Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Flow Sorting and Sequencing Active Environmental Viruses from Methane-Oxidizing Communities with Viral-BONCAT


Francisco Martinez-Hernandez1,2 (, Maria Alvarez-Sanchez2, Aitana Llorenç Vicedo2, Alon Philosof1, Aditi K. Narayanan1, Manuel Martinez-Garcia2, Victoria J. Orphan1


1California Institute of Technology–Pasadena; 2University of Alicante–Spain


The goals of this project are to (1) develop a mechanistic understanding of anaerobic oxidation of methane (AOM) syntrophic interactions; (2) define and functionally characterize the microbial community, including viruses, associated with methanotrophic consortia under changing environmental conditions; and (3) create an integrative modeling framework to explore the ecophysiology of AOM consortia and their community interactions in environmental context.


Beyond identifying the microorganisms present in the environment, characterizing their interactions and impact on other biological communities is becoming increasingly necessary to understand the functioning of ecosystems. One of the major regulators of biological communities are viruses, capable of infecting and killing a broad range of organisms across the tree of life. In recent years, metagenomics has significantly expanded the knowledge about the virosphere and its diversity (Laso-Pérez et al. 2023). In this project, researchers present the development of viral BONCAT-FACS coupled with metaviromic sequencing, where free viruses that have recently infected an active cell are specifically labeled with BONCAT and sequenced from complex environmental communities. This newly developed approach for sorting, quantifying, and sequencing active viral-like particles offers a new lens in which to track viral host dynamics and to characterize the selective pressure of distinct viral populations on microbial communities within diverse ecosystems.

Viral BONCAT-FACS is based on the BONCA methodology (Bioorthogonal noncanonical amino acid tagging), which marks newly application to the virosphere was demonstrated in a laboratory at Caltech, where free viruses were visualized using epifluorescence microscopy after incorporating newly synthesized peptides or amino acids from their active hosts (Pasulka et al. 2018). This optimization of viral BONCAT includes an enhancement in the fluorescence signal associated with active viruses, enabling detection by flow cytometry. Viral BONCAT-FACS, like the previously developed high-throughput FACS (HT-FACS) in the Martinez-Garcia group enables genomic analysis of flow sorted viral populations, allowing the differentiation of active, newly produced BONCAT labeled viruses, and the nonactive viral particles concurrently with active and inactive cell fractions (Martinez-Hernandez et al. 2017). The team successfully amplified and sequenced complete genomes using the WGA-X reaction, initially demonstrating the viability of this technique by sequencing more than 1,000 contigs from the active viral fraction in coastal waters followed by further optimization for sediment/rock associated microbial communities catalyzing the AOM (Stepanauskas et al. 2017; Garcia-Heredia et al. 2021).

Viral BONCAT-FACS was used on laboratory-maintained methane-fed sediment and AOM microbial mat incubations. After a 3-day BONCAT incubation in the presence of methane (CH4), active viruses and microorganisms were fluorescently labeled with the optimized click reaction and sorted, yielding ~3,500 active viral-like particles (VLPs) and ~500 active microbial cells. Sequencing and bioinformatic analysis of the amplified genomes from both viral fractions confirmed the recovery of diverse viral genomes. In a second BONCAT experiment, AOM mat samples enriched in ANME archaea and sulfate-reducing bacteria were incubated with either CH4 and SO-2 or CH4 and AQDS (e.g., decoupling ANME archaea from their sulfate-reducing partners; Scheller et al. 2016). After 4 weeks of incubation, higher cellular and viral BONCAT activity was observed in the AQDS treatment. Genomes of 75,000 active and 75,000 nonactive VLPs, along with more than 100,000 active and nonactive microbial cells were sorted and sequenced from the different treatment conditions. Sequencing is now underway and will help illuminate how viruses regulate the dynamics of these methane-fueled communities and how viral pressure is affected by cellular stress conditions.


Garcia-Heredia, I., et al. 2021. “Benchmarking of Single-Virus Genomics: A New Tool For Uncovering the Virosphere,” Environmental Microbiology 23(3), 1584–93. DOI:10.1111/1462-2920.15375.

Laso-Pérez, R., et al. 2023. “Evolutionary Diversification of Methanotrophic ANME-1 Archaea and their Expansive Virome,” Nature Microbiology 8(2), 231–45. DOI:10.1038/s41564-022-01297-4.

Martinez-Hernandez, F., et al. 2017. “Single-Virus Genomics Reveals Hidden Cosmopolitan And Abundant Viruses,” Nature Communications 8(1), 15892. DOI:10.1038/ncomms15892.

Pasulka, A. L., et al. 2018. “Interrogating Marine Virus-Host Interactions and Elemental Transfer with BONCAT and Nanosims-Based Methods,” Environmental Microbiology 20(2), 671–92. DOI:10.1111/1462-2920.13996.

Scheller, S., et al. 2016. “Artificial Electron Acceptors Decouple Archaeal Methane Oxidation from Sulfate Reduction,” Science 351(6274), 703–07.

Stepanauskas, R., et al. 2017. “Improved Genome Recovery and Integrated Cell-Size Analyses of Individual Uncultured Microbial Cells and Viral Particles,” Nature Communications 8(1), 84. DOI:10.1038/s41467-017-00128-z.

Funding Information

This work was supported by the DOE, Office of Science, BER program, Genomic Science Program under award number DE- SC0022991.