Reentrant DNA shells tune polyphosphate condensate size

Abstract

The ancient, inorganic biopolymer polyphosphate (polyP) occurs in all three domains of life and affects myriad cellular processes. An intriguing feature of polyP is its frequent proximity to chromatin, and in the case of many bacteria, its occurrence in the form of magnesium-enriched condensates embedded in the nucleoid, particularly in response to stress. The physical basis of the interaction between polyP and DNA, two fundamental anionic biopolymers, and the resulting effects on the organization of both the nucleoid and polyP condensates remain poorly understood. Given the essential role of magnesium ions in the coordination of polymeric phosphate species, we hypothesized that a minimal system of polyP, magnesium ions, and DNA (polyP-Mg²⁺-DNA) would capture key features of the interplay between the condensates and bacterial chromatin. We find that DNA can profoundly affect polyP-Mg²⁺ coacervation even at concentrations several orders of magnitude lower than found in the cell. The DNA forms shells around polyP-Mg²⁺ condensates and these shells show reentrant behavior, primarily forming in the concentration range close to polyP-Mg²⁺ charge neutralization. This surface association tunes both condensate size and DNA morphology in a manner dependent on DNA properties, including length and concentration. Our work identifies three components that could form the basis of a central and tunable interaction hub that interfaces with cellular interactors. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as the potential capacity of these mesoscale assemblies to remodel chromatin in response to diverse stressors at different length and time scales.

Figure 1.. PolyP-Mg²⁺ coacervates exhibit reentrant phase transition and are dynamic.

a Phase boundary curve for polyP-Mg²⁺ coacervates as determined by the solution turbidity ([polyP] = 1 mg/mL, 50mM HEPES-NaOH, pH 7.5). b Representative confocal fluorescence microscopy images of polyP-Mg²⁺ mixtures that correspond to 100mM MgCl₂ of the phase diagram. Images represent fusion of polyP-Mg²⁺ coacervates ([polyP] = 1 mg/mL, polyP-AF647 = 10% polyP, [Mg²⁺] = 100mM, 50mM HEPES-NaOH, pH 7.5; scale bar = 2μm). A movie showing a larger field of view of droplet fusion is available (SI Movie 1). c PolyP-Mg²⁺ coacervates recover to around 80% 50 minutes after photobleaching in Fluorescence Recovery After Photobleaching (FRAP) experiments (d_{bleached ROI} = 1.7μm, d_droplets = 8.4-8.5μm, n = 4). Representative images showing recovery at select timepoints are inset (scale bar = 2μm).

Figure 2.. DNA interacts with the surface of PolyP-Mg²⁺ coacervates and forms shells that exhibit reentrant behavior.

a Confocal fluorescence microscopy of polyP-Mg²⁺ coacervates and pUC19 (2.7kb) plasmid under different MgCl₂ conditions. DNA forms a shell on the surface of PolyP-Mg²⁺ coacervates within a Mg²⁺ concentration range of 50-200mM. Three channels corresponding to Alexa Fluor 647 (P700), YOYO-1 (DNA) and the merge of these two channels are shown ([polyP] = 1 mg/mL, polyP-AF647 = 10% polyP, 50mM HEPES-NaOH, pH 7.5; scale bar = 5 μm; P700, blue; DNA, yellow). b Intensity profiles across PolyP-Mg²⁺-DNA coacervates corresponding to [Mg²⁺]=100mM (other conditions described in panel a) showing the surface localization of DNA (scale bar = 5μm). c Confocal fluorescence microscopy images at different time-points of polyP-Mg²⁺-DNA coacervate fusion (for conditions described in b, scale bar = 2 μm). See Fig S2C for the full frame fusion and SI Movie 3 for a wider field of view video.

Figure 3.. Cryo-electron tomography.

(a-d) Representative tomographic slices of PolyP condensates incubated with different types of DNA. Red arrow highlights the dense edge of polyP, cyan arrows highlight DNA and yellow arrows highlight the dense edge+DNA surface (scale bar = 100 nm). (e-h) 3-dimensional renderings of tomograms shown in panels a-d, respectively. The dense edge of PolyP condensate is shown in red, the dense edge+DNA are shown in yellow, and DNAs are shown in cyan.

Figure 4.. Effect of DNA concentration and length on PolyP-Mg²⁺ size distribution and average droplet size.

a Representative sample confocal images of polyP-Mg²⁺ droplets given different DNA concentration (top & middle) and length (top & bottom) ([polyP] = 1mg/mL with ~10% P700-AF647, [DNA] = 10 μg/mL or 100 μg/mL, YOYO1 = 1μM, 50mM HEPES, scale bar = 2μm). Representative confocal images for each of the lengths tested and select concentrations in confocal and widefield are available in SI Figs 8–9, 13–14. b Scatter plot showing the average of mean droplet size across three experiments with respect to varied DNA concentrations (error bars = SD of mean diameters of each experiment) . At 30 μg/mL DNA, the average droplet size begins to decrease. c Scatter plot showing average droplet size as a function of time for three representative DNA concentrations. d Scatter plot showing the average of mean droplet size across three experiments with respect to different DNA lengths (error bars = SD of mean diameters of each experiment). DNA length used include circular plasmids of length 2.7kb (pUC19 used for panel a-c), 5kb, 8kb, 10kb, 15kb, 20kb, 24kb, 30kb and commercially available phage DNAs Lambda (49kb) and T4 (166kb). At longer DNA lengths, condensate size decreases. e Scatter plot showing average droplet size as a function of time for three representative DNA lengths.

Figure 5.. A framework for understanding polyP-chromatin interactions.

Left: Cryo-ET of nitrogen-starved P. aeruginosa cells with nucleoid region (ribosome depleted) delineated with dashed magenta line, polyphosphate granules shown as green spheres (image: Racki et al., 2017). Right: In this study we have developed a three-component polyP-Mg²⁺-DNA system (interactions represented by black arrows) which is a fundamental physicochemical interaction unit underlying the functional coupling between polyP granules and chromatin in cells. Red dashed arrow represents repulsive interactions between the polyanions, and polyvalent cationic species and proteinaceous partners, including NAPs, represent factors that mediate this interaction. Our results highlight the tunable nature of this minimal system, showing that DNA interacts with and forms reentrant shells around polyP-Mg²⁺ condensates, and modulates condensate size in a DNA length and concentration dependent manner. Future studies building on this framework to include relevant proteins such as nucleoid associated proteins (NAPs) known to associate with polyP in vivo (Hfq and AlgP, for example) are needed to understand how polyP affects chromatin structure and function in cells (gray arrows).