DOI: 10.5061/dryad.xwdbrv1kv
title: Anabaena circadian clock behavior under nitrogen-poor conditions from single-cell measurements of fluorescence intensity

description: 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.
description: # Anabaena circadian clock behavior under nitrogen-poor conditions from
 single-cell measurements of fluorescence intensity
 **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.