EDGE FGT: Genetic Tools for Picocyanobacteria that Dominate the Oceans

作为地球上最丰富的光合作用有机体，海洋蓝藻原氯球菌在为海洋食物网提供能量和营养方面发挥着核心作用。仅这一物种每年固定的二氧化碳就大约与全球农田一样多。由于我们无法修改原氯球菌的基因以检验各种假说，我们无法理解原氯球菌的各种基因与其在海洋新陈代谢中的核心作用之间的联系。在最近进展的基础上，该项目旨在制定两种不同但并行的方法来应对这一挑战。首先，通过利用在一些原氯球菌菌株中发现的新发现的遗传元件系统，将绕过将外来DNA引入原氯球菌的障碍。在第二项研究中，将开发触发机制的方法，允许细胞吸收新基因并将其整合到细胞机制中。一旦开发出来，这些方法将使人们能够更好地了解原氯球菌基因与海洋环境之间的联系，这对于描述海洋过程在调节全球碳循环中所起的作用至关重要。这些进展还将为开发研究其他微生物的类似方法提供蓝图。最后，开发这些技术是使原氯球菌成为人工光合作用潜在底座的关键第一步。由于原氯球菌的基因组是所有光合作用细胞中最小的，它已经是自然界中最基本的光合作用机器，使其成为开发生物工程工具的理想候选者。该项目旨在开发的工具将为新的调查和生物技术应用提供巨大的潜力。该项目为研究生、一名博士后研究助理和两名技术人员提供支持。在过去的三十年里，蓝藻原氯球菌以其在海洋中的数量优势和巨大的遗传多样性而闻名，已经发展成为跨尺度系统生物学的模式生物。虽然每个细胞有大约2000个基因，但全球原氯球菌群的独特基因总量可能超过8万个基因，其中大多数具有未知的功能。因此，尽管原氯球菌在海洋新陈代谢中起着核心作用，但人们才刚刚开始了解原氯球菌不同的遗传含量与塑造它的选择压力之间的关系。由于缺乏基因系统，这一挑战的进展一直停滞不前。在EDGE计划支持的之前的工作中，研究人员开发了一种将外源DNA导入原氯球菌细胞的方法，这代表着基因组操作方面向前迈出了重要的一步。在这个过程中，发现了一个可移动的遗传元件系统，这是一种很有前途的新工具，可以将外源DNA整合到原氯球菌体中，电穿孔传递由本地遗传元件构建的酪蛋白。将开发的第二种方法利用一种基于转座子的新型筛选来确定在原氯球菌中触发自然能力活性的条件，并将产生一张有希望的整合位点的全基因组图谱。这两种方法都具有利用原氯球菌本身的DNA的优势，这应该更有效地绕过针对外源DNA的细胞防御。这一奖项反映了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。

As the most abundant photosynthetic organism on the planet, the marine cyanobacterium Prochlorococcus plays a central role in providing energy and nutrients to ocean food webs. This species alone fixes approximately as much carbon dioxide annually as global croplands. Understanding the link between the diverse genes of Prochlorococcus and its central role in ocean metabolism is hindered by our inability to modify its genes to test various hypotheses. Building on recent progress, this project aims to develop two distinct but parallel approaches to the challenge. In the first, barriers to the introduction of foreign DNA to Prochlorococcus will be bypassed by exploiting a system of newly discovered genetic elements that are found in some Prochlorococcus strains. In the second, approaches to trigger mechanisms that allow cells to take up and integrate new genes into their cellular machinery will be developed. Once developed, these approaches would allow better understanding of the link between Prochlorococcus genes and the ocean environment, which is critical for characterizing the role ocean processes play in regulating the global carbon cycle. These advances will also provide a blueprint for the development of similar methodologies for studying other microbial organisms. Finally, developing these technologies is a critical first step in enabling Prochlorococcus as a potential chassis for artificial photosynthesis. Since it has the smallest genome of any photosynthetic cell, Prochlorococcus is already the most basic photosynthetic machine occurring in nature, making it the ideal candidate for developing the tools that would enable bioengineering. The tools this project aims to develop would provide immense potential for new inquiry and biotechnology applications. This project provides support for graduate students, a postdoctoral research associate, and two technicians. Over the past three decades, the cyanobacterium Prochlorococcus, notable for its numerical dominance of the oceans and enormous genetic diversity, has been developed as a model organism for cross-scale systems biology. While each cell has around 2000 genes, the total unique gene content of the global Prochlorococcus ‘collective’ likely exceeds 80,000 genes, most of which have unknown function. Thus, despite their central role in ocean metabolism, the relationship between Prochlorococcus’ diverse genetic content, and the selective pressures that have shaped it, is only beginning to be understood. Progress on this challenge has been stalled by the lack of a genetic system. In previous work supported by the EDGE program, the researchers developed a method of introducing exogenous DNA into Prochlorococcus cells, which represented a significant step forward in genomic manipulation. In the process, a system of mobile genetic elements was discovered, which represent a promising new tool for integration of exogenous DNA into the Prochlorococcus, electroporation delivered tycheposons that are constructed with native genetic elements. A second approach to be developed utilizes a novel transposon-based screen to identify conditions for triggering natural competence activity among Prochlorococcus and will result in a genome-wide map of promising integration sites. Both approaches have the advantage of utilizing DNA from Prochlorococcus itself, which should more effectively bypass cell defenses against exogenous DNA.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.