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

最近，人们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，促使人们对这些生物的研究产生了越来越大的兴趣。本研究旨在探讨蓝藻细胞分裂的分子调控。该项目将使用生化方法、先进的显微镜和计算机模拟的组合来研究蓝藻细胞分裂的核心机制。该项目将调查光线对这种有机体细胞分裂的影响。作为该项目的相关部分，将开发一种新的分子生物学方法，旨在通过实验控制蓝藻中目标蛋白的丰度，为研究控制分裂的重要蛋白质提供额外的工具，也为蓝藻的研究和工程提供一种潜在的强大的新方法。从该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。该计划还将通过本科研究经验为两名研究生和众多来自代表不足群体的本科生提供培训机会。本项目将使用模型单细胞杆状蓝藻细长聚球藻PCC7942来研究蓝藻细胞分裂的机制和调控方面。将结合定量成像、生化、分子工程和建模方法，了解蓝藻独特的细胞结构和光合作用生活方式如何影响它们的细胞分裂系统，并与经典异养模型中的研究相关联。具体地说，控制细胞分裂复合体(分裂体)定位的Min系统蛋白的长链球菌同源物将通过定位研究进行研究，该研究涉及仔细构建在内源和/或可调节启动子下表达的功能报告。蓝藻Min蛋白的动态、它们表现振荡行为的能力、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中以及在大的和潜在的混杂的类囊体膜系统的背景下进行研究。还将研究光、昼夜节律和光合代谢对最小因子和细胞分裂的控制和活性的上游影响。为了便于重要/必需分裂蛋白的功能分析，将开发一种基于降解子标签的系统，以允许目标蛋白的可诱导降解。这项技术可能广泛适用于对超出本项目范围的蛋白质丰度进行可预测的调节。最后，该项目将利用计算机模拟来检验预测的类囊体膜结构对Min系统自组织特性的影响，并将建模预测与在长柄链霉菌中实验得出的结果进行比较。

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.