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相当大的承诺，推动了对这些生物体的研究越来越感兴趣。本研究旨在探讨蓝藻细胞分裂的分子调控。该项目将使用生物化学方法，先进的显微镜和计算模拟相结合，以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，将开发一种新的分子生物学方法，用于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，同时也为蓝藻研究和工程提供了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。该项目将利用单细胞杆状蓝藻Synechococcus elongates（PCC 7942）的模型，研究蓝藻细胞分裂的机制和调控。定量成像，生物化学，分子工程和建模方法的组合将被用来了解蓝藻的独特的细胞结构和光合作用的生活方式如何影响他们的细胞分裂系统在经典的异养模型研究。具体来说，S.控制细胞分裂复合体（分裂体）定位的Min系统蛋白的细长体同源物将使用定位研究进行研究，所述定位研究涉及在内源性和/或可调启动子下表达的功能性报告基因的仔细构建。蓝藻Min蛋白的动态，他们的能力，显示振荡行为，他们的遗传和生化相互作用，以及他们对组织的divisome的影响将在相关的突变体和遗传背景，并在大的和潜在的混淆类囊体膜系统的背景下进行检查。光，昼夜节律，光合代谢的控制和活动的Min因素和细胞分裂的上游影响也将进行调查。为了促进重要/必需分裂蛋白的功能分析，将开发基于降解决定子标签的系统以允许靶蛋白的诱导降解。这种技术可能广泛适用于可预测地调节超出本项目范围的蛋白质丰度。最后，该项目将利用计算机模拟来研究类囊体膜结构对Min系统自组织特性的预测影响，并将模拟预测与S.细长的

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.