Metaviromes Reveal the Dynamics of Pseudomonas Host-Specific Phages Cultured and Uncultured by Plaque Assay.

Katrine Wacenius Skov Alanin,Witold Kot,Jacob Bruun Jørgensen,Laura Milena Forero Junco,Morten Arendt Rasmussen,Lars Hestbjerg Hansen,Tue Kjærgaard Nielsen

doi:10.3390/v13060959

Abstract

Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or those with the highest ability to make plaques in solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in environmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community’s shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome–host matrix to observe how enrichments in liquid media correspond to the metaviromic data. In this study, we observed a significant shift (p = 0.015) of the 47 identified putative Pseudomonas phages with a minimum twofold change above zero in read abundance when adding a Pseudomonas syringae DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the Pseudomonas strain, were highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlight how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.

Highlights

As bacteriophages are the most abundant entity across all environments, they serve as a major reservoir of genetic diversity and have an essential role in ecology and maintaining bacterial diversity as well as carbon and nutrient cycling [1,2,3,4]
These 12 assemblies were composed of the baseline, one assembly for each of the DC3000 + A–C viromes, as well as one assembly for the combined Illumina reads of DC3000 +
The last six assemblies were each of the DC3000 − A–C viromes, one of the combined reads Illumina of DC3000 −, one with all Illumina and Nanopore reads concatenated from the seven viromes, a baseline with the long reads for assembly and polished with all short reads, and a baseline with the long reads for assembly and polished with short reads from only the baseline

Summary

Introduction

As bacteriophages (phages) are the most abundant entity across all environments, they serve as a major reservoir of genetic diversity and have an essential role in ecology and maintaining bacterial diversity as well as carbon and nutrient cycling [1,2,3,4]. Phages keep dominant bacteria in check by ‘killing the winner’ through lysis, which results in the release of organic matter from the exterminated host used by other prokaryotes [5]. It is estimated that the number of phages is at least ten-fold larger than the number of bacteria in the sea and that the number of phage particles on Earth is 1031 [4,12,13,14]. The combined biomass of phages on Earth is assumed to correspond to 75 million blue whales [3,15,16]. Despite their importance and high number, phages remain understudied, research in phage genomics and viromics is on the rise. The number of available phage genomes in databases is rapidly increasing, especially uncultivated viral genomes (UViGs), but there is a lack of high-quality phage genome representatives in the databases [14]

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Results

Discussion

Conclusion