Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

In some organisms longevity, growth, and developmental rate are improved when they are maintained on a light/dark cycle, the period of which "resonates" optimally with the period of the endogenous circadian clock. However, to our knowledge no studies have demonstrated that reproductive fitness per se is improved by resonance between the endogenous clock and the environmental cycle. We tested the adaptive significance of circadian programming by measuring the relative fitness under competition between various strains of cyanobacteria expressing different circadian periods. Strains that had a circadian period similar to that of the light/dark cycle were favored under competition in a manner that indicates the action of soft selection.

We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem H protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAl message following light/dark entrainment supported the reporter data.The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock.Despite decades of study, the biochemical mechanism of circadian clocks remains a mystery. Circadian rhythms have been found in a wide spectrum of organisms (1) but, until recently, only in eukaryotes (2, 3). In the last few years circadian rhythms have been reported in the prokaryotic cyanobacteria (4-7). Unfortunately, these studies employed genetically intractable cyanobacterial strains and laborious assays to detect the rhythms. These difficulties have impeded the demonstration that the prokaryotic rhythms are equivalent to the circadian rhythms of eukaryotes.Proof that prokaryotes have circadian pacemakers has threefold significance. (i) With regard to the evolutionary emergence of circadian behavior: Can the simpler organization of prokaryotes support a circadian mechanism? Is circadian behavior adaptive for prokaryotic niches as well as for eukaryotic niches? (ii) The previous failure of attempts to discover circadian clocks in prokaryotes has led to a "eukaryotes-only" dogma which limited the types ofmodels that have been considered for the underlying clock mechanism (3). Now that prokaryotic cellular organization appears to be fully competent to generate circadian oscillations, a broader range of mechanisms can be seriously evaluated as candidates for the circadian pacemaker. (iii) The realization thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.prokaryotes express circadian behavior is significant from the perspective of designing an optimal s...

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green f luorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green f luorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.Interactions between proteins play a role in many biological processes. Current techniques to identify and characterize these interactions include in vitro-binding assays, library-based methods, and genetic methods (1). In this report, we introduce a method for assaying protein-protein interactions that takes advantage of a phenomenon that occurs in nature, namely, the Förster resonance energy transfer between a light-emitting luciferase and an acceptor fluorophore (2-5). The technique is related to an existing method for assessing protein-protein interaction, fluorescence resonance energy transfer (FRET). In this process, one fluorophore (the ''donor'') transfers its excited-state energy to another fluorophore (the ''acceptor''), which usually emits fluorescence of a different color. FRET efficiency depends on the spectral overlap, the relative orientation, and the distance between the donor and acceptor fluorophores. Generally, FRET occurs when the donor and acceptor are 10-100 Å apart (4), so it can be used to assay protein-protein proximity by attaching the donor and acceptor fluorophores to the candidate proteins. By using mutants of the green fluorescent protein (GFP; M r ϭ 27 kDa), it is possible to genetically attach donor and acceptor fluorophores to proteins (6-8). This GFP-based FRET assay allows protein interactions to be observed in the native organism under physiological conditions (9, 10). Moreover, compartmentalization of these interacting proteins is visible in the microscope (9, 10).As with any fluorescence technique, however, photobleaching and autofluorescence can limit the usefulness of FRET. It also can be complicated by direct excitation of the acceptor fluorophore. Furthermore, FRET may be impractical in tissues that are easily damaged by the excitation light or that are photoresponsive (e.g., retina). Our protein interaction assay, which we call bioluminescence resonance energy transfer (BRET), offers the advantages of FRET but avoids the consequences of fluorescence excitation. BRET is a naturally occurring phenomenon. For instance, when the photoprotein aequorin is purified from the...