Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria.

Abstract

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...

No Promoter Left Behind: Global Circadian Gene Expression in Cyanobacteria

“…By fusing the psbA1 promoter, which drives expression of a component of photosystem II, to a promoterless luxAB gene cassette, transcription from this promoter was shown to be under circadian control in S. elongatus PCC 7942 (Kondo et al, 1993). A similar approach was used to show that expression from the dnaK promoter was rhythmic in Synechocystis sp.…”

Differences in the control of the temperature‐dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803

“…After the colonies on plates had been exposed to n-decyl aldehyde vapour for 3 min, the luminescence was determined as described by Kondo et al (1993;.…”

A Cyanobacterial Circadian Clock Based on the Kai Oscillator

“…Cyanobacteria are the simplest organisms known to exhibit circadian rhythms. Thus, the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter, Synechococcus) is an experimental model for the study of molecular mechanisms underlying these types of clocks (Kondo et al 1993). Saturation mutagenesis revealed that three adjacent genes (kaiA, kaiB, and kaiC) and their protein products are essential for circadian rhythm generation.…”