Capturingin situVirus-Host Range and Interaction Dynamics through Gene Fusion with epicPCR

Abstract

Viruses impact microbial diversity, phenotype, and gene flow through virus-host interactions that in turn alter ecology and biogeochemistry. Though metagenomics surveys are rapidly cataloging viral diversity, capturing specific virus-host interactions in situ would identify hosts for novel viruses and reveal influential ecological or environmental factors. We leveraged metagenomics and a high-throughput, cultivation-independent gene fusion technique (epicPCR) to investigate viral diversity and virus-host interactions over time in a critical estuarine environment, the Chesapeake Bay. EpicPCR captured in situ virus-host interactions for viral clades with no closely related database representatives. Abundant freshwater Actinobacteria lineages were the most common hosts for these poorly characterized viruses, and observed viral interactions with one abundant Actinobacterial population (Rhodoluna) were correlated with environmental factors. Tracking virus-host interaction dynamics also revealed ecological differences between multi-host (generalist) and single-host (specialist) viruses. Generalist viruses had significantly longer periods with observed virus-host interactions but specialist viruses were observed interacting with hosts at lower minimum abundances, suggesting more efficient interactions. Together, these observations reveal ecological differences between generalist and specialist viruses that provide insight into evolutionary trade-offs. Capturing in situ interactions with epicPCR revealed environmental and ecological factors that shape virus-host interactions, highlighting epicPCR as a scalable new tool in viral ecology.

Uncovering bacterial hosts of class 1 integrons in an urban coastal aquatic environment with a single-cell fusion-polymerase chain reaction technology

“…Our hybrid epicPCR procedures were adapted from the procedures described by Sakowski et al 25 S2. To demonstrate the absence of false associations between 16S rRNA markers and class 1 integrons originating from different cells, we spiked one set of technical replicates with cells of a class 1 integron-free Escherichia coli MG1655 strain at a population frequency of 10%.…”

“…epicPCR (emulsion, paired isolation and concatenation PCR), also known as OIL-PCR (onestep isolation and lysis PCR), is a fusion PCR-based technique that overcomes these problems by co-amplifying bacterial taxonomic markers along with functional genes of interest and doing so from single uncultured bacterial cells. Single cells are trapped in droplets generated by emulsifying an aqueous phase suspension of environmental bacteria in an emulsion oil [21][22][23][24][25][26] . This compartmentalises the fusion PCR-type amplification process into parallel, single cell-level reactions.…”

“…To date, epicPCR has been successfully applied to target functional genes including the bacterial dissimilatory sulfite reductase dsrB gene 21,24 , the bacteriophage ribonucleotide reductase RNR gene 25 , antibiotic resistance genes 22,23,26,27 , and clinical class 1 integron genes intI1, sul1, and ΔqacE 23,27,28 . In all these studies, epicPCR amplicons comprised short fragments of genes of interest linked to the V4 hypervariable region of 16S rRNA markers.…”