Nitrate (Nar) and nitrite (Nir) reductase expression in PAO1 were quantified with fluorescently tagged promoter fusions (Supplementary Fig. 1), green for the NarK subunit (NarK-GFP) and red for the NirS subunit (NirS-dsRed)35. As the first two steps in the denitrification pathway, expression of these genes indicates a metabolic switch from aerobic respiration with O2 to anaerobic respiration via denitrification. These PAO1 reporter strains were grown embedded within 3 mm diameter14 agarose particle discs held within a custom-built gastight millifluidic device (Fig. 1; Supplementary Methods), permitting continual lateral nutrient supply of rich nutrient media (with O2 and NO3–) from the bulk media via diffusion while maintaining a constant boundary condition at the particle periphery (Fig. 1a, b). This created an analog of a particle replete with dissolved organic nutrients whereby the agarose acted as an inert polymeric matrix rather than as a carbon source. Over ~24 h of growth in Luria-Bertani Broth (LB) media supplemented with NO3–, PAO1 formed densely packed stationary microcolonies (Supplementary Video 1), similar to its growth morphology in model alginate beads15,36 and agar blocks34. Seeding bacterial cells within a 1% agarose matrix allows colony expansion due only to growth (passive movement) and prevents active aerotactic and chemotactic movement driven by flagellar motility37. A subset of agarose particles contained biocompatible O2 nanosensors that could faithfully report O2 conditions varying dynamically over the scale of minutes (Supplementary Fig. 2).
Agarose disc particles seeded with P. aeruginosa PAO1 were incubated with media in glass devices that permit only lateral diffusion into particles. a Topview and b sideview illustrating nutrient diffusion into particles as occurred in c, a millifluidic device with directional flow of air-saturated fluid, or a ‘domino’ device held within sealed glass bottles. Scale bar ~1.5 cm. a inset, PAO1 cells grew as dense microcolonies. d Air saturation across four particles as determined from microscopic signal of fluorescent oxygen nanosensors. Despite aerobic bulk fluid surrounding the particles, cell growth created anoxia and its onset depended on PAO1 seeding density, ranging 104–106 cells mL−1 (70–7000 cells particle−1; the 105 mL−1 cell density was tested for duplicate particles). Gray shading indicates time range shown in e and asterisks coincide with noted timepoints in e. e The air saturation for radial profiles across a single particle (105 cells mL−1 seeding) during the cellular respiration-driven transition from air-saturated to anoxic conditions. Suboxia first developed in the core (x > 1000 μm) then spread to the particle periphery (x < 600 μm). The 200 μm region closest to the particle edge was not resolved in this experiment.
PAO1 readily established anoxia within particles maintained in air-saturated bulk fluid. To determine this, agarose particles containing O2 nanosensors were co-seeded with cells at 3 different densities (~70–7000 cells particle−1, or 104–106 cells mL−1) and allowed to grow over 24 h. Coinciding with bacterial growth, suboxia developed within particles, and the timing of onset depended on bacterial density (Fig. 1d). Specifically, suboxia manifested first in the highest initial cell density particle (106 cells mL−1), followed by the intermediate (105 cells mL−1) then the lowest cell density (104 cells mL−1), respectively. In each case, the transition from the onset of suboxia to maximal anoxia occurred over ~12.5 h. Within a particle, suboxia rose first in the core then subsequently expanded outward to the particle periphery, such that it lagged behind the core, yet also itself eventually became anoxic (Fig. 1e). Meanwhile, microcolony sizes were inversely related to the seeding density, i.e., anoxia across the particle was equally able to manifest via both fewer larger microcolonies or more numerous smaller microcolonies.
Reduced O2 concentrations alone are insufficient for active denitrification, however; NO3– availability is essential. In fully anoxic bulk conditions conducive for denitrification, microcolony growth and expression of denitrification genes by PAO1 reflected NO3– availability in the bulk and depended on radial distance within the particle. Particles embedded with PAO1 denitrification reporter strains (106 cells mL−1) were incubated in anoxic LB media supplemented with NO3– across 3 orders of magnitude (40, 400, or 4000 µM). Here too, only lateral diffusion of nutrients into the particles occurred (Fig. 1a; Supplementary Fig. 3). After 40 h of growth, denitrification gene expression and size were quantified for >103 microcolonies within each particle (e.g., Fig. 2). Both Nar and Nir expression were skewed toward the particle edge for lower bulk NO3– concentrations (Fig. 2b, c, Supplementary Fig. 4), indicative of a low NO3– flux reaching microcolonies in the particle core. In contrast, homogenous Nar expression occurred across all radial distances with 4 mM bulk NO3– (Fig. 2b), which suggests NO3– was nonlimiting for expression throughout the particle at such high bulk NO3–. Meanwhile, reduced Nir expression across the particle at 4 mM NO3– compared with lower NO3– treatments (Fig. 2c) indicates PAO1 preferentially reduced NO3– over NO2– throughout the particle under such NO3–-replete conditions. Bulk NO3– also influenced microcolony size distributions (Fig. 2b, c), with larger microcolonies manifesting near the periphery closer to the NO3– supply from the bulk fluid. Higher bulk NO3– stimulated greater microcolony growth in the particle core, i.e., at the center, the mean microcolony radius r was 6.4 ± 0.1 µm (sd) at 40 µM bulk NO3– but 12.7 ± 0.1 µm (sd) at 4 mM. Notably, under 4 mM bulk NO3–, size was skewed toward the particle periphery even while Nar expression was not, suggesting maximum cell-specific Nar expression rates occurred across all radial locations despite biomass production in the particle core remaining NO3– limited.
a Example images showing PAO1 microcolonies expressing NarK-GFP (nitrate reductase) after 40 h of growth in bulk anoxic media amended with 3 nitrate concentrations. Scale bar = 700 μm. b Mean relative expression of NarK-GFP for microcolonies, and mean radii of those microcolonies, shown in relationship to the radial distance to the nearest particle edge. The mean NarK-GFP expression for microcolonies in each particle (3–4 replicates) is indicated by a thin green line and the mean for all particles by a thick line. Similarly, an exponential model fit of microcolony radii for each particle is indicated by a thin black line and the mean for all particles by a thick line inset, Results for one example particle in a, illustrating the data for n microcolonies relative to the respective fits for those data. c Same as b but for reporter strain NirS-dsRed (nitrite reductase) expression in separate particles incubated in parallel.
Not only did particle denitrification readily occur in anoxic bulk fluid as expected, it was also prevalent among particles in oxygenated bulk fluid. Moreover, the spatiotemporal expression of Nar and Nir coincided with the development and microscale distribution of suboxic conditions. In these experiments, agarose particles were embedded with either NarK-GFP or NirS-dsRed PAO1 (106 cells mL−1) as previously, then incubated in partially oxygenated LB media (50% air saturation) containing NO3– (40 µM). A subset of particles was co-embedded with oxygen nanosensors to enable imaging of the O2 landscape. Minimal Nar and Nir expression were detected for the first 14 h of incubation. Strikingly, Nar and Nir activation then occurred as a wave that traced the initiation and expansion of suboxia (Fig. 3a–c). At the earliest stage of the wave (following 16–18 h of growth), expression increased substantially in the vicinity of the particle core (Fig. 3d), crafting a transition zone of only 390 ± 50 µm (sd) width that differentiated low- from high-expression microcolonies (Fig. 3e, Supplementary Fig. 5, Supplementary Fig. 6). This upregulation of expression coincided spatially with the development of anoxia in the core (Fig. 4, Supplementary Fig. 7; (x) > 1000 µm). During the next stage of the wave (t = 20–22 h), expression near the core elevated further while also initiating farther afield, widening the transition zone and reflecting expansion of suboxia toward the particle periphery. As the wave progressed (t = 24–28 h), expression diminished near the core, and consequently, the transition zone width contracted (200 ± 15 µm (sd)) even while its outermost edge neared to within 200 µm of the particle edge, where O2 conditions had become anoxic (Fig. 4). Finally, in the final stage of the wave (exemplified at t = 40 h), high expression was confined to a fine band distantly from the core, yet not directly at the edge, as evidenced by a very narrow transition zone (57 ± 13 µm (sd) width). Notably, at the last stage, O2 throughout the particle was elevated above the minimal observed levels, in contrast to previous stages (t = 24–28 h; Fig. 4, Supplementary Fig. 7). This may reflect anoxic acclimation by PAO1 to preferentially perform denitrification over oxidative respiration thereby permitting O2 to diffuse more readily through the particle matrix.
Particles seeded with cells were incubated in LB media saturated with 50% air and supplemented with 40 μM NO3–, then stopped at various timepoints. a Example images showing PAO1 microcolonies expressing NarK-GFP (nitrate reductase). Scale bar = 700 μm and applies to all images. Separate particles with the reporter strain for NirS-dsRed (nitrite reductase) were incubated in parallel. b Relative expression of NarK-GFP and c NirS-dsRed in particles. All microcolonies from 3–4 replicate particles per timepoint are represented. Nar expression initiates at the particle core while Nir expression initiates just proximal to it. For both Nar and Nir, maximal expression migrates outward creating a wave over subsequent timepoints. d Relative microcolony expression (as in b and c) shown as a microcolony’s radial location within the particle. An expression intensity fit was calculated as mean fits of each strain at each timepoint (Supplementary Note). Here the maximum fit value for each reporter strain at t = 24 h was set equal to 1, and shown are the expression values for all microcolonies relative to 1. e The radial location and range of the transition zone for each timepoint, approximated as the sloped region closest to the particle edge in the expression profile for NarK-GFP (green) and NirS-dsRed (red) (see also Supplementary Fig. 6). The midpoint of the slope (white diamonds) and the inflection point of the slope (circles) are indicated. NarK-GFP shows significantly higher expression between timepoints for midpoints (one-way ANOVA; F 1,35 = 27.9, p = 4.0 × 10−11) and for inflection points (one-way ANOVA; F 1,35 = 17.2, p = 9.5 × 10−9). NirS-dsRed also showed significantly higher expression between timepoints for midpoints (one-way ANOVA; F 1,35 = 14.9, p = 4.5 × 10−8) and for inflection points (one-way ANOVA; F 1,35 = 21.0, p = 1.0 × 10−9).
Two-dimensional profiles of air saturation were generated from particles co-seeded with the NarK-GFP reporter strain (nitrate reductase) and oxygen nanosensors. Scale bar = 700 μm. Separate analogous particles with the NirS-dsRed reporter strain (nitrite reductase) were incubated in parallel (Supplementary Fig. 7). For both, suboxic conditions develop in the particle core and then migrate outward toward the particle periphery over time.
Throughout the evolution of particle suboxia, Nar and Nir activity corresponded with the estimated spatiotemporal availability of O2 and NO3– in particles. Curve fits for microcolony fluorescence signal data were generated by assuming that O2 and NO3– concentrations at each radial location were the only controlling factors on expression, wherein O2 inhibits expression exponentially and NO3– has a directly proportional relationship, i.e., (Epropto {e}^{-k[{{mathrm{O}}}_{2}]}times [{mathrm{N}}{mathrm{{O}}}_{3}^{-}]). Approximating the distributions of O2 and NO3– in the particle as simple logistic functions, denitrification gene expression, E, is governed by the following relationship:
$$E=alpha times {e}^{-beta left[2{[{{mathrm{O}}}_{2}]}_{{mathrm{bulk}}}left(1-frac{1}{1+{e}^{-gamma x}}right)right]}times {[{mathrm{N}}{{mathrm{O}}}_{3}^{-}]}_{{mathrm{bulk}}}left(1-frac{1}{1+{e}^{-delta (x-varepsilon )}}right)$$
whereby α, β, γ, δ, and ε are fitting parameters and x is the distance to the particle edge. (Supplementary Note). The shape of this fit prediction matches the observed empirical data remarkably well (Fig. 3d, Supplementary Fig. 5), and the midpoints and inflection points from one timepoint to the next were significantly different (one-way ANOVA, F 1,8 = 27.9, p = 4.0 (times) 10−11 for Nar midpoints, F 1,8 = 14.9, p = 4.5 (times) 10−8 for Nir midpoints, F 1,8 = 17.2, p = 9.5 (times) 10−9 for Nar inflection points, and F 1,8 = 21.0, p = 1.0 (times) 10−9 for Nir inflection points). These fits illustrate how the response of PAO1 nar and nir gene expression reflects the balance between bacterial consumption and diffusion of O2 and NO3– from bulk surrounding fluid. In this manner, the fluorescence signal diminishes after achieving its peak and microcolonies remain small behind the advancing fluorescence wave while colonies continue to expand ahead of it (Supplementary Fig. 5, Supplementary Fig. 8). Nar was downregulated in the wake of the wave, causing microcolony expansion to slow or cease in the absence of respiration. Putatively, continued O2 and then NO3– uptake by large microcolonies at the periphery created growth limitation for microcolonies in their shadows farther from the bulk fluid source. Nir expression also advanced as a wave but interestingly created an annulus of maximal expression bounded by lower expression toward both the periphery and center of the particle. This expression pattern likely reflects localized production and utilization of NO2– within the particle interior. Exterior to the ring, ample NO3– from the bulk favored nitrate reductase, but interior to the ring, NO3– and NO2– were diffusion-limited.
Importantly, the heterogeneous distribution of Nar and Nir expression across the particle also manifested among PAO1 cells at the scale of individual microcolonies. High magnification colony-scale images near the particle periphery and in the transition zone revealed a common phenotype reflecting the overall expression across the particle whereby the core of a single colony is expressive but the outer margin is not. As quantified for >103 microcolonies per particle, the subregion expressing Nar or Nir (i.e., “on”) varied with distance from the particle edge (Fig. 5). A thin radial zone within the particle (distance from the particle edge, x ~90–240 µm) harbored high heterogeneity with colonies ranging from 0–100% as shown (Fig. 5b, c). This narrow transition zone aligned with that quantified at lower magnification (Figs. 3, 4), indicative of a sharp transition from O2 to nitrate- and nitrite-driven respiration. In the flanking region exterior to this zone (x < 90 µm), Nar and Nir in microcolonies were uniformly off. In the region interior to this zone (x > 240 µm), Nar was predominately on (median colony fraction expression = 0.78 ± 0.40 interquartile range; Fig. 5b) and Nir was almost completely on (median colony fraction expression = 0.95 ± 0.25 interquartile range; Fig. 5c). These expression characteristics resulted in a significantly stronger population bimodality for Nir than for Nar (Fig. 5d); Kolmogorov–Smirnov nonparametric test for probability distribution similarity, n1 = 1554, n2 = 1354, p = 1.2 (times) 10−40, Dn = 0.25. Akin to the annular feature observed at lower magnification (Fig. 3), this binary Nir expression likely reflects localized endogenous production and utilization of NO2–. Since NO2– is not continuously supplied from bulk media via lateral external diffusion like NO3–, microcolonies in the interior use Nar to produce NO2–, which is then preferentially consumed via Nir within each microcolony as the next most available oxidant for generating energy.
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).
Respiratory shading by exterior colonies and cells was a key emergent feature among microcolonies within the agarose particles, occurring in a fractal-like geometry. At the particle-scale ((R) ~1500 µm), nutrient consumption by microcolonies at the periphery prevented uptake by those at the interior; similarly, at the microcolony-scale (r ~25 µm), cells at the margin prevented uptake by those at the center, with consumption generating gradients of uptake flux at both scales. Shading did not occur for microcolonies in the particle core through the early stages of suboxia, as Nar and Nir expression were absent and microcolony size distribution was uniform over the first 14 h (Fig. 3, Supplementary Fig. 8). Since microcolonies were small over this stage, the O2 flux to the center outpaced aerobic respiration, and cell biomass at the particle-scale had not yet substantially diminished diffusive O2 availability. Then, owing to cell growth and the onset of shading, the O2 concentrations decreased rapidly over ~2 h (Fig. 4). As such, respiratory shading should be diminished when the density of microcolonies is lower. We tested this hypothesis with particles seeded at very low density (~102 cells mL−1) resulting in 1–6 microcolonies particle−1. Indeed, after 40 h of growth, the resultant microcolonies were quite large (r = 62 ± 11 µm (sd)); Supplementary Fig. 9) regardless of radial location within the particle, reflecting low intercolony competition for oxidants that readily diffused throughout the particle. While here respiratory shading across scales occurred for a clonal population, natural multispecies communities may additionally distribute functional roles among diverse taxa, e.g., anammox aggregates spatially differentiate such that aerotolerant species encase the outer perimeter to respire O2, shielding Planctomycetes to perform oxygen-inhibited anammox in the interior10.
Source: Ecology - nature.com
