The itty-bitty time machine genetics of the cyanobacterial circadian clock.

The cyanobacterium Synechococcus elongatus PCC 7942 has been used as the prokaryotic model system for the study of circadian rhythms for the past two decades. Its genetic malleability has been instrumental in the discovery of key input, oscillator, and output components and has also provided monumental insights into the mechanism by which proteins function to maintain and dictate 24-h time. In addition, basic research into the prokaryotic system has led to interesting advances in eukaryotic circadian mechanisms. Undoubtedly, continued genetic and mutational analyses of this single-celled cyanobacterium will aid in teasing out the intricacies of the Kai-based circadian clock to advance our understanding of this system as well as other more "complex" systems.

A novel allele of kaiA shortens the circadian period and strengthens interaction of oscillator components in the cyanobacterium Synechococcus elongatus PCC 7942.

The basic circadian oscillator of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942, the model organism for cyanobacterial circadian clocks, consists of only three protein components: KaiA, KaiB, and KaiC. These proteins, all of which are homomultimers, periodically interact to form large protein complexes with stoichiometries that depend on the phosphorylation state of KaiC. KaiA stimulates KaiC autophosphorylation through direct physical interactions. Screening a library of S. elongatus transposon mutants for circadian clock phenotypes uncovered an atypical short-period mutant that carries a kaiA insertion. Genetic and biochemical analyses showed that the short-period phenotype is caused by the truncation of KaiA by three amino acid residues at its C terminus. The disruption of a negative element upstream of the kaiBC promoter was another consequence of the insertion of the transposon; when not associated with a truncated kaiA allele, this mutation extended the circadian period. The circadian rhythm of KaiC phosphorylation was conserved in these mutants, but with some modifications in the rhythmic pattern of KaiC phosphorylation, such as the ratio of phosphorylated to unphosphorylated KaiC and the relative phase of the circadian phosphorylation peak. The results showed that there is no correlation between the phasing of the KaiC phosphorylation pattern and the rhythm of gene expression, measured as bioluminescence from luciferase reporter genes. The interaction between KaiC and the truncated KaiA was stronger than normal, as shown by fluorescence anisotropy analysis. Our data suggest that the KaiA-KaiC interaction and the circadian pattern of KaiC autophosphorylation are both important for determining the period, but not the relative phasing, of circadian rhythms in S. elongatus.

Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus.

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.

Viral photosynthetic reaction center genes and transcripts in the marine environment.

Cyanobacteria of the genera Synechococcus and Prochlorococcus are important contributors to photosynthetic productivity in the open ocean. The discovery of genes (psbA, psbD) that encode key photosystem II proteins (D1, D2) in the genomes of phages that infect these cyanobacteria suggests new paradigms for the regulation, function and evolution of photosynthesis in the vast pelagic ecosystem. Reports on the prevalence and expression of phage photosynthesis genes, and evolutionary data showing a potential recombination of phage and host genes, suggest a model in which phage photosynthesis genes help support photosynthetic activity in their hosts during the infection process. Here, using metagenomic data in natural ocean samples, we show that about 60% of the psbA genes in surface water along the global ocean sampling transect are of phage origin, and that the phage genes are undergoing an independent selection for distinct D1 proteins. Furthermore, we show that different viral psbA genes are expressed in the environment.

Winding up the cyanobacterial circadian clock.

The endogenous circadian clock of the cyanobacterium Synechococcus elongatus controls many cellular processes and confers an adaptive advantage on this organism in a competitive environment. To be advantageous, this internal biological oscillator must be reset daily to remain in synchrony with its environment and to transduce temporal information to control behaviors at appropriate times of day. Recent studies have discovered new components of these input and output pathways of the clock that help to 'wind up' our understanding of the clock system as a whole. Here we review the mechanisms by which S. elongatus maintains internal time, discuss how external stimuli affect this oscillation, and evaluate the mechanisms underlying circadian controlled cellular events.

Detection of rhythmic bioluminescence from luciferase reporters in cyanobacteria.

The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying prokaryotic circadian rhythms. Although S. elongatus does not display an easily measurable overt circadian behavior, its gene expression is under circadian control; hence, a "behavior" is created by linking a cyanobacterial promoter to either the bacterial luxAB or firefly luc luciferase genes to create reporter fusions whose activity can be easily monitored by bioluminescence. Numerous vectors have been created in our lab for introducing luciferase reporter genes into the S. elongatus chromosome. A choice of methods and equipment to detect light production from the luciferase fusions provides a means for high-throughput, automated mutant screens as well as testing rhythms from two promoter fusions within the same cell culture.