Fig 1.
Enzymes related to c-di-GMP turnover in Anabaena and their corresponding mutants.
(A) Predicted domain architectures and consensus sequence motifs of 16 proteins for c-di-GMP synthesis and degradation. Abbreviations: GGDEF, diguanylate cyclase (DGC) domain; EAL, phosphodiesterase (PDE) domain; REC: receiver domain; PAS: Per–ARNT–Sim domain; GAF: cGMP phosphodiesterase/adenylate cyclase/FhlA domain; FHA: fork head-associated domain; orange rectangle: transmembrane domain. (B) Deletion mutants were generated in this study. The cdG0 strain (14ΔDGC) is a conditional mutant in which 13 genes were deleted, while for the 14th one (all1219), the promoter region was replaced by a Cu2+ and theophylline inducible platform (CT) at the native chromosomal locus.
Fig 2.
The phenotype and c-di-GMP level of all PDE deletion strains and the cdG0 strain.
(A) The intracellular c-di-GMP levels of the indicated strains. (B) Statistical analysis of cell length and cell width of the indicated strains based on images, as shown in S2 Fig, using a box plot. (C) The intracellular c-di-GMP levels of the cdG0 strain during the time course of Cu2+ and theophylline depletion. (D) The growth curves of the cdG0 strain and WT strain in BG11 medium with (+) or without (−) Cu2+ and theophylline. Absorbance at 750 nm was measured at the indicated time points. To better remove traces of Cu2+ and theophylline, the cultures were re-inoculated for the second time in a medium free of Cu2+ and theophylline after 8 days of incubation. All values are shown as mean ± standard deviation, calculated from triplicate data. (E) Micrographs of Anabaena filaments of WT and cdG0 strains cultured in BG11 medium with (+) or without (−) Cu2+ and theophylline at the indicated time points. Scale bars represent 15 µm. (F) Statistical analysis of cell size parameters of the cdG0 strain during the time course of Cu2+ and theophylline depletion. The cell length and cell width were measured based on images, as shown in (E). (G) The intracellular c-di-GMP level of the indicated strains at 96 h in BG11 medium without Cu2+ and theophylline. (H) Top panel: the micrographs of the indicated strains cultured in BG11 medium with (+) or without (−) Cu2+ and theophylline at 96 h. Bottom panel: the growth of different strains, as in the top panel tested in 24-well plates. All cultures started with a similar OD at 0.3 diluted from a pre-culture and imaged after 4 days of incubation. In (A), (C), and (G), the c-di-GMP concentrations of all mutants were normalized to the levels of the wild-type (WT) strain. The fold change numbers are shown as mean ± SD from three biological replicates. In (B) and (F), 150–200 cells of each strain from three independent experiments were measured. The boxplots enclose the 25th and the 75th percentile, with the black line representing the median value, red dot representing the mean value. The statistical significance in comparison to WT was carried out by a two-sided Student t test. The red asterisks indicate significance in comparison to WT as follows: *p < 0.05; **p < 0.01; ***p < 0.001. ****p < 0.0001, n.s.: not significant (p > 0.05). WT, wild-type Anabaena. The data underlying this Figure can be found in S1 Data. The raw images underlying this Figure can be found in S1 Raw Images.
Fig 3.
Alr2306 is the major PDE for cell size maintenance.
(A and C) Statistical analysis of cell size parameters of the indicated strains. The cell length (upper panel) and cell width (lower panel) were measured based on images, as shown in S4 Fig 200 cells of each strain from three independent experiments were measured. The boxplots enclose the 25th and the 75th percentile, with the black line representing the median value, red dot representing the mean value. (B and D) The intracellular c-di-GMP level of the indicated strains. The c-di-GMP concentrations of all mutants were normalized to the levels of the WT strain. The fold change numbers are shown as mean ± SD from three biological replicates. The red asterisks indicate significance in comparison to WT or between the two strains connected by a bracket at the top as follows: *p < 0.05; **p < 0.01; ***p < 0.001. ****p < 0.0001, n.s.: not significant (p > 0.05). The data underlying this Figure can be found in S1 Data. The raw images underlying this Figure can be found in S1 Raw Images.
Fig 4.
The phenotype and c-di-GMP levels of cdgS, alr3599, and alr1219-related mutant strains.
(A) The growth curves of the indicated conditional mutant strains and the WT strain in BG11 medium with (+) or without (−) Cu2+ and theophylline. The promoter region of all1219 in the ΔcdgSCT-all1219 strain, the Δ8DGC-1 strain, and the ΔcdgSΔalr3599CT-all1219 strain was replaced by a Cu2+ and theophylline inducible platform (CT) at the native chromosomal locus. Absorbance at 750 nm was measured at the indicated time points. All values are shown as mean ± standard deviation, calculated from triplicate data. (B) The intracellular c-di-GMP levels of the ΔcdgSCT-all1219 strain and the Δ8DGC-1 strain during the time course of Cu2+ and theophylline depletion. (C and D) Micrographs of Anabaena filaments of WT, ΔcdgSpICT-all1219 strains (C), and Δ8DGC-1 strain (D) cultured in BG11 medium with (+) or without (−) Cu2+ and theophylline at the indicated time points. Scale bars represent 15 µm. (E) The growth of indicated strains was tested in 24-well plates. All cultures started with a similar OD at 0.3 diluted from a pre-culture and imaged after 4 days of incubation. The purple triangle indicated the decreasing levels of the inducers added to the culture media (from top to bottom column): 0.3 μM copper and 2 mM theophylline, 0.125 μM copper and 0.5 mM theophylline, and 0 μM copper and 0 mM theophylline. (F) The intracellular c-di-GMP levels of the indicated strains at 0 h, 48 h, and 96 h of Cu2+ and theophylline depletion. The green and red dotted line represents the 50% and 80% of the c-di-GMP level to WT, respectively. The data underlying this Figure can be found in S1 Data. The raw images underlying this Figure can be found in S1 Raw Images.
Fig 5.
The Interplay among c-di-GMP, CdgR, and cellular morphology in Anabaena.
(A) Correlation between intracellular c-di-GMP levels and cell length (left) and cell width (right) across different mutant strains. The red dot and dashed line indicate the lethal phenotype of the corresponding strains. Two concentration-dependent regulatory thresholds are indicated by purple dashed lines. (B) Dose-dependent effects of intracellular c-di-GMP levels on cell parameters (cell length (left) and width (right)) in the cdG0 strain under varying concentrations of inducers (Cu2+ and theophylline). (C) CdgR abundance in the indicated variant strains and the cdG0 strain during the time course of Cu2+ and theophylline depletion. Total proteins were visualized with Coomassie Brilliant Blue (CBB), and CdgR was probed with a polyclonal antibody against CdgR (Anti-CdgR). CdgR levels corresponding to those of the WT were quantified with ImageJ from three biological replicates. (D) Correlation between the molar ratios of CdgR to c-di-GMP calculated for the indicated strains and intracellular c-di-GMP levels. In (A) (B) (D), data points represent the mean ± SD from biological replicates. The data underlying this Figure can be found in S1 Data. The raw images underlying this Figure can be found in S1 Raw Images.
Fig 6.
A dual-threshold c-di-GMP regulatory system governs cell growth and survival in Anabaena.
Under our experimental conditions, the c-di-GMP levels are regulated by three enzymatic modules: (1) Basal module: maintains basal c-di-GMP pool (~40%) through the activity of 13 enzymes, including the dominant PDE Alr2306. This module is analogous to a battery in an electromechanical relay. (2) Primary module: comprises the DGCs CdgS and All1219, which operate in a manner like an electromagnetic coil. Their signal-dependent contribution (~60% of total c-di-GMP under our test conditions), together with the basal c-di-GMP pool, defines three phenotypic states: >80 ± 5% c-di-GMP: normal Anabaena morphology (steel piece engaged); 50 ± 5–80 ± 5% c-di-GMP: cell size decreases proportionally with c-di-GMP level; < 50% ± 5 c-di-GMP: lethal effect in Anabaena (steel piece disengaged). (3) Emergency module: the dormant DGC Alr3599 activates when c-di-GMP drops to the viability threshold (50 ± 5% c-di-GMP), functioning as a backup coil to save cells from death. A dual-threshold control mechanism (morphological threshold: 80 ± 5%; viability threshold: 50 ± 5%) ensures precise regulation of cellular c-di-GMP homeostasis. The phenotypic outcomes, including reduced cell size and lethality, are ultimately determined by the molar ratio of CdgR to c-di-GMP. Arrows indicate threshold values. cdG: c-di-GMP.