Raman‐activated cell sorting and metagenomic sequencing revealing carbon‐fixing bacteria in the ocean

Abstract

It is of great significance to understand CO fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for β-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

An automated Raman-based platform for the sorting of live cells by functional properties

“…After a short incubation, the microbial cells that have consumed the compound and have incorporated the isotope into their biomass are detected by microautoradiography (for radioactive isotopes) 13 , NanoSIMS [14][15][16] or Raman microspectroscopy (for stable isotopes) [17][18][19][20] .…”

“…Upon clicking button (4), the program starts the RACS process: a Raman spectrum with 2-s acquisition time is taken every 2 s and the calculated PC value is displayed at window (7) in real time. When the program recognizes an optically captured cell (on the basis of the PC criterion), the spectrum is shown at window(18), and the optical tweezers move to the evaluation location (their current lateral position is displayed at window (13)). After calculating PL using a new Raman spectrum (5-s acquisition time), the program displays the PL value at window(8) and makes a decision according to the threshold set by the user at window(6).…”

Toward the Integrated Marine Debris Observing System

“…Popular and powerful direct hyperspectral/FT-IR techniques used on filtered samples (e.g., Karlsson et al, 2016) and being increasingly automated may be problematic in situ due to the optical absorption of water in preferred spectral ranges (e.g., 1,000-2,500 nm, Karlsson et al, 2016). However, Raman spectroscopy has previously been applied in situ for other applications (Kirkwood et al, 2013;Peltzer et al, 2016;Guo et al, 2017;Jing et al, 2018;Li et al, 2018), and for microplastics can be operated in a spectral range with low absorption in water (e.g., 785 nm laser, Frere et al, 2016). Imaging (Nelson et al, 2018) and flow cytometry (Sgier et al, 2016;Long et al, 2017) can also be used, currently in the lab to analyze small plastic particles.…”

Raman‐activated sorting of antibiotic‐resistant bacteria in human gut microbiota

“…Single-cell Raman micro-spectroscopy is a noninvasive profiling technology to reveal metabolic activity of single cells without externally tagging molecules (Huang et al, 2009;Zhang et al, 2015a;McIlvenna et al, 2016;Song et al, 2016;Song et al, 2017b;Jing et al, 2018;Lee et al, 2019). Raman microscopy with deuterium isotope probing (Raman-DIP) has been demonstrated to in situ identify the metabolic activity and pathway of single cells of bacterial cultures, intestinal bacteria in mouse caecum, bacteria in human faeces, and ARB in river water (Henk-Jan et al, 2008;Berry et al, 2015;Wang et al, 2016;Xu et al, 2017;Wang et al, 2019).…”