CTP synthase forms cytoophidia in archaea

Shuang Zhou^{a, b, c},
Hua Xiang^{b, d},
Ji-Long Liu^{a
, ,}

a.
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
b.
University of Chinese Academy of Sciences, Beijing, 100049, China
c.
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
d.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

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Corresponding author: E-mail address: liujl3@shanghaitech.edu.cn (Ji-Long Liu)

摘要

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Abstract: CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.
- Archaea /
- CTP synthase /
- Cytoophidium

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Fig. 1. Neighbor-joining phylogenetic trees of CTPS proteins. A: Representative CTPS proteins from the three life domains are selected and aligned for creating the phylogram. The CTPS proteins from eucarya, bacteria and archaea are respectively shaded in wathet blue, pale green, and pink. Proteins from halobacteria are indicated in carmine. B: Almost all CTPS proteins in Euryarchaeota were used for building the phylogenetic tree, which contains clusters from Halobacteria, Thermoplasmata, Thermococci, Tethanobacteria, Methanomicrobia, Methanococci. CTPS, CTP synthase.

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Fig. 2. CTPS forms cytoophidia in H. hispanica. A: C-terminal targeting of endogenous H. hispanica CTPS (HhCTPS) with GFP. B: Confocal images of H. hispanica show the compartmentalized capability of HhCTPS in the late log phase. Arrows point to the punctate structure. The DIC image represents the cell contour.C: In H. hispanica cell, a compartmented CTPS structure (green, upper pane) observed under conventional confocal microscopy shows an elongated shape (red, lower panel) under stimulated emission depletion (STED) microscopy. This elongated filamentous structure in the archaeal cell resembles cytoophidia described in bacteria and eukaryotes. D: Heterologous expression of HhCTPS in E. coli. Homogenous E. coli CTPS (EcCTPS) forms longer cytoophidia in more E. coli cells than heterogeneous HhCTPS. Both HhCTPS and EcCTPS are tagged with mCherry. H. hispanica is collected in the late log phase; E. coli is collected in stationary phase. Scale bars, 5 μm in B and D, 1 μm in C. CTPS, CTP synthase; DIC, differential interference contrast.

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Fig. 3. Low salinity and DON treatment change cell shapes in H. hispanica. A: DIC images of HhCTPS-GFP in normal conditions, low-salinity medium, DON-treated medium, on the third day of cultivation.B–D: Images were analyzed for cell size (B), circularity (C), and length (the longer side of minimum bounding rectangle) (D). n = 1897 cells in normal conditions, 1181 cells in low-salinity medium, and 1874 cells in DON-treated medium. Scale bars, 5 μm. DIC, differential interference contrast; DON, 6-diazo-5-oxo-l-norleucine.

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Fig. 4. Low salinity promotes the compartmentation of CTP synthase in H. hispanica. A: Many compartmentalized structures appear in the low-salinity medium. B: Compartmentation ratio increases in the low-salt medium. Cells were collected at the first day (n = 1117 cells in AS168, 1553 cells in 1.5 M NaCl AS168), the second day (n = 2538 cells in AS168, 2180 cells in 1.5 M NaCl AS168), the third day (n = 3069 cells in AS168, 2118 cells in 1.5 M NaCl AS168), and the fifth day (n = 2639 cells in AS168, 1001 cells in 1.5 M NaCl AS168) of cultivation. Mean ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.C and D: H. hispanica cannot grow normally in 1.5 M NaCl AS168 medium. There are red colonies in the AS168 plate (left pane), but no colonies in 1.5 M NaCl AS168 after 10 days of incubation (right pane) (C). The growth curve of H. hispanica in the two media with a log scale for y axis (D). E–H: Salt supplementation recovers cell growth but decreases the abundance of punctate structures. Confocal images (E), ratio of compartmentalized structures (F) (n = 1001 cells for low-salt culture, 1072 cells for normal AS168 culture), growth curve (G), and plates (H) show that salinity recovery diminishes compartmentation of HhCTPS-GFP. Positions of four repeat inoculations per plate are at the four corners of the red squares. Mean ± SD, ∗∗∗∗P < 0.0001. Scale bars, 5 μm. SD, standard deviation.

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Fig. 5. Overexpressing CTPS promotes cytoophidium assembly in H. hispanica. A: Confocal images of OE-GFP and OE-HhCTPS-GFP in late log phase in AS168 medium. B: STED images of OE-HhCTPS-GFP. C: Images of OE-HhCTPS-GFP sampled at different time points. D: Growth curve of DF60, OE-GFP, and OE-HhCTPS-GFP with a log scale for y axis. E and F: Quantification analysis of images of OE-HhCTPS-GFP for the abundance of compartmentalized structures and foci size. Mean ± SD inD, mean with 95% CI in E and F. n = 1210 cells for 6 h, 1247 cell for 1 day, 1378 cells for 2 days, 1540 cells for 3 days, 1961 cells for 5 days, and 1938 cells for 7 days in E. n = 814 cells for 6 h, 478 for 1 day, 619 cells for 2 days, 740 cells for 3 days, 1230 cells for 5 days, and 1330 cells for 7 days in F. Scale bars, 5 μm in A and C. CI, confidence interval; CTPS, CTP synthase; HhCTPS, H. hispanica CTPS; SD, standard deviation; STED, stimulated emission depletion.

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Fig. 6. Yeast extract deprivation promotes cytoophidium formation in H. hispanica. A: Confocal images of HhCTPS-GFP in normal and yeast extract–subtracted AS168 on the third day of cultivation. B: Quantification of the cells containing compartmentalized structures. n = 985 cells for 1 day, 1322 cell for 3 days, 1445 cells for 5 days, and 1259 cells for 7 days in AS168. n = 1316 cells for 1 day, 1436 cells for 3 days, 1042 cells for 5 days, and 1172 cells for 7 days in yeast extract–subtracted AS168. Mean ± SD. C: Comparison of confocal and STED images of HhCTPS-GFP in yeast extract–subtracted AS168 on the third day of cultivation. D: Growth curve of HhCTPS with a log scale for y axis. Scale bars, 5 μm in A and the upper panes of C. 1 μm in the lower panes in C. SD, standard deviation; STED, stimulated emission depletion. Mean ± SD, ∗∗P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

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