Cyanobacterial Cell Division: Mechanisms and Inputs Towards the Decision to Divide

最近，认识到蓝藻作为生物工业和可持续生物能源解决方案的工程平台的巨大前景，推动了对这些生物研究的兴趣日益浓厚。本研究项目旨在研究蓝藻细胞分裂的分子调控。该项目将结合生化方法、先进的显微镜和计算模拟来研究蓝藻细胞分裂的核心机制。该项目将研究光对这种生物细胞分裂的影响。作为该项目的一个相关部分，一种新的分子生物学方法，旨在允许实验控制蓝藻中目标蛋白的丰度，将被开发，提供了一个额外的工具来研究重要的蛋白质控制分裂，但也允许一个潜在的强大的蓝藻研究和工程的新方法。从这个项目中获得的基本知识将广泛涉及进化和生态学的科学问题，以及蓝藻工程和“绿色”生物能源的应用。该提案还将通过本科生研究经验，为两名研究生和众多来自代表性不足群体的本科生提供培训机会。本项目将采用单细胞棒状蓝藻长聚球菌模型PCC7942，研究蓝藻细胞分裂的机制和调控方面。定量成像、生化、分子工程和建模方法的结合将被用来了解蓝藻独特的细胞结构和光合生活方式如何影响它们的细胞分裂系统，与那些在经典异养模型中研究的细胞分裂系统有关。具体来说，控制细胞分裂复合体（分裂体）定位的Min系统蛋白的S. elongatus同源物将通过定位研究进行研究，包括在内源性和/或可调启动子下表达的功能报告子的仔细构建。蓝藻Min蛋白的动力学，它们显示振荡行为的能力，它们的遗传和生化相互作用，以及它们对分裂体组织的影响，将在相关的突变体和遗传背景下，以及在大的和可能混淆的类囊体膜系统的背景下进行研究。光、昼夜节律和光合代谢对Min因子的控制和活性以及细胞分裂的上游影响也将被研究。为了促进重要/必需分裂蛋白的功能分析，将开发一种基于降解标签的系统来诱导降解目标蛋白。这项技术可能广泛适用于可预测地调节蛋白质丰度，超出了本项目的范围。最后，该项目将利用计算机模拟来检验类囊体膜结构对Min系统自组织特性的预测影响，并将模型预测与长形螺的实验推导结果进行比较。

Recently, recognition of the considerable promise of cyanobacteria as engineering platforms for bioindustrial and sustainable bioenergy solutions has fueled growing interest in research on these organisms. This research project aims to address the molecular regulation of cyanobacterial cell division. The project will use a combination of biochemical methods, advanced microscopy, and computational simulation to investigate core mechanisms of cell division in cyanobacteria. The project will investigate the influence that light has on this organism's cell division. As a related part of the project, a new molecular biology approach designed to allow experimental control of the abundance of target proteins in cyanobacteria will be developed, providing an additional tool to study the important proteins controlling division, but also permitting a potentially powerful new method for cyanobacterial research and engineering. The fundamental knowledge to be gained from this project will be broadly relevant to scientific questions of evolution and ecology, as well as to cyanobacterial engineering and applications in "green" bioenergy. The proposal will also provide training opportunities for two graduate students and numerous undergraduates from underrepresented groups through undergraduate research experiences.This project will employ the model unicellular rod-shaped cyanobacterium Synechococcus elongates, PCC7942, to investigate mechanistic and regulatory aspects of cell division in cyanobacteria. A combination of quantitative imaging, biochemical, molecular engineering, and modeling approaches will be used to understand how the unique cellular architecture and photosynthetic lifestyle of cyanobacteria impact their cell division systems in relation to those studied in classic heterotrophic models. Specifically, the S. elongatus homologs of the Min system proteins, which control the positioning of the cell division complex (divisome), will be investigated using localization studies involving careful construction of functional reporters expressed under endogenous and/or tunable promoters. The dynamics of the cyanobacterial Min proteins, their capacity to display oscillatory behavior, their genetic and biochemical interactions, and their influences on the organization of the divisome will be examined in relevant mutants and genetic backgrounds, and in the context of the large and potentially confounding thylakoid membrane system. The upstream influences of light, circadian rhythms, and photosynthetic metabolism on the control and activity of Min factors and cell division will also be investigated. To facilitate functional analysis of important/essential division proteins, a degron tag-based system will be developed to allow inducible degradation of target proteins. This technique may be broadly applicable to predictably regulating protein abundance beyond the scope of this project. Finally, the project will utilize computer simulations to examine the predicted effects of thylakoid membrane structure on the self-organization properties of the Min system and to compare modeling predictions to the experimentally derived results in S. elongatus.