RUI: Characterization of the gene regulatory network promoting hormogonium development in the filamentous cyanobacterium Nostoc punctiforme

丝状蓝藻是大约25亿年前首次进化的一群光合作用微生物，使它们成为地球上最早的多细胞生物之一。这些相对简单的多细胞生物为研究发育生物学的基本方面提供了一个极好的、成本效益高的模型系统。此外，由于这些细菌相对容易进行基因改造，并且能够将阳光转化为能源，并为某些植物提供氮肥来源，它们在生物燃料生产和农业中具有相当大的开发兴趣。这个项目的重点是利用模式菌株点状念珠菌来表征控制能够移动的特化细丝发育的遗传机制。作为更广泛影响的一部分，该项目还将为本科生和研究生提供最先进的基因和分子技术方面的培训，并向当地学校提供外展计划，这些学校服务于不同的学生群体，其中许多人处于经济上的不利地位。该项目的方方面面将被纳入教学实验室，几名本科生和研究生将参与研究，包括从社区参与计划(C.I.P.)招募的学生。太平洋大学，为来自种族代表性不足和经济困难背景的第一代大学生提供服务。从CI.P.招募的学生将获得暑期研究助学金，并参加当地学校5-8年级教室的外展计划，在那里他们将传播一个使用便携式荧光显微镜探索细胞生物学的学习模块。丝状蓝藻是发育最复杂的原核生物之一，对这些生物的研究为发育生物学提供了宝贵的见解。许多物种发育出一系列特殊的细胞类型，包括被称为角质细胞的活动细丝，这些细胞有助于扩散。对于形成超细胞结构和建立对全球固氮有重要贡献的植物和真菌-蓝藻共生体，触角细胞也是必不可少的。目前，控制角质层发育和生态上重要的角质层依赖过程的潜在分子相互作用在很大程度上是未知的。根据最近的研究和初步数据，工作假说是Hormogonium的发育是由一个保守的基因调控网络控制的，该网络包括sigma因子、伙伴转换调控系统、HMP趋化系统和几个额外的未表征的成分。该项目有两个具体目标：1)确定和表征促进羊角藻发育的基因调控网络的组成部分；2)确定西格玛因子和伙伴转换调控系统在羊角藻发育中的作用。该项目使用正向和反向遗传方法，结合对突变菌株的一系列表型分析，包括形态分析和霍尔木素特异蛋白和多糖表达的免疫学分析，以及转录转录研究，以确定在丝状蓝藻模式点状念珠藻中促进霍莫霍尔库发育的基因调控网络。该项目的完成将为促进羊草属植物发育的基因调控网络模型奠定基础，然后该模型可用于合理设计丝状蓝藻的转基因菌株，用于各种应用，包括生物燃料生产和新型植物-蓝藻共生。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The filamentous cyanobacteria are a group of photosynthetic microorganisms that first evolved about 2.5 billion years ago, making them one of the first multicellular organisms on earth. These relatively simple multicellular organisms provide an excellent, cost-effective model system for studying fundamental aspects of developmental biology. Additionally, because these bacteria are relatively easy to modify genetically, and are capable of both converting sunlight into energy and providing a source of nitrogen-based fertilizer to certain plants, they are of considerable interest for development in biofuel production and agriculture. This project is focused on characterizing the genetic mechanism controlling the development of specialized filaments that are capable of movement, using the model strain Nostoc punctiforme. As part of the broader impacts, this project will also provide training for both undergraduate and graduate students in state-of-the art genetic and molecular techniques, as well as an outreach program to local area schools which serve a diverse student body, many of whom are economically disadvantaged. Aspects of this project will be incorporated into teaching labs and several undergraduate and graduate students will participate in the research, including students recruited from the Community Involvement Program (C.I.P.) at University of the Pacific, which serves first generation college students from ethnically underrepresented and economically disadvantaged backgrounds. Students recruited from C.I.P. will be provided support for summer research assistantships and also participate in an outreach program for 5th-8th grade classrooms at local schools where they will disseminate a learning module using a portable fluorescence microscope to explore cell biology.Filamentous cyanobacteria are among the most developmentally complex prokaryotes and the study of these organisms has provided valuable insight into developmental biology. Many species develop an array of specialized cell types including motile filaments termed hormogonia that facilitate dispersal. Hormogonia are also essential for the formation of supracellular structures and the establishment of nitrogen-fixing plant- and fungal-cyanobacterial symbioses that contribute significantly to global nitrogen fixation. Currently, the underlying molecular interactions controlling hormogonium development and ecologically important hormogonium-dependent processes are largely undefined. Based on recent studies and preliminary data, the working hypothesis is that hormogonium development is governed by a conserved gene regulatory network that includes sigma factors, partner-switching regulatory systems, the hmp chemotaxis system, and several additional uncharacterized components. This project has two specific aims: 1) Identify and characterize components of the gene regulatory network promoting hormogonium development, and 2) Define the role of sigma factors and partner-switching regulatory systems in hormogonium development. The project uses forward and reverse genetic approaches combined with a range of phenotypic analyses of mutant strains including morphological analyses and immunological assays of hormogonium-specific protein and polysaccharide expression, as well as transcriptomic studies to define the gene regulatory network promoting hormogonium development in the model filamentous cyanobacterium Nostoc punctiforme. Completion of this project will lay the foundation for a model of the gene regulatory network promoting hormogonium development that can then be used to rationally design genetically modified strains of filamentous cyanobacteria for various applications including biofuel production and novel plant-cyanobacterial symbioses.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.