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. 2021 May 13;4:570. doi: 10.1038/s42003-021-02102-4

Fig. 5. Heterogenous denitrification gene expression within individual microcolonies.

Fig. 5

a PAO1 NarK-GFP (top) or NirS-dsRed (bottom) were grown in separate particles in LB media saturated with 50% air and supplemented with 40 μM NO3–. Arrows indicate the particle edge; scale bars = 100 μm. For hundreds of microcolonies, the fraction expressing either NarK or NirS was quantified, and example microcolonies (right) illustrate ‘on’ fractions ranging from 0 to 0.90, with ‘on’ subregions noted by blue dotted lines. b Microcolony fraction expression for NarK-GFP and c NirS-dsRed, as a function of distance to the nearest particle edge. Shown are the median (blue crosses) and quartiles binned over 25 μm of radial particle space. Colonies were primarily ‘off’ in the aerated zone nearest bulk fluid (x < 100 μm) and primarily ‘on’ in anoxic conditions (x > 250 μm). In the aerobic zone, the median expression fraction of NarK-GFP is 3.1 × 10−5 (interquartile range 5.5 × 10−5) whereas the NirS-dsRed expression fraction is 2.7 × 10−5 (interquartile range 1.5 × 10−5). Expression of both genes are not significantly different from each other (Wilcoxon rank sum; p = 0.1). In the anaerobic zone, the median expression fraction of NarK-GFP and NirS-dsRed are 0.70 (interquartile 0.39) and 0.94, (interquartile 0.24), respectively. These anoxic fractional expressions are significantly different from each other (n1 = 1160, n2 = 1078, Wilcoxon rank sum; p = 2.2 × 10−32, w = 2724). The occurrence of heterogenous partially-on microcolonies reflects a sharp transition zone between presumptive aerobic and anoxic conditions (x ~ 100–250 μm). d Probability density functions for each reporter strain indicate stronger bimodality and higher binary expression for NirS-dsRed than for NarK-GFP. The distribution of NarK v. NirS expression are significantly different from each other (two sample Kolmogorov–Smirnov test; n1 = 1554, n2 = 1354 p = 1.2 × 10−40, Dn = 0.25) with significantly different medians (n1 = 1554, n2 = 1354, Wilcoxon rank sum; p = 1.9 × 10−33, w = 1.9 × 106).