Anabaena circadian clock behavior under nitrogen-poor conditions from single-cell measurements of fluorescence intensity
Data files
Dec 07, 2023 version files 942.05 KB
Abstract
Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.
README: Anabaena circadian clock behavior under nitrogen-poor conditions from single-cell measurements of fluorescence intensity
https://doi.org/10.5061/dryad.xwdbrv1kv
Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments
Rinat Arbel-Goren, Bareket Dassa, Anna Zhitnitsky, Ana Valladares, Antonia Herrero,
Enrique Flores and Joel Stavans
Deposited data includes the following data files used to generate different figures in the main text, with their respective description. Files of .opju type can be opened with OriginPro software (OriginLab Corporation).
Noise and pecB expression in Anabaena filaments under nitrogen poor conditions. Origin file containing the data of Figure S2 and the autofluorescence of photosynthetic pigments in Anabaena (AF) (column D). Column A represents time in minutes following nitrogen deprivation. Temporal dependence of noise (column left axis) of the expression intensity of PpecB-gfp for vegetative cells along a filament (column D) or within individual vegetative intervals (column F); Noise of autofluorescence AF as a function of time (column E); Fluorescence intensity from PpecB-gfp in a filament as a function of time for a wild-type genetic background under nitrogen-poor conditions (Column B).
Cell lineage of Anabaena cells and their autofluorescence.opju Origin file containing the lineages of three cells each in one of three contiguous vegetative cell intervals in Fig. 5B. The data correspond to the fluorescence intensity from PpecB-gfp as a function of time in heterocysts and vegetative cell lineages (PecB_Fluorescence_Intensity), as well as the autofluorescence AF of photosynthetic pigments (Autofluorescence (AF)). Time corresponds to Frame column, with intervals of 30 min between frames.
PecB expression in heterocysts.opju Origin file corresponding to Fig. 5A. The fluorescence intensities from PpecB-gfp as a function of time (column time) in heterocysts are given in columns Int1-Int14).
Delta Kai mutant growth curves in Anabaena.opju Origin file containing the data used to plot Fig. 6B of growth curves of delta Kai mutant and wild-type strains grown in liquid BG110 medium with and without antibiotics (neomycin or with neomycin). Column A indicates the time of growth under photoautotrophic culture conditions.
Gating of differentiation in Anabaena.opju Origin file containing the data used to generate the two panels in Fig. 4. Panel A was generated with data in Book Lineage. Time (column A), fluorescence intensities of two neighboring, sister cells bearing a P*pecB-gfp* fusion (columns C and D, green) one of which eventually becomes a heterocyst (columns D), and their respective autofluorescence intensity traces (columns F and G). Histogram data on the regulation of differentiation timing along a circadian cycle (Book Bootstraphist), with bin centers (column A), normalized histogram height per bin (column B) and errors determined using bootstrap methods (column C). The data of phases of individual differentiation events (column B) from different experiments (column A) is given in book Phasediffere.
Synchronization index R of cells in Anabaena filaments.xlsx Contains bare data used to calculate the synchronization index R (Eq. 1 in main text), of different cells in different filaments from independent experiments, as reported in the main text (section: Discoordinated expression of a clock-controlled gene along filaments under constant light conditions.) as well as in Table 1. Each sheet corresponds to a the indicated experiment (Exp) and the indicated vegetative interval (Int). The interval between consecutive frames in column A is 30 min. Each column represents the fluorescence intensity from PpecB-gfp in the indicated cell.
Scatter plots.opju Origin file containing the data of the two panels in Fig. 2 on the dis-coordinated expression of the pecB promoter PpecB-gfp between vegetative cell intervals. (A) Scatter plot of the fluorescence intensities f of nearest-neighbor cells (?(i,j),f(i+1,j)) within vegetative cell intervals ?. (B) Scatter plot of the fluorescence intensities of corresponding cells in adjacent vegetative cell intervals (?(i,j),f(i,j+1)). Each color in A, B represents data from three independent experiments. The Pearson statistics of the separate experiments is included in book Pearsonstats.
Code/Software
The software for image analysis developed in the lab and its scripts for data analysis, were deposited in Zenodo. All software develpment was carried out using Matlab (MathWorks). The instructions of how to use the software for image analysis are included in file Anabaena_Manual.pdf, together with all the needed scripts.