Journal: Current biology : CB
Article Title: The cyanobacterial circadian clock couples to pulsatile processes using pulse amplitude modulation.
doi: 10.1016/j.cub.2024.10.047
Figure Lengend Snippet: Figure 4. RpoD4 levels modulate cell size (A and B) (A) Mean birth length and (B) mean pulse amplitude of WT (cyan, same data as in Figure 1F) and DrpoD4 (blue) strains carrying PrpoD4-EYFP-LVA reporters grown on agarose pads under constant light (18 mmol m2 s1), plotted against time of day and repeated for two cycles to highlight periodicity. White and gray backgrounds represent subjective day and night, respectively. 1,140 WT cells (19 colonies) and 1,310 DrpoD4 cells (18 colonies) were analyzed. (C) Violin plots of birth length of WT (cyan, median = 3.77 mm) and 100 mM IPTG induced Ptrc-rpoD4 (magenta, abbreviated as rpoD4OX, median = 5.29 mm) strains (carrying a clock reporter, see Figure S4) and grown on agarose pads under constant light (15 mmol m2 s1). 239 WT cells (4 colonies) and 161 rpoD4OX cells (4 colonies) were analyzed. (D) Violin plots of cell length of WT and inducible strains for 10 different sigma factors (Ptrc-sigma) incubated with 1 mM IPTG (sigmaOX) or without any IPTG for 3 days under constant light (20–25 mmol m2 s1) in liquid cultures (3 independent experiments). Central marks in violin plots indicate the median. Strains with significantly different cell sizes compared with WT are highlighted with different colors (**p < 0.01 [yellow], ****p < 0.0001 [red], Student’s t test). (E) Cell length of Ptrc-rpoD4 cells incubated with increasing concentration of IPTG for 3 days. Data shown as mean ± SEM of three independent experiments. A Hill equation is fitted to the data, with EC50 = 37 mM and Hill coefficient = 0.758. (F) High-level schematics of cell size control points in S. elongatus. The clock regulates the cell-division-dependent expression of rpoD4 through PAM and RpoD4 levels modulate cell size. On the other hand, levels of environmental light determine the average division frequency rate, which is modulated by the circadian clock, effectively an FM process that results in cell sizes that vary throughout the day. See also Figures S3 and S4.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains Synechococcus elongatus PCC 7942 ATCC ATCC 33912 Chemicals, peptides, and recombinant proteins OmniPur Low-Melting Agarose Merk Millipore Cat# 2070-100GM NaNO3 Sigma-Aldrich Cat# 221341-500G CaCl2,2H2O Sigma-Aldrich Cat# 223506-25G Ferric ammonium citrate Sigma-Aldrich Cat# RES20400-A702X EDTA disodium salt Sigma-Aldrich Cat# E4884-100G K2HPO4 Sigma-Aldrich Cat# 450200-25G-A MgSO4,7H2O Sigma-Aldrich Cat# 230391-25G NaCO3 Sigma-Aldrich Cat# 221341-500G H3BO3 Sigma-Aldrich Cat# B0394-100G MnCl2,4H2O Sigma-Aldrich Cat# 221279-100G ZnSO4,7H2O Sigma-Aldrich Cat# 221376-100G NaMoO4,2H2O Sigma-Aldrich Cat# 331058-5G CuSO4,5H2O Sigma-Aldrich Cat# 939315-100G Co(NO3)2,6H2O Sigma-Aldrich Cat# 239267-5G Critical commercial assays QIAprep Spin Miniprep Kit (50) QIAGEN Cat# 27104 Direct DNA Extraction Kit (Bacteria) Geneflow Cat# P4-0390 Q5 Site-Directed Mutagenesis Kit New England Biolabs Cat# E0554S Deposited data Data and code used for generating figures in the paper Zenodo https://doi.org/10.5281/zenodo.13912391 Experimental models: Organisms/strains 7942_CY120: WT PrpoD4-EYFP-LVA_pAM2314 (used in Fig. 1A-D&F, 3A-C, 4A&B, S1B, S2A-C, S3B) This paper N/A 7942_CY278: DkaiBC::GentR PrpoD4-EYFP-LVA_pAM2314 (used in Fig. 1E&G, S1A&C, S3A&B) This paper N/A 7942_CY140: WT PrpoD4-RpoD4-EYFP_pAM1303 (used in Fig. S1D-F&H-J) This paper N/A 7942_CY131: DkaiBC::GentR PrpoD4-RpoD4-EYFP_pAM1303 (used in Fig. S1G,H, K & J) This paper N/A 7942_CY117: WT PkaiBC-EYFP-fsLVA_pAM1579 (used in Fig. 4C, S4C-E) This paper N/A 7942_CY286: KaiCR393C::SptR (endogenous locus) PkaiBC-EYFP-fsLVA_pAM1579 (used in Fig. S2D) This paper N/A 7942_JLS339: KaiCR393C::SptR (endogenous locus) PrpoD4-EYFP-LVA_pAM1579 (used in Fig. 3D) This paper N/A 7942_CY259: DrpoD4::GentR PkaiBC-EYFP-fsLVA_pAM1579 (used in Fig. S4A&B) This paper N/A 7942_CY273: WT Ptrc-rpoD4_pAM2991 PkaiBC-EYFP-fsLVA_pAM1579 (used in Fig. 4C, S3C-E, S4C&D) This paper N/A 7942_CY133: DrpoD4::KanR PrpoD4-EYFP-LVA_pAM2314 (used in Fig. 4A&B, S3B) This paper N/A (Continued on next page) Current Biology 34, 1–8.e1–e6, December 16, 2024 e1
Techniques: Incubation, Concentration Assay, Control, Expressing
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![Figure 4. RpoD4 levels modulate cell size (A and B) (A) Mean birth length and (B) mean pulse amplitude of WT (cyan, same data as in Figure 1F) and DrpoD4 (blue) strains carrying PrpoD4-EYFP-LVA reporters grown on agarose pads under constant light (18 mmol m2 s1), plotted against time of day and repeated for two cycles to highlight periodicity. White and gray backgrounds represent subjective day and night, respectively. 1,140 WT cells (19 colonies) and 1,310 DrpoD4 cells (18 colonies) were analyzed. (C) Violin plots of birth length of WT (cyan, median = 3.77 mm) and 100 mM IPTG induced Ptrc-rpoD4 (magenta, abbreviated as rpoD4OX, median = 5.29 mm) strains (carrying a clock reporter, see Figure S4) and grown on agarose pads under constant light (15 mmol m2 s1). 239 WT cells (4 colonies) and 161 rpoD4OX cells (4 colonies) were analyzed. (D) Violin plots of cell length of WT and inducible strains for 10 different sigma factors (Ptrc-sigma) incubated with 1 mM IPTG (sigmaOX) or without any IPTG for 3 days under constant light (20–25 mmol m2 s1) in liquid cultures (3 independent experiments). Central marks in violin plots indicate the median. Strains with significantly different cell sizes compared with WT are highlighted with different colors (**p < 0.01 [yellow], ****p < 0.0001 [red], Student’s t test). (E) Cell length of Ptrc-rpoD4 cells incubated with increasing concentration of IPTG for 3 days. Data shown as mean ± SEM of three independent experiments. A Hill equation is fitted to the data, with EC50 = 37 mM and Hill coefficient = 0.758. (F) High-level schematics of cell size control points in S. elongatus. The clock regulates the cell-division-dependent expression of rpoD4 through PAM and RpoD4 levels modulate cell size. On the other hand, levels of environmental light determine the average division frequency rate, which is modulated by the circadian clock, effectively an FM process that results in cell sizes that vary throughout the day. See also Figures S3 and S4.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1971/pm39591971/pm39591971__page6_image1.jpg)