Susan S. Golden

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.

Genetic engineering of the cyanobacterial chromosome.

Publisher Summary This chapter describes methods for the direct genetic engineering of the cyanobacterial chromosome and techniques for the analysis of DNA and RNA from the resulting transformants. The DNA in lane B described in the chapter is a by-product of the RNA isolation procedure. The genetic engineering of the cyanobacterial chromosome includes the techniques described in the chapter. The chapter focuses on methods that employ wholly homologous transforming DNA or heterologous sequences, which are flanked by cyanobacterial DNA on either side. The analysis of DNA from transformants is necessary to determine the chromosomal structure following recombination. The recombination events illustrated in the chapter are those that are usually seen, but aberrant events have been observed when the cloned region homologous to the chromosome is small. These events include the deletion of a chromosomal segment and integration at a related locus in the case of a gene family. Southern hybridization can determine whether the expected event has in fact, occurred. It is also important to analyze RNA from transformants, which have been engineered by these methods. A homologous recombination event, which replaces a wild-type gene with another functional allele, should result in the production of a full-length mitochondrial RNA (mRNA), which initiates and terminates properly. The chapter also mentions the instructions of the procedure that describe the preparation of glass fines used in the cyanobacterial total DNA miniprep procedure, which was adapted from the method of Vogelstein and Gillespie.

A KaiC-Interacting Sensory Histidine Kinase, SasA, Necessary to Sustain Robust Circadian Oscillation in Cyanobacteria

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp. strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.

Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: A potential clock input mechanism

In the cyanobacterium Synechococcus elongatus (PCC 7942) the proteins KaiA, KaiB, and KaiC are required for circadian clock function. We deduced a circadian clock function for KaiA from a combination of biochemical and structural data. Both KaiA and its isolated carboxyl-terminal domain (KaiA180C) stimulated KaiC autophosphorylation and facilitated attenuation of KaiC autophosphorylation by KaiB. An amino-terminal domain (KaiA135N) had no function in the autophosphorylation assay. NMR structure determination showed that KaiA135N is a pseudo-receiver domain. We propose that this pseudo-receiver is a timing input-device that regulates KaiA stimulation of KaiC autophosphorylation, which in turn is essential for circadian timekeeping.

Light-Driven Changes in Energy Metabolism Directly Entrain the Cyanobacterial Circadian Oscillator

Cyanobacterial circadian clock components are directly coupled to the metabolic status of the cell through interactions with adenine nucleotides. Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock.

Circadian clocks in prokaryotes

Prokaryotes have long been thought incapable of expressing circadian (daily) rhythms. Recently, however, such biological ‘clocks’ have been discovered in several species of cyanobacteria. These endogenous timekeepers control gene expression on a global level in cyanobacteria. Even in cyanobacterial cultures that are growing with average doubling times more rapid than one per 24 h, the circadian clock controls gene expression and cell division. We have isolated mutants of the cyanobacterial circadian pacemaker and are currently characterizing the loci responsible for their altered period phenotypes.

A sigma factor that modifies the circadian expression of a subset of genes in cyanobacteria.

We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp. strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype. The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16). We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70‐like transcription factors in Synechococcus. We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor. Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus. This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

How a cyanobacterium tells time.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator composed of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. An input pathway of redox-sensitive proteins uses photosynthetic function to relay light/dark information to the oscillator, and signal transduction proteins of well-known families broadcast temporal information to the genome, where global changes in transcription and a compaction of the chromosome are clock regulated.

LdpA: a component of the circadian clock senses redox state of the cell

The endogenous 24‐h (circadian) rhythms exhibited by the cyanobacterium Synechococcus elongatus PCC 7942 and other organisms are entrained by a variety of environmental factors. In cyanobacteria, the mechanism that transduces environmental input signals to the central oscillator of the clock is not known. An earlier study identified ldpA as a gene involved in light‐dependent modulation of the circadian period, and a candidate member of a clock‐entraining input pathway. Here, we report that the LdpA protein is sensitive to the redox state of the cell and exhibits electron paramagnetic resonance spectra consistent with the presence of two Fe4S4 clusters. Moreover, LdpA copurifies with proteins previously shown to be integral parts of the circadian mechanism. We also demonstrate that LdpA affects both the absolute level and light‐dependent variation in abundance of CikA, a key input pathway component. The data suggest a novel input pathway to the circadian oscillator in which LdpA is a component of the clock protein complex that senses the redox state of a cell.

Circadian gating of the cell cycle revealed in single cyanobacterial cells

Cycle Entrainment Cells manage many cyclic processes that must coordinate with each other for best cellular performance. Yang et al. (p. 1522) present a general theoretical framework that quantitatively describes coupled cyclic processes and then apply this to the interaction between the circadian and cell-division cycles in single cyanobacteria. Simultaneously tracking individual cell divisions and circadian phases and fitting the data with the model suggest that cell-cycle progression slows down dramatically during a specific circadian interval, whereas cell-cycle progression is independent of the cell-cycle phase. Modeling and observation of cyanobacteria show entrainment of the cell cycle by their biological clock. Although major progress has been made in uncovering the machinery that underlies individual biological clocks, much less is known about how multiple clocks coordinate their oscillations. We simultaneously tracked cell division events and circadian phases of individual cells of the cyanobacterium Synechococcus elongatus and fit the data to a model to determine when cell cycle progression slows as a function of circadian and cell cycle phases. We infer that cell cycle progression in cyanobacteria slows during a specific circadian interval but is uniform across cell cycle phases. Our model is applicable to the quantification of the coupling between biological oscillators in other organisms.