Admin. Supplement for Equipment: Molecular and cellular mechanisms of circadian timekeeping in a prokaryote model

行政。

基本信息

批准号：
10811051
负责人：
SUSAN S GOLDEN
金额：
$ 19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-04 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10811051
关键词：
Address Anacystisnidulans Animals Bar Codes Binding Biochemical Biological Biological Assay Biological Clocks Biological Models Biological Rhythm Biotechnology Cardiovascular Diseases Cell physiology Cells Circadian Dysregulation Circadian Rhythms Clock protein Complex Cryo-electron tomography Cues Cyanobacterium Cytology Disease Environment Equipment Escherichia coli Eukaryota Event Functional disorder Genes Genetic Genomics Growth Health Human In Vitro Ions Libraries Malignant Neoplasms Mammals Mediating Mental disorders Metabolic Metabolic syndrome Metabolism Modeling Molecular Nucleotides Organism Personal Satisfaction Phase Phenotype Phosphotransferases Phylogenetic Analysis Physiological Physiology Preparation Production Prokaryotic Cells Property Protein Biochemistry Proteins Regulation Resolution Sasa Signal Transduction Site Sleep Disorders System Time Trees Visualization Work circadian circadian pacemaker experimental study fitness model organism pathogen promoter reconstitution transcription factor

项目摘要

This project leverages a cyanobacterial model system to answer the following questions: what are the molecular interactions that mark the passage of time in a cell, where do they occur in the cell, how do they mediate temporal regulation of events, and why does biological timing matter for fitness? The circadian biological clock is an oscillatory timer that drives 24-h rhythms of biological activities. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. However, the circadian clock is pervasive well beyond mammals, promoting fitness in diverse organisms throughout the phylogenetic tree. The circadian clock of the cyanobacterium Synechococcus elongatus generates bona fide circadian rhythms of genetic, physiological, and metabolic activities that fulfill all criteria that define circadian clocks in eukaryotes. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. A new in vitro preparation comprising the oscillator proteins KaiA, KaiB, and KaiC, along with the kinases CikA and SasA and the transcription factor RpaA, reconstitutes the circadian rhythm of binding of RpaA to its target promoter with a real-time readout. This project will apply the in vitro clock and other technical and conceptual advances towards biochemical, cytological, genomic, and physiological objectives that will answer the target questions. The in vitro clock will reveal the molecular events that occur when the clock resets to an environmental timing cue, identify the sites of action of nucleotides that modulate the timing circuit, and determine how RpaA and a second transcription factor that is regulated by environmental signals, RpaB, work together to influence circadian phasing. The discovery that the kinases SasA and CikA impart tolerance to fluctuating oscillator component concentrations will overcome past hurdles for establishing a circadian circuit in Escherichia coli as a naïve model system for exploring clock connections to cellular physiology and for biotechnology applications. High-resolution cryo-electron tomography and focused ion-beam milling will be used to visualize clock-controlled daily changes in intracellular organization and the clock complex itself. The molecular basis and fitness advantage of circadian control of natural transformation will be determined. A bar-coded transposon library first used to identify all genes required for photoautotrophic growth will be used to identify new loci that contribute to fitness in a day-night cycle. Paired with physiological and metabolic assays, these experiments will answer the question: why does the timing of molecular events matter? Together, these approaches will elucidate clock mechanisms and the value of the clock to diurnal physiology, and will advance biotechnological opportunities for controlling metabolism in both photosynthetic and traditional bacterial production systems.

该项目利用蓝细菌模型系统回答以下问题：分子是什么标志着细胞中时间的流逝，它们在细胞中发生的何处，它们如何介导临时性的相互作用事件的调节，为什么生物时机对健身至关重要？昼夜的生物钟是驱动生物活动的24小时节奏的振荡计时器。人类的时钟功能障碍与心血管疾病，癌症，代谢综合征，精神疾病等健康状况范围睡眠障碍。但是，昼夜节律的钟表远远超出了哺乳动物，促进潜水员的健身整个系统发育树的生物。蓝细菌的昼夜节律钟表 Elongatus产生真正的遗传，生理和代谢活动的昼夜节律节奏定义真核生物的昼夜节律的标准。在这种遗传上的模型生物中，有可能系统地改变时钟蛋白的物理和生化特性，并追踪这些影响通过蛋白质互动网络从其代理效应到表达的昼夜节律的变化表型。一项新的体外制剂，完成振荡器蛋白kaia，kaib和kaic，以及激酶Cika和SASA以及转录因子RPAA，重构RPAA结合的昼夜节律通过实时读数到其目标启动子。该项目将应用体外时钟和其他技术以及将回答生化，细胞学，基因组和物理对象的概念进步目标问题。体外时钟将揭示时钟重置为时发生的分子事件环境时机提示，确定调节正时电路的核动肽的作用部位，并确定 RPAA和第二个转录因子如何受环境信号（RPAB）调节影响昼夜节律。激酶Sasa和Cika赋予了波动的耐受性的发现振荡器成分浓度将克服过去的障碍，以在大Escherichia中建立昼夜节律大肠杆菌是一种幼稚的模型系统，用于探索与细胞生理和生物技术的时钟连接申请。高分辨率的冷冻电子层析成像和聚焦离子束铣削将用于可视化时钟控制的细胞内组织和时钟复合物本身的每日变化。分子基础和将确定昼夜节律控制自然转化的适应性优势。条形编码的转座子库首先用于识别光自营养生长所需的所有基因在昼夜周期中有助于健身。这些实验将与物理和代谢测定法配对回答问题：为什么分子事件的时机很重要？这些方法将共同阐明时钟机制和时钟对昼夜生理学的价值，并将提高生物技术在光合和传统细菌生产系统中控制新陈代谢的机会。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit.

DOI：
10.1016/j.lssr.2022.11.001
发表时间：
2023-03
期刊：
LIFE SCIENCES IN SPACE RESEARCH
影响因子：
2.5
作者：
Bishe, Bryan;Golden, Susan S.;Golden, James W.
通讯作者：
Golden, James W.

An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium.

DOI：
10.1073/pnas.2211789119
发表时间：
2022-11-08
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
通讯作者：

A Hard Day's Night: Cyanobacteria in Diel Cycles.

DOI：
10.1016/j.tim.2018.11.002
发表时间：
2019-03
期刊：
Trends in microbiology
影响因子：
15.9
作者：
D. Welkie;Benjamin E. Rubin;Spencer Diamond;Rachel D. Hood;David F Savage;S. Golden
通讯作者：
D. Welkie;Benjamin E. Rubin;Spencer Diamond;Rachel D. Hood;David F Savage;S. Golden

Comparative Genomics of Synechococcus elongatus Explains the Phenotypic Diversity of the Strains.

DOI：
10.1128/mbio.00862-22
发表时间：
2022-06-28
期刊：
mBio
影响因子：
6.4
作者：
通讯作者：

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus

DOI：
10.1073/pnas.1812871115
发表时间：
2018-12
期刊：
Proceedings of the National Academy of Sciences
影响因子：
0
作者：
Yiling Yang;Vinson Lam;M. Adomako;Ryan Simkovsky;A. Jakob;N. Rockwell;Susan E. Cohen;Susan E. Cohen;A. Taton;Jingtong Wang;J. Lagarias;A. Wilde;David Nobles;J. Brand;S. Golden
通讯作者：
Yiling Yang;Vinson Lam;M. Adomako;Ryan Simkovsky;A. Jakob;N. Rockwell;Susan E. Cohen;Susan E. Cohen;A. Taton;Jingtong Wang;J. Lagarias;A. Wilde;David Nobles;J. Brand;S. Golden