Formation and functionalization of membraneless compartments in Escherichia coli

Abstract

Membraneless organelles formed by liquid–liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid–liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.

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Fig. 1: Formation of cellular compartments upon spider silk expression in E. coli.
Fig. 2: Formation of cellular compartments via LLPS.
Fig. 3: Functionalization of cellular compartments via targeted colocalization of cargo proteins.
Fig. 4: Functionalized intracellular compartments catalyze a cascade of two reactions.

Data availability

All data that support the findings of this study are available within the paper, the supplementary information and the source data or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

Financial support was provided by the National Natural Science Foundation of China (grant no. 21674061 to X.-X.X, grant no. 21406138 to Z.-G.Q. and grant no. 31470216 to X.-X.X) and the National Key Research and Development Program of the Ministry of Science and Technology of China (grant no. 2016YFE0204400 to X.-X.X). X.-X.X. acknowledges the Program for Professor of Special Appointment at Shanghai Institutions of Higher Learning. We thank H. Tang (Shanghai Jiao Tong University) for his generous gift of P. putida KT2440 genomic DNA.

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Contributions

X.-X.X., Z.-G.Q. and S.-P.W. conceived the project. Z.-G.Q. and X.-X.X. designed the research, analyzed the data and wrote the manuscript. S.-P.W. performed plasmid construction, cell imaging, droplets fusion and photobleaching experiments; analyzed the data; and wrote the draft. C.-F.H. performed recombinant protein production, purification and reconstitution experiments. F.P. constructed recombinant strains for 1,3-diaminopropane biosynthesis, performed urea perturbation experiments and aided plasmid construction. M.-T.C. performed 1,3-diaminopropane production experiments and aided plasmid construction. S.Y.L. analyzed the data and revised the manuscript.

Corresponding author

Correspondence to Xiao-Xia Xia.

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Competing interests

X.-X.X., S.-P.W., M.-T.C., F.P. and Z.-G.Q. have filed a patent application (‘Construction and applications of membraneless organelles in prokaryotic Escherichia coli’, Chinese Patent Application No. 201910929503.X) on the basis of this contribution.

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Extended data

Extended Data Fig. 1 Formation of compartments was adjustable by varying the spidroin length.

a, Confocal images of E. coli cells expressing I4-GFP and I32-GFP. Cell samples were taken 6 or 12 h after induction at 30 °C for protein production. Scale bars, 5 µm. b, SDS-PAGE analysis of I4-GFP and I32-GFP expression. Lanes: M, molecular weight marker; W, whole cell extract; S, supernatant of whole cell extract; P, pellets of whole cell extract. The induced cells were taken at 6 h after exposure to the inducer IPTG for silk gene expression, and the uninduced cells were included as a control. The arrows indicate the respective target proteins, and uncropped gel is displayed in Source Data. Data in a, b are representative of n = 3 independent experiments. Source data

Extended Data Fig. 2 Formation of compartments was adjustable by modulating the induction temperature and duration of post-induction.

a, Confocal images of the cellular compartments in E. coli cells expressing silk I16-GFP at diverse temperatures. Cell samples were taken 6 or 12 h after inducing protein expression. Scale bar in the upper left panel, 8 µm; scale bars in the other panels, 5 µm. Note that clear compartmentalization was observed for the cells induced at 16 °C for 12 h, but not at 6 h. The condensates do not form as rapidly as at higher temperatures, which might be due to that lower temperature leads to the lower rate of protein expression. Data are representative of n = 3 independent experiments. b, Specific fluorescence per OD600 (arbitrary units, a.u.) of the cells with evenly dispersed or compartmented I16-GFP at the indicated induction temperature for 6 h. Data are presented as mean ± s.d. of n = 3 biologically independent samples, with individual data points shown as black dots. c, SDS-PAGE analysis of the cellular protein extracts from the cells described in b. The uninduced cells were included as a control, and uncropped gel is displayed in Source Data. Data in b and c are representative of n = 2 independent experiments. Source data

Extended Data Fig. 3 Characterization of the dynamic compartments within E. coli cells by urea perturbation.

a, Images of the I16-GFP expressing cells upon treatment with varying levels of urea. The recombinant E. coli cells were induced with 200 µM IPTG at 30 °C for 6 h, harvested, and resuspended in PBS buffer without and with the addition of urea. Following incubation at 25 °C for 20 min, the cells were imaged. Scale bars, 2.5 µm. b, Relative fluorescence of the cells upon treatment with varying levels of urea. Data are presented as mean ± s.d. of n = 3 biologically independent samples, with individual data points shown as black dots. The asterisks represent statistical significance with P‐value < 0.05 based on one-way analysis of variance (ANOVA). Data in a, b are representative of n = 3 independent experiments. Note that the data reveals disassembly of the I16-GFP condensates for the urea-treated cells in a dose-dependent manner, with the cell morphology and fluorescence well retained. These results indicate that the molecular interactions of forming spidroin condensates are relatively weak and easy to be broken with the hydrogen bonding disruptor (urea), thus offering a new line of evidence proving the condensates are liquid-like.

Extended Data Fig. 4 TEM analyses revealed the formation of silk-based compartments in the cytoplasm.

a, TEM image of fixed control cells without silk I16 expression. TEM images of microtome slices of E. coli cells without (b) and with expression of silk I16 protein (c). The cell samples were taken at 6 h after exposure to the inducer IPTG for silk gene expression at 37 °C. Note that clear compartmentalization was observed in the silk-expressing cells, but not in the control, non-expressing cells. Data in ac are representative of n = 3 independent experiments.

Extended Data Fig. 5 Effects of formation of compartments on the cell growth and morphology.

The growth (a), length (b), and width (c) of E. coli cells expressing recombinant major ampullate spidroin 1 (I16) and resilin-like protein (R32), and the control cells harboring empty vector. All cells were induced with 200 µM IPTG at 30 °C, and cell OD600 measured at the indicated time after induction. For evaluation of cell size, the cell samples were taken at 6 h post-induction for bright field light microscopy analyses. The data in a are presented as mean ± s.d. of n = 3 biologically independent samples, with individual data points shown. The data in b and c represent the average of 20 cells and error bars correspond to the standard deviations. Statistical significance was determined using one-way ANOVA for P values. Note that neither the cell length nor width was significantly altered upon protein production. Data in a-c are representative of n = 2 independent experiments.

Extended Data Fig. 6 In vitro reconstitution of the purified proteins with macromolecular crowder dextran-70.

a, Reconstituted mixtures containing 50 mg/ml dextran-70 and I16-GFP were visualized by bright-field light microscopy, and spun by centrifugation to show separation into two liquid phases. Included as controls were reconstituted GFP at a final concentration of 50 mg/ml (no phase separation) or 75 mg/ml (aggregation). The red arrow indicates the coexistence of two liquid phases, whereas the black indicates the coexistence of a liquid phase and a solid phase of GFP aggregates. b, Reconstituted mixtures containing dextran-70 and each of the indicated proteins at 50 mg/ml were visualized by light microscopy, and spun by centrifugation to show separation into two liquid phases. Scale bars in a, b, 100 µm. Data in a, b are representative of n = 2 independent experiments.

Extended Data Fig. 7 Liquid-liquid phase separation (LLPS) of the spider silk I16 protein in vitro.

a, Reconstitution of the purified I16 protein at varying final concentrations with the macromolecular crowding agent, dextran-70 at room temperature. The turbid mixtures were spun by centrifugation to show the co-existence of two liquid phases, as indicated by the red arrows in the photograph. b, Turbidity profiles of silk I16 as a function of protein concentration in the reconstituted mixtures at diverse temperatures. Data are presented as mean ± s.d. of n = 3 biologically independent samples, with individual data points also shown. Note that LLPS of I16 occurred at concentrations beyond critical values of approximately 6 mg/ml. c, Reconstituted mixtures containing I16 and macromolecular crowder Ficoll 70 were visualized by light microscopy, and spun to show separation into two liquid phases. Recombinant GFP was also included as a control. Scale bars in c, 100 µm. Data in ac are representative of n = 2 independent experiments.

Extended Data Fig. 8 Reversible liquid-liquid phase separation (LLPS) of the spider silk I16 protein upon dilution in vitro.

Turbidity of the reconstituted mixtures with I16 and dextran 70 at the indicated concentrations was monitored by measuring optical density at 350 nm (OD350). Data are presented as mean ± s.d. of n = 3 biologically independent samples (individual data points also shown), and are representative of n = 2 independent experiments.

Extended Data Fig. 9 Formation of cellular compartments in E. coli cells expressing recombinant resilin-like protein (R32) and major ampullate spidroin 2 (II16).

To visualize expression, both R32 and II16 were fused N-terminally to GFP. E. coli cells harboring the corresponding plasmid constructs were induced with 200 µM IPTG at 30 °C, and cell samples were taken at 6 h post-induction for fluorescence and bright field microscopy analyses. Scale bars, 5 µm. Data are representative of n = 3 independent experiments.

Extended Data Fig. 10 Functional membraneless compartments for bioproduction of nanoparticles (NPs) within E. coli cells.

a, b, and c, TEM images of individual cells expressing I16-MT for the production of Se NPs (left panel). The dotted boxes (left panels) are shown at a higher magnification at the right (scale bars, 200 nm). Data in ac are representative of n = 3 independent experiments.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Note and Figs. 1–7.

Reporting Summary

Supplementary Video 1

A time-lapse video showing fusion of two droplets formed via LLPS of spider silk I16–GFP fusion protein in vitro. Scale bar, 10 µm.

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Wei, S., Qian, Z., Hu, C. et al. Formation and functionalization of membraneless compartments in Escherichia coli. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0579-9

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