Oceanic Crustal Fluid Single Cell Genomics Complements Metagenomic and Metatranscriptomic Surveys With Orders of Magnitude Less Sample Volume.

Timothy D’Angelo,Ramunas Stepanauskas,Nicole J Poulton,Beth N Orcutt,Julie A Huber,Jacqueline Goordial,Lauren Seyler

doi:10.3389/fmicb.2021.738231

Abstract

Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 103 cells ml–1) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis.

Highlights

Oceanic crustal fluids may be significant habitats for life on Earth, yet relatively little is known about the composition of life in these habitats, and even less is known about the functional potential of their microbial communities (Orcutt B. et al, 2020)
The sample used in this study was collected in October 2017 during cruise AT39-01 on R/V Atlantis using remotely operated vehicles (ROVs) Jason II to access the subseafloor borehole observatories previously installed at North Pond by the Integrated Ocean Drilling Program (IODP; Edwards et al, 2012b)
We evaluated the next-gen physiology approach of sorting of cells after short incubation with RedoxSensorTM Green cells from a low biomass (∼103 cells ml−1) deep biosphere crustal fluid, and compared the resulting interpretation of taxonomy and function of cells sorted with the redox-active dye with bulk metagenomes and metatranscriptomes from the same location sampled across multiple years (Seyler et al, 2020)

Summary

Introduction

Oceanic crustal fluids may be significant habitats for life on Earth, yet relatively little is known about the composition of life in these habitats, and even less is known about the functional potential of their microbial communities (Orcutt B. et al, 2020). Large volumes (101–104 L) of crustal fluids are often required to obtain sufficient biomass for sequencing nucleic acids (i.e., DNA or RNA) to determine community structure, functional potential, and transcriptomic activity using current metagenomic and metatranscriptomic technologies. Collecting these large volumes requires in situ fluid pumping systems (Cowen et al, 2012; Li et al, 2020) that necessitate considerable payload and time on the seafloor with remotely operated vehicles (ROVs) for sampling. Considering the infrequency of expeditions along with the above sampling limitations, only a handful of metagenomic datasets have been reported from these environments, and only one metatranscriptomic dataset has been reported (Jungbluth et al, 2017; Tully et al, 2018; Seyler et al, 2020)

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Discussion

Conclusion