Targeted genomic analysis of a predominant uncultured marine pelagiphage-host model via microfluidics and semipermeable capsule technology

Microbes and their viruses drive central biogeochemical cycles on a global scale. Understanding the biology and ecology of virus-host interactions and their impact on ecosystems depends on our ability to develop tools that enable high-throughput screening of ecologically relevant, uncultured virus-host pairs. Viruses infecting Pelagibacterales, the predominant bacteria in surface oceans, have been studied through computational analyses and cultivation efforts. Here, we employ an accessible microfluidics and semi-permeable capsule (SPC) technology to investigate the uncultured pelagiphage vSAG 37-F6-host interactions since it is one of the most abundant and ubiquitous viruses in the marine virosphere. First, we validated this technology using cultured virus-host pairs. Then, marine single cells were microfluidically encapsulated in SPCs, lysed, whole-genome amplified, and screened using fluorescent PCR for the presence of a hallmark gene of vSAG 37-F6. Data indicate that ~ 30% of the targeted cell population (cell fraction ≤ 0.45 μm) contained the virus vSAG 37-F6-like. A total of ~ 500 putatively infected cells were sorted, combined, and sequenced. Data showed that most reads (~60%) and assembled genome fragments (~85%) were identified as viral, indicating that the sorted host cells were likely in the final stages of infection. Two major viral clusters were detected: one corresponding to vSAG 37-F6 and another mixed viral cluster consisting of cyanophages, pelagiphages, and vibriophages. A significant proportion of total reads (~20%) were assigned to Pelagibacter spp. TMED287, a bacterium reported to be abundant in the Mediterranean Sea. This flexible microfluidic-SPC technology holds enormous potential for exploring uncultured microbial and viral communities across various perspectives and microbiology fields.

This research has been funded by Generalitat Valenciana (ref. “Virhost” CIPROM/2021/006 PROMETEO2022) and by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0022991 and subaward No. S591062, with additional support from the Spanish Ministry of Universities (ref. Agencia Estatal de Investigación ref. PID2021-125175OB-I00).