(l to r) Sophie de Buyl, Shu-Wen Teng, Jeffrey R. Moffitt, and Shankar Mukherji with Nikhil
Living organisms tightly coordinate their physiology with the day and night cycle. The time-keeping mechanism used to perform this coordination, named the circadian clock, can maintain oscillation phase, frequency and amplitude for many cycles, even in the absence of external cues. In the model cyanobacterial species Synechococcus elongatus, single cells generate remarkably stable circadian oscillations, enabling a population of growing cells to maintain synchrony with a correlation time of many weeks in constant environmental conditions.
The O’Shea group, in a recent paper published in Science, identified the mechanism underlying the remarkable stability of the circadian oscillations in growing cyanobacteria. The answer turned out to lie in the precise way the biochemical circuits that generate and regulate the circadian oscillations interact with each other. Circadian oscillations in S. elongatus are generated by a network architecture that consists of a posttranslational regulation (PTR) circuit and a transcriptional-translational regulation (TTR) circuit. The PTR circuit generates circadian oscillations in the phosphorylation state of the clock protein KaiC. The TTR circuit, layered on top of the PTR circuit and common to all circadian systems, regulates expression of the operon containing the genes for KaiC and the clock protein KaiB. Previous to this study, it remained unclear how PTR and TTR circuits integrate in vivo and what the functions of PTR and TTR circuits are in growing cells.
To address the contributions of the PTR and TTR circuits in generating the in vivo oscillation, they performed fluorescence microscopy on cells grown in a custom-built microfluidic device to study three strains: (i) a wild-type strain in which both the TTR and PTR circuits are intact, (ii) a PTR-only strain in which the TTR circuit was abrogated by making expression of the kaiBC operon constitutive, and (iii) a TTR-only strain which expresses an unphosphorylatable KaiCS431E/T432E, thus lacking the PTR circuit but leaving an intact TTR circuit.
They experimentally demonstrated that while the PTR circuit is sufficient to generate oscillations in growing cyanobacteria, the absence of the TTR circuit causes populations of cyanobacteria to lose synchrony due to phase drift of individual oscillators. Using mathematical modeling, they reproduced the ability of the PTR circuit alone to sustain oscillations, but demonstrated that the TTR is crucial for oscillator synchrony. They showed that the coupling of PTR and TTR generates robustness against fluctuations in gene expression and growth rate that enables the clock to maintain accurate oscillations in constant environment for several weeks. The general architecture of coupled PTR and TTR circuits is shared by circadian circuits in many species, so this work likely represents a general solution used in other circadian circuits.
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