Abstract
Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.
Highlights
Binding selectivity, based on the anticipated high affinity of some cellulose-degrading microbes for cellulose, was monitored using flow cytometry by first identifying the labeled cellulose particles, and identifying the fraction that became bound by bacteria over time (Fig. 1a, b)
In an exponentially growing culture of C. hutchinsonii we observed that ~40% of the cells were substrate bound after 2 h, and ~65% after 7 h of incubation, while for C. cellulolyticum, ~2.5% of the cells were bound to cellulose after 2 h and ~8% after 7 h (Fig. 1b)
The noncellulolytic organism E. coli showed no binding in similar experiments and binding was undetectable in heat-killed C. hutchinsonii (Fig. 1b)
Summary
New techniques and instrumentation are being developed including the application of ‘click’ chemistry to bioorthogonally label active cells in conjunction with fluorescence-activated cell sorting (FACS) [10] and the pairing of substrate-independent isotopic labeling (e.g., heavy water) with Raman imaging and isolation [11, 12] Another highly relevant approach is to couple the fluorescent labeling of substrate molecules to FACS and shotgun sequencing. We identified putative cellulases from the genomes of the cellulose-bound microorganisms, expressed these enzymes and evaluated their activity using oxime derivatization of the sugar products and nanostructure-initiator mass spectrometry (NIMS) [15] These data revealed cellulase activity across several taxonomic groups including Goldbacteria, a poorly understood candidate phylum found in the rare biosphere. Our findings emphasize the potential of a function-driven genomics approach to directly link function to the individual, highlighting its ability to enrich uncultivated microbes that would otherwise be too rare to capture with shotgun metagenomics of a bulk sample
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