EDGE FGT: Genome-editing tools for keystone freshwater heterotrophs

微生物活动推动了淡水环境中的碳排放和埋藏，淡水环境只占地球表面的4%，但同时排放和储存了不成比例的大量碳。淡水中高达60%的细菌属于特定的放线菌群，这一群可能控制着这些生态系统中有机物向二氧化碳和埋藏的生物量的转化。尽管这些细菌的基因不到2000个，但它们在沼泽、藻类泛滥的湖泊、原始的高山池塘、河流、河口和水库中茁壮成长。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操作的淡水放线菌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有助于这些过程的基因。这项工作反过来将提供将基因与生态系统功能联系起来的实验数据。淡水放线菌分支的生理学和新陈代谢的实验室研究一直受到阻碍，因为只有几个栽培物种，没有一个拥有可用的基因组编辑工具。为了了解这样一组表面上同质的细菌如何在如此多样化的生态环境中促进营养循环，我们建议开发在乳红菌和金抗微生物中进行定向和随机突变的系统。菌株MWH-MO1，两个易于培养的淡水放线菌的代表种。定向突变不仅可以使特定基因失活，还可以插入来自染色体的异源表达基因。基于随机转座子的突变将使构建全面的突变体文库成为可能，这些突变文库可以在各种不同的环境相关压力下进行适应性筛选。这些工具将促进将简单基因组与复杂表型、表型与微生物群落中的生化网络、微生物群落代谢与生态系统碳收支联系起来的研究。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.