Robust, coherent, and synchronized circadian clock-controlled oscillations along Anabaena filaments
- Department of Physics of Complex Systems, Weizmann Institute of Science, Israel
- Dipartimento di Fisica e Astronomia, Università di Firenze, INFN and CSDC, Italy
- Consiglio Nazionale delle Ricerche, Istituto dei Sistemi Complessi, Italy
- Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Spain
Figures
Figure 1
Circadian oscillation in Anabaena.
(A) GFP fluorescence in a filament of an Anabaena strain bearing a promoter fusion, growing under nitrogen-replete conditions. The snapshots were chosen near maxima and minima of the circadian …
Figure 2 with 1 supplement
Characterization of a clock-controlled gene in Anabaena.
(A) Average cell fluorescence intensity from in a filament as a function of time for a wild-type genetic background (full black circles) and for a kaiABC background (empty black circles); …
Figure 2—figure supplement 1
Effects of perturbation of cell-cell communication on the expression of PpecB−gfp.
Figure 3 with 2 supplements
Transcriptional oscillations in the core clock genes, rpaA and pecB.
(A) Relative expression of kaiA (green), kaiB (red), and kaiC (blue) as a function of time measured by RT-qPCR (Materials and methods). A persistence homology analysis of these data is presented in F…
Figure 3—figure supplement 1
Persistent homology analysis of periodic behavior in the trascriptionaltime series of kai genes of Anabaena.
Figure 3—figure supplement 2
Schematic representation and regulatory sequences of the kaiABC, rpaA,pecB and ftsZ promoter regions in Anabaena.
Figure 4
Circadian oscillations in Synechococcus.
(A) Growth and lineage of a cell in patterned agarose, expressing YFP from the kaiBC promoter. The snapshots were chosen near maxima and minima of the circadian oscillations. (B) Fluorescence …
Figure 5 with 1 supplement
Stochastic model for circadian oscillations in Synechococcus.
(A) Schematic representation of interconversion between KaiC phosphoforms modulated by the activity of KaiA in an individual clock. The different phosphoform states of KaiC are denoted by U …
Figure 5—figure supplement 1
Typical shape of the nonlinear function f.
Figure 6
Stochastic model for circadian oscillations in Anabaena.
(A) Schematic representation of the Anabaena filament showing coupling of circadian clocks via cell-cell communication (red arrows). (B) Gillespie simulations of quasi-cycles of T-KaiC in a …
Appendix 1—figure 1
Schematic representation of an Anabaena filament.
The parameter measures the connectivity of each node.
Appendix 1—figure 2
Stability diagram.
Stability of the equilibrium points for the three species as a function of and . The values of all the other parameters are specified in Appendix 1—table 1. Continuous lines denote stable …
Appendix 1—figure 3
Deterministic limit cycle region.
The portion of the plane delimited by the blue line marks the region of the parameter's space where deterministic regular oscillations occur.
Appendix 1—figure 4
Deterministic simulation.
Results of the numerical integration of system (Equation 23) for and . The other parameters are assigned as specified in Appendix 1—table 1. for stands here for the relative abundance of …
Appendix 1—figure 5
Comparison between deterministic and stochastic simulations.
The blue lines denote the result of the numerical integration of Equation (23) while the noisy red lines represent the stochastic simulation of the system through the Gillespie, 1977 algorithm. For …
Appendix 1—figure 6
The complex coherence function: comparison between theory and experiments.
Complex coherence function measuring the correlation of 35 cell segments at the frequency of temporal oscillations. Red circles correspond to experimental data, and the squares represent fits to the …
Appendix 1—figure 7
Construction of an AnabaenakaiABC deletion mutant in the PpecB-gfp genetic background.
PCR, DNA restriction/ligation, and transformation into Escherichia coli were performed by standard techniques. Conjugation from E. coli to Anabaena was performed as described by Elhai et al., 1997, …
Videos
Video 1
Real-time expression of a clock-controlled gene and filament autofluorescence during circadian oscillations in WT Anabaena.
Tables
Table 1
Synchronization of expression of a clock-controlled gene in cells within and between Anabaena filaments.
The synchronization index for strains with the indicated genotypes (Materials and methods) was measured from the fluorescence intensities of expression in the same cells followed over a full …
| Genotype | Cell cluster | R (mean ± SEM) | n | Comparison with strain | p-Value |
|---|---|---|---|---|---|
| WT | Contiguous | 0.89 ± 0.04 | 3 | WT (separate) | 0.117 |
| WT | Separate | 0.85 ± 0.01 | 2 | ||
| WT | Different filaments | 0.75 ± 0.04 | 2 | WT (separate) | 0.026* |
| Contiguous | 0.73 ± 0.05 | 4 | WT (contiguous) | 0.001* | |
| Contiguous | 0.71 ± 0.03 | 3 | WT | 0.001* |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Anabaena) | , WT | This paper | Anabaena PCC 7120 WT, bearing a pecB promoter fusion to gfp | |
| Strain, strain background (Anabaena) | , | This paper | Anabaena PCC 7120 deletion mutant of the genes, bearing a pecB promoter fusion to gfp | |
| Strain, strain background (Anabaena) | doi: 10.1371/journal.pgen.1005031 | CSL64 | Anabaena PCC 7120 WT, bearing a hetR promoter fusion to gfp | |
| Strain, strain background (Anabaena) | , | This paper | Anabaena PCC 7120 deletion mutant of the , , genes (CSVM141), bearing a pecB promoter fusion to gfp | |
| Strain, strain background (Synechococcus elongatus) | YFP-SsrA | This paper | PCC 7942 | Synechococcus elongatus PCC 7942 (wild-type) expressing YFP- SsrA |
| Recombinant DNA reagent | EB2316 (plasmid) | Addgene plasmid | 87753 | http://n2t.net/addgene: 87753 |
| Recombinant DNA reagent | pSpark (plasmid) | Canvax | C0001 | https://lifescience.canvaxbiotech.com/wpcontent/uploads/sites/2/2015/08pSpark-DNA-Cloning.pdf |
| Recombinant DNA reagent | pCSRO containing cloning vector | doi: 10.1128/JB.00181-13 | ||
| Commercial assay or kit | Fast SYBR Green Master Mix | Applied Biosystems | 4385612 | |
| Commercial assay or kit | QuantiTect Reverse Transcription kit | QIAGEN | 205311 |
Appendix 1—table 1
Parameters used in the simulations.
| 0 h−1 | 0.479077 h−1 | ||
| 0 h−1 | 0.212923 h−1 | ||
| 0 h−1 | 0.505692 h−1 | ||
| 0 h−1 | 0.0532308 h−1 | ||
| 0.21 h−1 | 0.0798462 h−1 | ||
| 0 h−1 | 0.1730000 h−1 | ||
| 0.31 h−1 | −0.319885 h−1 | ||
| 0.11 h−1 | −0.133077 h−1 | ||
| 0.43 µM | [KaiC] | 3.4 µM |
-
All the values are from Lambert et al., 2016.
Appendix 1—table 2
Oligodeoxynucleotide primers used in this work.
| Name | Sequence 5′–3′ |
|---|---|
| alr0523-EcoRI-Fw | TTTTGAATTCGCTTATAAACAGCAGTTAACAGGCT |
| alr0523-Rev | TGCTACCTCCACCGCCTGCCTGTTCAACTACTTTGGA |
| 4G-GFP-Fw | GCGGTGGAGGTAGCAAAGGAGAAGAACTTTTCAC |
| GFP-Rev | GCCTGAATTCTTATTTGTATAGTTCATCCATGCC |
| alr0523-1-Fw | GAATTCGCTTATAAACAGCAGTTAACAGGCT |
| alr0523-1-Rev | CTAGCACCTCCACCGCCTGCCTGTTCAACTACTTTGGA |
| 4G-GFP-Fw in plasmid PCSV3 | ATTTGAAACTGCGCCACGGATC |
| Rev plasmid PCSV3 | GACCATGACGGATTAGCTCAGTAG |
| alr0523(7120)–1 | CGT GAG TCT CCA ACG GAG GC |
| Kai-1 | GAAACTGCAGGCAGAATAGGAAATCTCTAC |
| Kai-2 | CCAAATGATATCGTGCTGACAAACCTACAGTGC |
| Kai-3 | CAGCACGATATCATTTGGTATCGTACTATATTC |
| Kai-4 | CTTTCTGCAGGTTGTCCAGCCAGCAGGGTAG |
| kaiA-2 | CAGGGTGAGGCGATAATCCAT |
| kaiA-1 | GCCAGAGTACTTGTTTCTAAGCAAC |
| CK1-R | CGATTCCGAAGCCCAACCT |
| kaiC-4 | CGAGCTACCAACCGAAAG |
| kaiB-1 | CGGCAATACTCCAAACTCAG |
| PkaiBC-1 | GGTCTATCCCACGAGAAACC |
| YFP-2 | GGTAGCTCAGGTAGTGGTTGTC |
| all5167-1q (forward) | GCTCAAGCAATTCGTCACTGTTCC |
| all5167-2q (reverse) | AAAGATTGCGTCGGTCTGGTGT |
| rnpB-1q (forward) | CTCTTGGTAAGGGTGCAAAGGTG |
| rnpB-4q (reverse) | GGCTCTCTGATAGCGGAACTGG |
| kaiC-3q (forward) | ATGAAGCAGTGGGAGTGGTG |
| kaiC-8q (reverse) | ACGTTACGGGCTATGACCAC |
| kaiB-1q (forward) | ACCAAATTCAGTCAGGGCGT |
| kaiB-4q (reverse) | GCCAATCAGAACTCTTTCCCG |
| kaiA-3q (forward) | CAACTCAAATCAGATTATCGCCA |
| kaiA-4q (reverse) | CTGCCCTCTAGTCGTAGCTG |
| pecB-1q (forward) | ATATTTAATGCTGGTGGTGCTTGTT |
| pecB-4q (reverse) | GCAGCGATCGTCCATGACACTAC |
| rpaA-1q (forward) | TTTAACGCCGGAGCAGATGA |
| rpaA-4q (reverse) | TGTCCGTGACGTTGTAGCAA |
