Abstract
Viral metagenomics (viromics) has reshaped our understanding of DNA viral diversity, ecology, and evolution across Earth’s ecosystems. However, viromics now needs approaches to link newly discovered viruses to their host cells and characterize them at scale. This study adapts one such method, sequencing-enabled viral tagging (VT), to establish “Viral Tag and Grow” (VT + Grow) to rapidly capture and characterize viruses that infect a cultivated target bacterium, Pseudoalteromonas. First, baseline cytometric and microscopy data improved understanding of how infection conditions and host physiology impact populations in VT flow cytograms. Next, we extensively evaluated “and grow” capability to assess where VT signals reflect adsorption alone or wholly successful infections that lead to lysis. Third, we applied VT + Grow to a clonal virus stock, which, coupled to traditional plaque assays, revealed significant variability in burst size—findings that hint at a viral “individuality” parallel to the microbial phenotypic heterogeneity literature. Finally, we established a live protocol for public comment and improvement via protocols.io to maximally empower the research community. Together these efforts provide a robust foundation for VT researchers, and establish VT + Grow as a promising scalable technology to capture and characterize viruses from mixed community source samples that infect cultivable bacteria.
Highlights
Viruses that infect bacteria have been studied for over a century as model systems to establish the fundamentals of molecular biology and genetics, as well as their value as reagents or tools in these disciplines [1,2,3,4,5]
Improving our understanding of “viral tagging” flow cytometric signals VT is a deceptively simple idea whereby a mixture of natural viruses are labeled with a DNA-binding fluorescent dye and ‘bait’ hosts infected by these stained viruses can be detected with flow cytometry via the fluorescent shift of “viral-tagged cells” [38, 39] (Fig. 1A, B)
To gain a better understanding of the biology behind VT signatures, we examined how H71 interacts with HM1, a phage specific for this host, and HS8, a phage that does not adsorb to this host – both assayed via flow cytometry and microscopy
Summary
Viruses that infect bacteria (phages) have been studied for over a century as model systems to establish the fundamentals of molecular biology and genetics, as well as their value as reagents or tools in these disciplines [1,2,3,4,5]. We are well into the third age of phage [7] whereby ecologists seek to better map the “virosphere” (the diversity of virus sequence space that exists in nature) and elucidate the roles viruses play in complex communities. In this age, microbiologists have revealed that microbes support healthy ecosystems functioning in natural systems (e.g., global biogeochemical processes [8], climate change feedback loops [9], and human systems [10]), there is increasing awareness that viruses that infect these microbes are abundant and impactful. The modern viral ecogenomic toolkit [29] is capable of resolving multiple levels of diversity over scales as vast as the global oceans
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