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

Abstract

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 13 C-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.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging

“…using Raman active cell sorting SIP identified, sorted and isolated the active toluene degrader single cell Pigmentiphaga. 23 Li Menqui et al . have shown the potential of RSIP using phenylalanine and thymine as Raman spectral biomarkers to track the carbon flow in the predator–prey model at the single-cell level.…”

“…22 Li J. et al using Raman active cell sorting SIP identified, sorted and isolated the active toluene degrader single cell Pigmentiphaga. 23 Li et al have shown the potential of RSIP using phenylalanine and thymine as Raman spectral biomarkers to track the carbon flow in the predator-prey model at the single-cell level. 20 Although these studies explored the possibilities of RSIP, we considered RrSIP a cost-effective approach with respect to RSIP, as preparing the labelled cell for inoculation, and growing in unlabelled media will require an extremely low amount of stable isotope.…”

Raman cell sorting for single-cell research

“…They successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells. Additionally, the genotypes and phenotypes of this degrader were directly correlated at the single-cell level ( Li et al, 2023 ). Li et al used 13C-labeled phenanthrene as the target and developed a new method coupling MMI−SIP and RACS.…”

“…Consequently, Raman cell sorting has become a widely embraced analytical method for rapid, non-destructive, and non-labeled single-cell characterization ( Wagner, 2009 ). Raman cell sorting technology will generate suitable live cells for subsequent single-cell genomics, metagenomics, and gene sequencing, and this technology will link the phenotype and genotype of live cells ( Song et al, 2017 ; Jing et al, 2018 ; Li et al, 2022 ; Li et al, 2023 ).…”

Raman-Activated Cell Ejection for Validating the Reliability of the Raman Fingerprint Database of Foodborne Pathogens

“…Furthermore, the heat generated by the pulse laser of appropriate power was extremely limited; it was almost harmless to cells [ 16 ]. So far, RACE has been applied to investigate the genome and metabolic mechanism of microorganisms from oral cavity [ 17 ], soil [ 18 ], ocean [ 19 ], intestinal tract [ 20 ] and so on. Therefore, RACE has tremendous untapped potential in the field of rapid identification for foodborne pathogens.…”