Cell Cycle Regulation in Caulobacter Crescentus

描述(由申请人提供)：细胞周期的适当的空间和时间调节对所有生物的生存和增殖至关重要。复杂的调节电路已经进化，以确保在细胞周期中稳定、有序地进行。令人惊讶的是，这些电路背后的分子机制和设计原理仍然不完全清楚，特别是在细菌中。α-蛋白细菌新月芽孢杆菌是一个实验上易于操作的系统，用于阐明细菌细胞周期调控的基本机制，并在系统水平上理解调控。铜绿假单胞菌的细胞周期主要由两个重要的调节因子驱动：CTRA和DNAA。CTRA是一种反应调节蛋白和双组分信号转导蛋白，直接调节近100个基因的表达，其中许多基因协调细胞分裂。CTRA还直接结合和沉默子代细胞的复制起点，但在子代柄细胞中被失活和降解。因此，CTRA主宰着子代细胞的不对称复制命运，这是Caulbacter细胞周期的一个标志。然而，CtrA并不显著影响DNA复制的基本周期。相反，Dna A，一种几乎普遍存在的启动DNA复制的细菌蛋白质，控制着复制的周期。在富媒体中，Dna A蛋白水平是恒定的，表明它主要在 活跃度。尽管在理解CtrA的监管方面取得了实质性进展，在较小程度上理解DNAA，但仍然存在重大差距。该项目的目标是确定在营养充足和饥饿后控制CtrA和Dna A的完整分子回路。我们的目标是识别这个回路的新组件，阐明它们与DNAA和CtrA的联系，并结合遗传、生化、生物信息学、基因组和细胞生物学方法来了解它们的动态。具体地说，我们的目标是(I)确定CtrA的激活是如何与DNA复制启动相耦合的，(Ii)鉴定和表征在富含介质中生长过程中调节Dna A活性的因素，以及(Iii)阐明营养饥饿后控制CtrA和Dna A活性的分子机制。这些研究将揭示细菌细胞如何在一系列生长条件下严格调控DNA复制和调节细胞周期进展。更好地了解细菌是如何调节细胞周期的，将指导新抗生素的开发，以减缓或阻止病原体的增殖。最后，通过将这里公布的调控策略与真核生物中使用的调控策略进行比较，我们的工作将有助于揭示整个生物学中调控电路的设计原则。

DESCRIPTION (provided by applicant): Proper spatial and temporal regulation of the cell cycle is crucial to the survival and proliferation of all organisms. Complex regulatory circuits have evolved to ensure stable, orderly progression through the cell cycle. Surprisingly, the molecular mechanisms and design principles underlying these circuits remain incompletely understood, particularly in bacteria. The alpha-proteobacterium Caulobacter crescentus is an experimentally tractable system for elucidating the fundamental mechanisms underlying cell cycle regulation in bacteria, and for understanding regulation at the systems level. The Caulobacter cell cycle is driven largely by two essential regulators: CtrA and DnaA. CtrA is a response regulator and two-component signal transduction protein that directly regulates the expression of nearly 100 genes, many of which coordinate cell division. CtrA also directly binds to and silences the origin of replication in daughter swarmer cells, but is inactivated and degraded in daughter stalked cells. CtrA thus governs the asymmetric replicative fates of daughter cells, a hallmark of the Caulobacter cell cycle. However, CtrA does not significantly influence the fundamental periodicity of DNA replication. Instead, DnaA, a nearly universal bacterial protein that initiates DNA replication, governs the periodicity of replication. In rich media, DnaA protein levels are constant, indicating that it is regulated predominantly at the level of activity. Although substantial progress has been made in understanding the regulation of CtrA and, to a lesser extent, DnaA, major gaps remain. The goal of this project is to define the complete molecular circuitry controlling CtrA and DnaA in both nutrient replete conditions and following starvation. We aim to identify new components of this circuitry, to elucidate their connections to DnaA and CtrA, and to understand their dynamics using a combination of genetic, biochemical, bioinformatic, genomic, and cell biological approaches. Specifically, we aim to (i) determine how the activation of CtrA is coupled to DNA replication initiation, (ii) idenify and characterize factors that regulate DnaA activity during growth in rich media, and (iii) elucidate the molecular mechanisms governing CtrA and DnaA activity following nutrient starvation. These studies will unveil how bacterial cells tightly regulate DNA replication and modulate cell cycle progression in a range of growth conditions. A better understanding of how bacteria regulate their cell cycle will guide the development of new antibiotics that slow or halt the proliferation of pathogens. Finally, by comparing the regulatory strategies unveiled here to those employed in eukaryotes our work will help to reveal the design principles of regulatory circuits throughout biology.