Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment

Abstract

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

Fig. 1. Site location and workflow for BONCAT-FACS identification of active cells from environmental samples.

Location of hot spring FS5 in YNP. Microscopy image of unsorted hot spring cells visualized with general nucleic acid stain, 4′,6-diamidino-2-phenylindole (DAPI). Scale bar represents 5 µm. Samples were incubated in the presence of L-homopropargylglycine (HPG) under various treatments (Supplementary Table 1) for 48 h. Cells were detached from particle material and newly synthesized proteins were dye-stained via azide-alkyne click chemistry. Translationally active, fluorescent cells were sorted using fluorescence-activated cell sorting (FACS) from the presort, total extractable cell community. Cells were lysed and 16S rRNA genes were amplified with PCR. Black squares (■) in cells represent new proteins containing HPG. Map of YNP provided by the US National Park Service. Satellite image obtained from Google Maps.

Fig. 2. 16S rRNA gene relative abundances in averaged incubations.

Top panel, unamended FS5 community at the time of sampling (T₀). Middle panel, community composition of presort samples, representing the extractable microbial community, after the incubation experiments. Bottom panel, composition of the sorted active cell fraction after incubation with substrate amendment. Only taxa that were represented above 1% in at least one sample are shown. Additional taxa were combined into “Other” category. Taxon level represents highest taxonomic resolution for each taxa (Silva 128). Some replicates did not pass the quality control steps of our bioinformatics pipeline (<5000 reads), which precluded including them in this figure.

Fig. 3. Change in Shannon’s diversity indices of each treatment standardized to HPG-only controls.

The Shannon’s diversity indices for presort and sorted samples were compared relative to their respective HPG-only controls. The value for HPG-only was set to 0 and the difference for each sample is plotted. Overall, presort samples exhibited less variability than sorted samples. Sorted samples for cellobiose and anoxic conditions (100% N₂ headspace) were significantly different from the sorted HPG-only (atmospheric air) control (p < 0.05) and marked with an asterisk (*). Samples marked with a circle (●) indicate no data available.

Fig. 4. Log2-fold change (LFC) of active taxa in cellobiose substrate amendment and N2 100% headspace condition.

Each treatment was compared to the HPG-only control for sorted samples and LFC was calculated. The value for HPG-only was set to 0 and the LFC for each taxon is plotted. Only taxa significantly different from the control are shown (p < 0.1). Taxon level represents highest taxonomic resolution for each taxa (Silva 128).