EDGE FGT: Genome-editing tools for keystone freshwater heterotrophs

EDGE FGT：关键淡水异养生物的基因组编辑工具

• 批准号: 2118003
• 负责人: Julia Maresca
• 金额: $ 39.87万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2118003&HistoricalAwards=false
• 关键词: EDGE; FGT; Genome; editing; tools

Microbial activity drives carbon emission and burial in freshwater environments, which cover only 4% of Earth's surface but both emit and store disproportionally large amounts of carbon. Up to 60% of the bacteria in fresh waters belong to a specific group of Actinobacteria, and this group likely controls conversion of organic matter to CO2 and buried biomass in these ecosystems. Although these bacteria have fewer than 2000 genes, they thrive in bogs, algae-choked lakes, pristine alpine ponds, rivers, estuaries, and reservoirs. To understand how bacteria with so few genes are able to dominate in such a diverse range of environments, we propose to develop genome-editing tools in two easy-to-work-with species of freshwater Actinobacteria. The work proposed here, and the research that builds on it, will enable scientists working on freshwater carbon cycling, bacterial-algal interactions, heterotrophy, and light capture to identify the genes in freshwater Actinobacteria that contribute to these processes. That work, in turn, will provide experimental data linking genes to ecosystem functions. Laboratory research on the physiology and metabolism of freshwater clades of Actinobacteria has been hampered by having only a few cultivated species, none with available genome editing tools. To understand how such an apparently homogeneous group of bacteria contributes to nutrient cycling in such a diverse range of ecological settings, we propose to develop systems for targeted and random mutagenesis in Rhodoluna lacicola and Aurantimicrobium sp. strain MWH-Mo1, two easily cultivated, representative species of freshwater Actinobacteria. Targeted mutagenesis will enable not just inactivation of specific genes, but also insertion of genes for heterologous expression from the chromosome. Random transposon-based mutagenesis will enable construction of comprehensive mutant libraries that can be screened for fitness under a variety of different environmentally relevant stresses. These tools will catalyze research that connects simple genomes to complex phenotypes, phenotypes to biochemical networks in microbial communities, and microbial community metabolism to ecosystem carbon budgets.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.

微生物活动导致淡水环境中的碳排放和埋藏，淡水环境仅覆盖地球表面的 4%，但排放和储存的碳量却异常巨大。淡水中高达 60% 的细菌属于特定的放线菌群，该菌群可能控制着这些生态系统中有机物向二氧化碳和埋藏生物量的转化。尽管这些细菌只有不到 2000 个基因，但它们在沼泽、藻类堵塞的湖泊、原始高山池塘、河流、河口和水库中繁衍生息。为了了解基因如此少的细菌如何能够在如此多样化的环境中占据主导地位，我们建议在两种易于使用的淡水放线菌物种中开发基因组编辑工具。这里提出的工作以及在此基础上进行的研究将使致力于淡水碳循环、细菌-藻类相互作用、异养和光捕获的科学家能够识别淡水放线菌中促进这些过程的基因。反过来，这项工作将提供将基因与生态系统功能联系起来的实验数据。对淡水放线菌分支生理学和代谢的实验室研究受到了阻碍，因为只有少数栽培物种，而且没有可用的基因组编辑工具。为了了解如此明显同质的细菌群如何在如此多样化的生态环境中促进营养循环，我们建议开发用于 Rhodoluna lacicola 和 Aurantimicrobium sp 的定向和随机诱变系统。 MWH-Mo1 菌株，两个易于培养的淡水放线菌的代表种。定向诱变不仅可以使特定基因失活，还可以插入基因以从染色体进行异源表达。基于随机转座子的诱变将能够构建全面的突变体库，可以在各种不同的环境相关胁迫下筛选其适应性。这些工具将促进将简单基因组与复杂表型、表型与微生物群落生化网络以及微生物群落代谢与生态系统碳预算联系起来的研究。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。