SummaryCellular life emerged ~3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles due to the Earth’s rotation. The advantage conferred upon organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterising their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular time-keeping with redox homeostatic mechanisms following the Great Oxidation Event ~2.5 billion years ago.

The environment significantly influences the dynamic expression and assembly of all components encoded in the genome of an organism into functional biological networks. We have constructed a model for this process in Halobacterium salinarum NRC-1 through the data-driven discovery of regulatory and functional interrelationships among approximately 80% of its genes and key abiotic factors in its hypersaline environment. Using relative changes in 72 transcription factors and 9 environmental factors (EFs) this model accurately predicts dynamic transcriptional responses of all these genes in 147 newly collected experiments representing completely novel genetic backgrounds and environments-suggesting a remarkable degree of network completeness. Using this model we have constructed and tested hypotheses critical to this organism's interaction with its changing hypersaline environment. This study supports the claim that the high degree of connectivity within biological and EF networks will enable the construction of similar models for any organism from relatively modest numbers of experiments.

BackgroundThe circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS→pTS→pTpS→TpS→TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC.Methodology and Principal FindingsThe conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP γ-phosphate. T432 is phosphorylated first because it lies consistently closer to Pγ. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation.Conclusions and SignificanceWe provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.

Abstract. Circadian oscillators are known to regulate the timing of cell division in many organisms. In the case of Chlamydomonas reinhardtii, however, this conclusion has been challenged by several investigators. We have reexamined this issue and find that the division behavior of Chlamydomonas meets all the criteria for circadian rhythmicity: persistence of a cell division rhythm (a) with a period of ~ 24 h under free-running conditions, (b) that is temperature compensated, and (c) which can entrain to light/dark signals. In addition, a mutation that lengthens the circadian period of the phototactic rhythm similarly affects the cell division rhythm. We conclude that a circadian mechanism determines the timing of cell division in Ch/amydomonas reinhardtii.

The effectiveness of drugs active in phase-shifting the circadian rhythm of bioluminescent glow in the unicellular dinoflagellate Gonyaulax polyedra differs, depending upon the time of drug exposure (as pulses). For two drugs tested--cycloheximide and anisomycin, both inhibitors of cytosolic protein synthesis--this function, referred to as the drug phase response curve (dPRC), differs, depending upon the ambient temperature. Since dPRCs may differ at different drug concentrations, the effects observed may be attributable to differences in the effectiveness of or recovery from the drugs at different temperatures.