Timing the day: what makes bacterial clocks tick?

Abstract

Chronobiological studies of prokaryotic organisms have generally lagged far behind the study of endogenous circadian clocks in eukaryotes, in which such systems are essentially ubiquitous. However, despite only being studied during the past 25 years, cyanobacteria have become important model organisms for the study of circadian rhythms and, presently, their timekeeping mechanism is the best understood of any system in terms of biochemistry, structural biology, biophysics and adaptive importance. Nevertheless, intrinsic daily rhythmicity among bacteria other than cyanobacteria is essentially unknown; some tantalizing information suggests widespread daily timekeeping among Eubacteria and Archaea through mechanisms that share common elements with the cyanobacterial clock but are distinct. Moreover, the recent surge of information about microbiome-host interactions has largely neglected the temporal dimension and yet daily cycles control important aspects of their relationship.

Figure 1. Many bacteria are exposed to daily selective pressures

Free-living bacteria can be exposed to daily cycles of light and temperature that affect viability (for example, exposure to UV light) and/or provide energy (for example, through photosynthesis). Even the gut microbiota is often exposed to daily cycles of nutrients, owing to the rhythmic eating habits of the host, and temperature, as most animal hosts have daily rhythms of body temperature that are metabolically controlled in endotherms and behaviourally controlled in ectotherms. These same bacteria may be exposed to daily environmental cycles of light and temperature following excretion from the gut.

Figure 2. Circadian molecular and genetic oscillators in cyanobacteria

a | A self-sustained post-translational oscillator (PTO) is embedded in a transcription–translation feedback loop (TTFL). The synthesis of non-phosphorylated KaiC monomers feeds into the molecular oscillator, which undergoes a cycle of association into, and disassociation from, the KaiA–KaiB–KaiC nanocomplex. KaiC interacts with competing KaiB and SasA proteins to mediate the activity of transcriptional factors (such as RpaA) and rhythmic DNA torsion to control global transcription levels, including those that drive the expression of the essential clock genes kaiA, kaiB and kaiC. b | The core clock reaction that is embedded in the PTO is ATP hydrolysis, which is primarily catalysed by the ATPase in the CI domain of KaiC. In this view, the phosphorylation and nanocomplex cycles enhance and regulate the ATPase activity to promote robustness and temperature compensation. Part a is adapted with permission from REF. 42, AAAS.

Figure 3. The post-translational oscillator (PTO)

The post-translational oscillator (PTO) is composed of rhythmic associations between KaiC (purple), KaiA (blue) and KaiB (green or black) to form a nanocomaplex that ATPase and phosphorylation activities of KaiC. KaiC forms two hexameric rings, CI and CII. KaiB monomers undergo a slow transition from ground-state KaiB (gsKaiB; green diamonds) to the fold-shifted state (fsKaiB; black diamonds), which binds to the CI domain of KaiC hexamers. KaiA associates with hypophosphorylated KaiC and stimulates the autophosphorylation of KaiC (red circles indicate the added phosphates), which accelerates the rate of ATP hydrolysis. The association and dissociation of the nanocomplex coincide with changes in the phosphorylation state of KaiC; residue T432 is phosphorylated first, followed by S431, and dephosphorylation occurs in the opposite order. Adapted with permission from REF. 37, Elsevier.

Figure 4. KaiC phosphorylation temporal patterns differ between complete circadian clocks that consist of KaiA–KaiB–KaiC and proto-circadian KaiB–KaiC systems

The phosphorylation of KaiC in light and dark cycles, constant darkness and constant light is shown for three different species of bacteria: the two cyanobacteria Synechococcus elongatus and Prochlorococcus marinus and the purple bacterium Rhodopseudomonas palustris. S. elongatus harbours kaiABC and has a complete circadian system, whereas P. marinus and R. palustris only harbour kaiBC.

Figure 5. Criteria for testing the adaptive fitness of daily timekeeping mechanisms

a | The fitness advantages of timekeeping can be assessed by competition between strains in mixed cultures under different environmental conditions. b | In competition between rhythmic and arrhythmic strains of Synechococcus elongatus, the arrhythmic strain is rapidly out-competed by the rhythmic wild-type strain in light–dark cycles, but slowly outgrows the wild-type strain in constant light, which provides no selective pressure for circadian timekeeping. c | In competition among strains that are rhythmic, a strain that has an endogenous rhythm (free-running period (FRP)) that closely matches the environmental light–dark cycle out-competes strains with non-optimal free-running periods. Under continuous light, all strains are maintained in the population. d | Fitness can also be inferred from single-strain growth rates of cultures of wild-type or kaiC-knockout cells in the purple bacterium Rhodopseudomonas palustris growing phototrophically. Under continuous light, KaiC-dependent timekeeping confers no advantage to the growth of R. palustris. e | A cycle of 12 h of light and 12 h of darkness, which matches the environmental conditions to which R. palustris has adapted, provides wild-type cells with a fitness advantage over the kaiC-knockout strain. f | Under a cycle of 1 h of light and 1 h of darkness, both the wild-type and kaiC-knockout cells grow at the same rate. Parts a–c are adapted from REF. 101, Elsevier. Parts d–f are adapted from REF. 14.

Figure 6. A hypothesis for the evolution of Kai-based timekeepers in bacteria

The type of time keeper (that is, hourglass timer, damped oscillator or self-sustained circadian oscillator) that evolves is primarily determined by the regularity of the environmental daily cycle of the bacterium, as this is the dominant selective pressure. In very regular environments, an hourglass timer that is based on KaiC alone is sufficient. In fluctuating environments, which require more flexibility of the timer, a sustained oscillator based on KaiA–KaiB–KaiC is advantageous. In intermediate environments, a damped oscillator that is based on KaiB–KaiC is sufficient.