No promoter left behind: global circadian gene expression in cyanobacteria

Abstract

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a clock system that appears to be similar to those of eukaryotes in many ways. On the other hand, the KaiABC-based core cyanobacterial clockwork is clearly different from the transcription-translation feedback loop model of eukaryotic clocks in that the cyanobacterial clock system regulates gene expression patterns globally, and specific clock gene promoters are not essential in mediating the circadian feedback loop. A novel model, the oscilloid model, proposes that the KaiABC oscillator ultimately mediates rhythmic changes in the status of the cyanobacterial chromosome, and these topological changes underlie the global rhythms of transcription. The authors suggest that this model represents one of several possible modes of regulating gene expression by circadian clocks, even those of eukaryotes.

Figure 1

Global circadian regulation of transcriptional activities in cyanobacteria. Representations of rhythmic waveforms from a promoter trap experiment (Liu et al., 1995) and the effect of KaiC overexpression on promoter activities (Nakahira et al., 2004) are shown in (A) and (B). Promoter activity was measured as luminescence from a bacterial luciferase (luxAB) reporter. (A) “Clock-dominated” genes. Waveforms representing both class 1 and class 2 promoter activities are shown. Promoters of clock-dominated genes display a high peak-to-trough amplitude with a relatively low basal level of activity. Overexpression of KaiC (lower panel, ± KaiC Ox) causes the high-amplitude waveform of these promoters to be abolished, and only low basal activity is observed (dashed line). (B) “Clock-modulated” genes. Promoters of clock-modulated genes display waveforms of luxAB expression with a low peak-to-trough amplitude but with a relatively high basal activity. Overexpression of KaiC abolishes the rhythmic expression, but a relatively high, nonrhythmic basal promoter activity remains (dashed line).

Figure 2

Circadian rhythms of plasmid supercoiling and cyanobacterial promoter activity. (A) Plot of plasmid DNA mobility versus time in constant light. Wild-type cyanobacterial cells were given a 12-h dark pulse prior to release into constant light, and plasmid DNA from cells was isolated; luminescence from a chromosomal psbaIp::luxAB reporter was measured in the same culture at various times after cells had been released into constant light. The mobility of an endogenous plasmid (pANS) was determined by chloroquine agarose gel electrophoresis (solid curve), and luminescence from the chromosomal reporter (dashed curve) is plotted versus time in constant light (Woelfle et al., manuscript in preparation). (B) A schematic representation of cyanobacterial promoter activity when the promoters driving a luxCDABE reporter are contained on a plasmid. Luminescence versus time in constant light is shown for kaiC⁺ and ΔkaiC cyanobacterial strains carrying the plasmid reporters. In kaiC⁺ cells, the psbAI promoter (a class 1 promoter) drives rhythmic transcription of luxCDABE in the same phase as when the promoter is located in the chromosome (upper panel), but promoter activity is arhythmic from the plasmid in cells in which kaiC is deleted (ΔkaiC). The purF promoter (class 2) drives transcription of luxCDABE from a plasmid displaying a rhythm that is in antiphase to that of psbAIp in kaiC⁺ cells (lower panel); this rhythmic promoter activity is abolished in ΔkaiC cells (Woelfle et al., manuscript in preparation).

Figure 3

The oscilloid model for the circadian system of cyanobacteria. KaiA, KaiB, and KaiC are transcribed and translated from the kaiABC gene cluster. KaiA (white cylinders) and KaiB (white cubes) interact with hexamers of KaiC (gray) to form KaiC-containing protein complexes forming the minimal KaiABC timing loop, which is capable of displaying rhythms of KaiC phosphorylation both in vivo and in vitro. The level of KaiC phosphorylation (indicated by stars on KaiC hexamers) in these complexes increases during the subjective day, reaching its peak around dusk; the level of phosphorylated KaiC decreases during the subjective night, reaching its lowest level around dawn. This minimal KaiABC-based timing loop is inactive in terms of regulating promoter activities and is subject to actions of light-dependent or metabolic “clutch,” presumably due to the interaction of an unknown protein(s). This clutch activates KaiC-containing protein complexes for either direct (inset A) or indirect (inset B) interaction with the cyanobacterial chromosome. The exact protein composition of “active” KaiC-containing protein complexes is not known. Inset A: Direct action of KaiC-containing complexes on DNA. Active KaiC-containing protein binds directly to chromosomal DNA to modulate levels of supercoiling. Inset B: Indirect action of KaiC-containing complexes on DNA. KaiC-containing complexes activate an intermediate factor or factors (e.g., second component pathways, DNA gyrases, topoisomerases, or other nucleoid-associated proteins) that in turn modulate levels of chromosomal supercoiling. Regardless of whether interaction of KaiC-containing protein complexes is direct or indirect, changes in chromosomal supercoiling affect the activity of both “clock-dominated” and “clock-modulated” promoters. Clock-dominated promoter activity is turned on and off by changes in chromosomal supercoiling. For clock-modulated promoters, the peak-to-trough amplitude, but not the basal level of activity (determined by the “basal input”), is affected by changes in chromosomal supercoiling. Gene products from the clock-dominated genes or other protein factors may influence the activity of clock-modulated genes.