Circadian Rhythms of Gene Expression in Cyanobacteria

Texas A&M Research Foundation, College Station TX

Investigators

Abstract

Abstract: Golden, MCB 9982852 An endogenous timekeeping mechanism, called the circadian clock, allows organisms from animals to cyanobacteria to regulate their physiological processes temporally and to coordinate these with the environment. The hallmarks of circadian-controlled processes are that these rhythms show a periodicity of approximately 24 h in the absence of environmental cues, their phasing can be reset by light/dark signals, and they are temperature compensated. These intrinsic properties of circadian clocks allow metabolic and behavioral events to be synchronized with the earth's daily cycles, acommodating seasonal changes in day length and daily changes in temperature. Defining the molecular components and biochemical mechanisms that give rise to the properties of circadian clocks is the central goal of circadian research in general, and this project specifically. Cyanobacteria are the only prokaryotes that have been shown to exhibit circadian rhythms. The ease of genetic manipulation in Synechococcus sp. strain PCC 7942 has resulted in rapid progress toward understanding the cyanobacterial circadian clock. The period, amplitude, and phasing of the cyanobacterial circadian rhythm of gene expression can be readily monitored from any Synechococcus promoter by using luciferase gene fusions (Vibrio harveyi luxAB, or firefly luc), such that light production reports transcription in real time. Previous research using this strategy identified a three-gene locus (kaiA, kaiB, kaiC) that is fundamentally important for cyanobacterial circadian rhythms. Mutations in any of the kai genes can cause a change in circadian period, and inactivation of any of them results in arrhythmia. The ability to generate mutants easily, to grow cyanobacteria with a short generation time, and to work with large populations of cells has allowed some important questions to be addressed very directly. For example, experiments with Synechococcus demonstrated that the circadian clock is operative even when cells are doubling much faster than once per day. Populations of mixed wild-type and mutant cells were used to show definitively that a circadian clock whose intrinsic period closely matches the light/dark cycle in the environment improves the fitness of cyanobacterial cells. This project has two aims. The first is to test the hypothesis that circadian rhythmicity depends on a transcriptional feedback loop involving the kai genes as a component of the timekeeping mechanism. This model is consistent with proposed mechanisms for the clocks of animals and fungi. However, phenotypes from mutants that desynchronize the normal phasing of kaiA and kaiBC suggest that the expected model may not be correct. Heterologous promoters will be used to bypass the natural transcriptional regulation of the kai genes and the strains will be monitored to determine whether timekeeping is still operational. The second aim is to understand output pathways that allow the circadian clock to orchestrate the timing of gene expression in the organism. This will be accomplished by: analysis of cis elements that allow some genes (such as purF and opcA) to be expressed in an exceptional phase (expression peaking near dawn, unlike most genes in the organism); identification of suppressors of known genes that affect the circadian timing of expression from subsets of genes; and isolation of new mutants that affect the circadian control of promoters such as that of kaiBC, for which no output pathway components have yet been identified. These experiments address both the mechanism of the clock itself, and the means by which circadian timekeeping is conveyed to the biological processes that the clock controls.