Circadian Rhythms of Gene Expression in Cyanobacteria

摘要：Golden，MCB 9982852一种内源性的计时机制，称为生物钟，允许从动物到蓝藻的生物体在时间上调节其生理过程，并使其与环境协调。昼夜节律控制过程的特点是，这些节律在没有环境线索的情况下显示出大约24小时的周期性，它们的相位可以通过亮/暗信号重置，并且它们是温度补偿的。生物钟的这些固有特性使得新陈代谢和行为活动与地球的日周期同步，从而协调了日长的季节性变化和温度的日变化。定义产生生物钟特性的分子组成和生化机制是生物钟研究的中心目标，特别是这个项目。蓝细菌是唯一显示出昼夜节律的原核生物。聚球藻属菌株PCC 7942的遗传操作的容易性导致了对蓝藻生物钟的理解的快速进展。通过使用荧光素酶基因融合体（哈氏弧菌luxAB或萤火虫luc），可以容易地从任何聚球藻启动子监测基因表达的蓝细菌昼夜节律的周期、幅度和定相，使得光产生以真实的时间报告转录。 以前的研究使用这种策略确定了一个三基因位点（kaiA，kaiB，kaiC），这是至关重要的蓝藻昼夜节律。任何kai基因的突变都能引起昼夜节律的改变，任何一个基因的失活都会导致心律失常。容易产生突变体的能力，以短的世代时间生长蓝藻，并与大量的细胞一起工作，使一些重要的问题得到了非常直接的解决。例如，对聚球藻的实验表明，即使细胞加倍的速度比每天一次快得多，生物钟也是有效的。混合野生型和突变体细胞的群体被用来明确地表明，生物钟的内在周期密切匹配的光/暗周期在环境中提高了蓝藻细胞的健身。该项目有两个目标。第一个是测试的假设，昼夜节律依赖于一个转录反馈回路，涉及作为计时机制的一个组成部分的kai基因。这个模型与动物和真菌的生物钟机制是一致的。然而，来自使kaiA和kaiBC的正常定相去突变的突变体的表型表明预期的模型可能不正确。异源启动子将用于绕过kai基因的天然转录调节，并且将监测菌株以确定计时是否仍然可操作。第二个目标是了解生物钟控制生物体基因表达时间的输出途径。这将通过以下方式实现：顺式元件的分析，（如purF和opcA）在异常阶段表达（与生物体中的大多数基因不同，表达在黎明附近达到峰值）;从基因子集中鉴定影响表达的昼夜节律定时的已知基因的抑制子;以及分离影响启动子如kaiBC的昼夜节律控制的新突变体，其输出途径成分尚未确定。这些实验既解决了生物钟本身的机制，也解决了生物钟控制的生物过程中昼夜节律计时的方法。

Abstract: Golden, MCB 9982852An 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.