Denitrification in low oxic environments increases the accumulation of nitrogen oxide intermediates and modulates the evolutionary potential of microbial populations

Abstract

Denitrification in oxic environments occurs when a microorganism uses nitrogen oxides as terminal electron acceptors even though oxygen is available. While this phenomenon is well-established, its consequences on ecological and evolutionary processes remain poorly understood. We hypothesize here that denitrification in oxic environments can modify the accumulation profiles of nitrogen oxide intermediates with cascading effects on the evolutionary potentials of denitrifying microorganisms. To test this, we performed laboratory experiments with Paracoccus denitrificans and complemented them with individual-based computational modelling. We found that denitrification in low oxic environments significantly increases the accumulation of nitrite and nitric oxide. We further found that the increased accumulation of these intermediates has a negative effect on growth at low pH. Finally, we found that the increased negative effect at low pH increases the number of individuals that contribute to surface-associated growth. This increases the amount of genetic diversity that is preserved from the initial population, thus increasing the number of genetic targets for natural selection to act upon and resulting in higher evolutionary potentials. Together, our data highlight that denitrification in low oxic environments can affect the ecological processes and evolutionary potentials of denitrifying microorganisms by modifying the accumulation of nitrogen oxide intermediates.

FIGURE 1

Dynamics of nitrogen oxide production and reduction by strain PD1222 in oxic and anoxic liquid batch cultures. (A) ¹⁵NO₃ ⁻ reduction in anoxic (upper) and oxic (lower) liquid cultures at pH 7.5. Data points are averages of three independent biological replicates and the shaded regions are ± one standard deviation. Symbols and colours are for nitrate (square, yellow), nitrite (circle, yellow‐green), nitric oxide (upper triangle, green), nitrous oxide (lower triangle, blue) and nitrogen gas (diamond, purple). For incubations in oxic liquid cultures, the oxygen concentrations in the headspace were ~10% (v/v) throughout the duration of the experiment as determined by gas chromatography (Figure S1). (B) Single‐cell analysis of the activities of the nar, nir, nor and nos promoters quantified by flow cytometry. Each promoter‐reporter strain was incubated individually and the promoter activity levels were measured for 100,000 cells. The normalized count is the number of events at each fluorescence intensity normalized by the maximum number of events. Lines and colours are for O₂(+) NO₃ ⁻(−) (dotted line, yellow), O₂(+) NO₃ ⁻(+) (solid line, green) and O₂(−) NO₃ ⁻(+) (dashed line, purple). Data were taken after 10 h of incubation at 30°C when cells were in the exponential growth phase.

FIGURE 2

Effect of pH on growth in oxic and anoxic environments. (A) The maximum specific growth rate of strain PD1222 (μ _max) in oxic or anoxic liquid batch cultures amended with or without nitrate. The μ _max was calculated from five consecutive data points coinciding with the most rapid period of growth for each individual culture. Each data point is the measurement for an independent experimental replicate (n = 3). The middle horizontal line is the mean and the upper and lower horizontal lines are ± one standard deviation. Asterisks indicate statistically significant differences in the means of the labelled groups calculated with two‐sample two‐sided t tests (ns, no significant difference; *, p < 0.05). (B) Colony growth as a function of the nitrate concentration. Colonies were grown on LB agar plates (one colony per plate) at pH 6.5 or 7.5 and incubated in an oxic or anoxic atmosphere for 5 days at 30°C. Data points are the means and error bars are ± one standard deviation from three independent biological replicates. LB, lysogeny broth.

FIGURE 3

Effect of pH on spatial intermixing during colony growth in an oxic or anoxic atmosphere. (A) Representative CLSM images of quartiles from colonies growing on LB agar plates amended with or without 50 mM nitrate in an oxic or anoxic atmosphere. The colonies consist of two subpopulations of strain PD1222, where one expresses red (appears magenta here) while the other expresses green fluorescent protein. The images were taken after 5 days of incubation at 30°C. (B) The intermixing index of the two subpopulations at the final colony periphery (50 μm circular band positioned at the colony periphery). Each data point is the measurement for an independent experimental replicate (n = 5). The middle horizontal line is the mean and the upper and lower horizontal lines are ± one standard deviation. Asterisks indicate statistically significant differences in the means of the labelled groups calculated with two‐sample two‐sided t tests (***, p < 0.001; *****, p < 0.00001). CLSM, confocal laser scanning microscopy; LB, lysogeny broth.

FIGURE 4

Effect of interactions between and within subpopulations and the magnitude of intermediate toxicity on spatial intermixing during colony growth. (A) The four models that we investigated in this study. We simulated growth inhibition by producing and receiving signals (intermediates) within cells of the same colour (I), as well as cells with different colours (II). Additionally, we incorporate a detoxification factor by producing and receiving signals within cells of the same colour (III) and cells with different colours (IV), both based on the inhibition process itself. (B) Representative simulations for the four different models as a function of the strength of the toxic effect of the intermediate. (C) The intermixing index at the final colony periphery for the simulation results. For (B) and (C), the toxic effects were set by 1/K _tox and the detox factor (consumption of the intermediate) was consistent across all simulations (K _detox = 0.001). Data points are the averages from five independent simulations and the shaded regions are ± one standard deviation.