Bistability in a Metabolic Network Underpins the De Novo Evolution of Colony Switching in Pseudomonas fluorescens
Fig 5
Changes in pyrimidine biosynthetic pathway flux alter proportion of Cap+ cells.
(A) Addition of 2 mM uracil to 1B4 growth medium causes a reduction in the proportion of Cap+ cells (two-sample t test, p < 0.001); addition of 2 mM arginine has no effect (two-sample t test, p > 0.1). The addition of both uracil and arginine reduces capsulation levels below those seen with uracil alone, possibly as a result of complex regulatory interactions. Each point is the mean of five replicates, and error bars show +/- 1 SE. Asterisks indicate a significant difference between each genotype in the presence and absence of uracil. (B) Seven independently isolated switcher genotypes (see Table 1) show varying proportions of Cap+ cells. Addition of 2 mM uracil significantly reduces Cap+ levels in carB switchers (two-sample t tests, p < 0.05) but has no effect on Cap+ in the pyrH switcher (Re1_4, p > 0.1) or 1A4 (p > 0.1). Bars are means of five replicates, and error bars show +/- 1 SE. (C–D) The proportion of Cap+ cells when overexpressing carB, pyrH, ndk, galU, or pyrG in 1B4 (C) and SBW25 (D). “pSX” bars are means of 18 replicates (six replicates from each of three biological replicates), while others are means of nine or six replicates (three replicates from each of two or three biological replicates). Significance tests are for differences between the relevant genotype and empty pSX. (E) Light microscope image of 1B4 + pSX (left) and 1B4 + pSX-galU (right). Overexpression of galU results in cell chains. Bar ~5 μm. The saturation of the images was altered. (F) Fluorescence microscope image of 1B4 (left) and JG49 (right) cells stained with Fluorescent Brightener 28 (cellulose binding dye). Inactivation of galU gives short cells. Bar ~5 μm. The exposure of the second image was altered.