Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

Biofilms are accumulations of microorganisms embedded in extracellular matrices that protect against external factors and stressful environments. Cyanobacterial biofilms are ubiquitous and have potential for treatment of wastewater and sustainable production of biofuels. But the underlying mechanisms regulating cyanobacterial biofilm formation are unclear. Here, we report the solution NMR structure of a protein, Se0862, conserved across diverse cyanobacterial species and involved in regulation of biofilm formation in the cyanobacterium Synechococcus elongatus PCC 7942. Se0862 is a class α+β protein with ααββββαα topology and roll architecture, consisting of a four-stranded β-sheet that is flanked by four α-helices on one side. Conserved surface residues constitute a hydrophobic pocket and charged regions that are likely also present in Se0862 orthologs.

The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB-KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB-KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA-KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA-KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiBs favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.

The circadian oscillator of cyanobacteria is composed of only three proteins, KaiA, KaiB, and KaiC. Together, they generate an autonomous ~24-h biochemical rhythm of phosphorylation of KaiC. KaiA stimulates KaiC phosphorylation by binding to the so-called A-loops of KaiC, whereas KaiB sequesters KaiA in a KaiABC complex far away from the A-loops, thereby inducing KaiC dephosphorylation. The switch from KaiC phosphorylation to dephosphorylation is initiated by the formation of the KaiB-KaiC complex, which occurs upon phosphorylation of the S431 residues of KaiC. We show here that formation of the KaiB-KaiC complex is promoted by KaiA, suggesting cooperativity in the initiation of the dephosphorylation complex. In the KaiA-KaiB interaction, one monomeric subunit of KaiB likely binds to one face of a KaiA dimer, leaving the other face unoccupied. We also show that the A-loops of KaiC exist in a dynamic equilibrium between KaiA-accessible exposed and KaiA-inaccessible buried positions. Phosphorylation at the S431 residues of KaiC shift the A-loops toward the buried position, thereby weakening the KaiA-KaiC interaction, which is expected to be an additional mechanism promoting formation of the KaiABC complex. We also show that KaiB and the clock-output protein SasA compete for overlapping binding sites, which include the B-loops on the CI ring of KaiC. KaiA strongly shifts the competition in KaiB's favor. Thus, in addition to stimulating KaiC phosphorylation, it is likely that KaiA plays roles in switching KaiC from phosphorylation to dephosphorylation, as well as regulating clock output.

As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as a model system for detailed mechanistic studies of day-night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the "bulk" and "edge" KaiBC sites in stoichiometric and substoichiometric KaiB_iC₆ complexes (i = 1-5). We provide kinetic evidence to support the intermediacy of the "edge" KaiBC sites as bridges and nucleation sites between free KaiB and the "bulk" KaiBC sites. Furthermore, we show that the relative abundance of "edge" and "bulk" sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.

The recurrent pattern of light and darkness generated by Earth's axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins, KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a nighttime metabolic program that is essential for S. elongatus to survive in the dark.

The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.

Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of Earths rotation. Once KaiB switches folds, it binds phosphorylated KaiC and captures KaiA, which initiates a phase transition of the circadian cycle, and it regulates components of the clock-output pathway, which provides the link that joins the timekeeping and signaling functions of the oscillator.

Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post-adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.