Jing Xiaoyan, Gong Yanhai, Xu Teng, Meng Yu, Han Xiao, Su Xiaolu, Wang Jianmei, Ji Yuetong, Li Yuandong, Jia Zhongjun, Ma Bo, Xu Jian
Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics and Shandong Institute of Energy Research, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China.
mSystems. 2021 Jun 29;6(3):e0018121. doi: 10.1128/mSystems.00181-21. Epub 2021 May 27.
Soil harbors arguably the most metabolically and genetically heterogeneous microbiomes on Earth, yet establishing the link between metabolic functions and genome at the precisely one-cell level has been difficult. Here, for mock microbial communities and then for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) platform, which identifies, sorts, and sequences precisely one bacterial cell via its anabolic (incorporating D from heavy water) and physiological (carotenoid-containing) functions. We showed that (i) metabolically active cells from numerically rare soil taxa, such as spp., spp., spp., spp., and Pseudomonas spp., can be readily identified and sorted based on DO uptake, and their one-cell genome coverage can reach ∼93% to allow high-quality genome-wide metabolic reconstruction; (ii) similarly, carotenoid-containing cells such as spp., spp., spp., Pseudomonas spp., and spp. were identified and one-cell genomes were generated for tracing the carotenoid-synthetic pathways; and (iii) carotenoid-producing cells can be either metabolically active or inert, suggesting culture-based approaches can miss many such cells. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at exactly one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems. Soil is home to an enormous and complex microbiome that features arguably the highest genomic diversity and metabolic heterogeneity of cells on Earth. Their metabolic activities drive many natural processes of pivotal ecological significance or underlie industrial production of numerous valuable bioactivities. However, pinpointing "who is doing what" in a soil microbiome, which consists of mainly yet-to-be-cultured species, has remained a major challenge. Here, for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) method, which identifies, sorts, and sequences at the resolution of precisely one microbial cell via its catabolic and anabolic functions. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems.
土壤中可能存在着地球上代谢和基因最具异质性的微生物群落，但要在精确到单细胞水平上建立代谢功能与基因组之间的联系却很困难。在这里，我们先针对模拟微生物群落，然后针对土壤微生物群，建立了一个拉曼激活重力驱动单细胞封装与测序（RAGE-Seq）平台，该平台通过其合成代谢功能（从重水中掺入氘）和生理功能（含类胡萝卜素）来识别、分选并精确测序单个细菌细胞。我们发现：（i）来自数量稀少的土壤分类群（如 spp.、 spp.、 spp.、 spp.和假单胞菌属）的代谢活跃细胞，可以根据氘摄取情况轻松识别和分选，其单细胞基因组覆盖率可达约93%，从而实现高质量的全基因组代谢重建；（ii）同样，含类胡萝卜素的细胞（如 spp.、 spp.、 spp.、假单胞菌属和 spp.）也被识别出来，并生成了单细胞基因组以追踪类胡萝卜素合成途径；（iii）产生类胡萝卜素的细胞可能是代谢活跃的，也可能是惰性的，这表明基于培养的方法可能会遗漏许多此类细胞。作为一种能够在土壤中精确到单细胞分辨率建立代谢-基因组联系的拉曼激活细胞分选仪（RACS）家族成员，RAGE-Seq有助于在复杂生态系统中精确查明“谁在做什么”。土壤是庞大而复杂的微生物群落的家园，其具有地球上细胞最高的基因组多样性和代谢异质性。它们的代谢活动驱动着许多具有关键生态意义的自然过程，或构成众多有价值生物活性物质工业生产的基础。然而，在主要由尚未培养的物种组成的土壤微生物群落中，查明“谁在做什么”仍然是一项重大挑战。在这里，针对土壤微生物群，我们建立了一种拉曼激活重力驱动单细胞封装与测序（RAGE-Seq）方法，该方法通过其分解代谢和合成代谢功能在精确到单个微生物细胞的分辨率上进行识别、分选和测序。作为一种能够在土壤中以单细胞分辨率建立代谢-基因组联系的拉曼激活细胞分选仪（RACS）家族成员，RAGE-Seq有助于在复杂生态系统中精确查明“谁在做什么”。
