony morphotypes with opposing motility phenotypes.

Fig 2

(A) C. difficile R20291 liquid culture was spotted on BHIS-1.8% agar and grown for 48 hours to allow spreading growth (panel 1). Bacteria collected from the center of a spot yield mostly smooth colonies (panel 2), whereas bacteria from the edge yield almost exclusively rough colonies (panel 3). (B) Rough and smooth colony isolates obtained as in (A) were passaged in 0.5× BHIS-0.3% agar (panel 4), and samples were collected from the edge of motile growth. Both rough and smooth colony isolates only gave rise to smooth colonies (panels 5 and 6). Shown are representative images of colonies from four independent experiments. All images were taken at ×2 magnification. (C) Quantification of colony morphology for rough and smooth colony isolates after passage on a BHIS agar surface (surf) as in (A) or in motility medium (swim) as in (B), with samples collected from the edges of growth. R, S indicate rough and smooth starting inocula, respectively. Rough and smooth isolates were also directly passaged through BHIS broth medium or struck on a BHIS agar plate and then enumerated. Symbols indicate individual values from three to four independent experiments, and bars indicate means and standard deviations. (D, E) C. difficile R20291, a TFP-null control (pilB1), and rough and smooth colony isolates were assayed for surface motility on BHIS-1.8% agar. (D) A representative of four experiments is shown. (E) Surface motility was quantified by measuring the diameter of growth after 48 hours. (F, G) C. difficile R20291, a nonmotile control (sigD), and rough and smooth colony isolates were assayed for swimming motility in 0.5× BHIS-0.3% agar. (F) A representative of four experiments is shown. (G) Swimming motility was quantified by measuring the diameter of growth after 48 hours. (E,G) **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA and Tukey’s posttest. Data can be found in supplemental file S1 Data. BHIS, brain heart infusion plus yeast; R, rough; S, smooth; sigD, sigma factor D gene; surf, surface; TFP, type IV pilus; WT, wild-type.