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In Situ Discrimination and Cultivation of Active Degraders in Soils by Genome-Directed Cultivation Assisted by SIP-Raman-Activated Cell Sorting

稳定同位素探测 基因组 基因组 单元格排序 微生物 富集培养 生物 鼠李糖脂 计算生物学 生物修复 基因 遗传学 细菌 细胞 铜绿假单胞菌
作者
Jibing Li,Dayi Zhang,Chunling Luo,Bei Li,Gan Zhang
出处
期刊:Environmental Science & Technology [American Chemical Society]
卷期号:57 (44): 17087-17098 被引量:13
标识
DOI:10.1021/acs.est.3c04247
摘要

The identification and in situ cultivation of functional yet uncultivable microorganisms are important to confirm inferences regarding their ecological functions. Here, we developed a new method that couples Raman-activated cell sorting (RACS), stable-isotope probing (SIP), and genome-directed cultivation (GDC)─namely, RACS-SIP-GDC─to identify, sort, and cultivate the active toluene degraders from a complex microbial community in petroleum-contaminated soil. Using SIP, we successfully identified the active toluene degrader Pigmentiphaga, the single cells of which were subsequently sorted and isolated by RACS. We further successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells, which was confirmed by gyrB gene comparison and average nucleotide identity determination. Additionally, the genotypes and phenotypes of this degrader were directly linked at the single-cell level, and its complete toluene metabolic pathways in petroleum-contaminated soil were reconstructed. Based on its unique metabolic properties uncovered by genome sequencing, we modified the traditional cultivation medium with antibiotics, amino acids, carbon sources, and growth factors (e.g., vitamins and metals), achieving the successful cultivation of RACS-sorted active degrader Pigmentiphaga sp. Our results implied that RACS-SIP-GDC is a state-of-the-art approach for the precise identification, targeted isolation, and cultivation of functional microbes from complex communities in natural habitats. RACS-SIP-GDC can be used to explore specific and targeted organic-pollution-degrading microorganisms at the single-cell level and provide new insights into their biodegradation mechanisms.
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