The Isolation and Genome Sequencing of Five Novel Bacteriophages From the Rumen Active Against Butyrivibrio fibrisolvens.

Jessica C A Friedersdorff,Christopher J Creevey,David Rooke,Justin A Pachebat,Alison H Kingston-Smith,Alan R Cookson

doi:10.3389/fmicb.2020.01588

Abstract

Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium Butyrivibrio fibrisolvens. The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes.

Highlights

Despite the abundance of viruses in the rumen, little is known about the bacteriophage population in the rumen microbiome compared to other predominant microbes in this environment (Gilbert et al, 2020)
The screening of bacteriophages from a total of eight rumenassociated samples resulted in the isolation of five phage samples active against Butyrivibrio fibrisolvens DSM 3071
The implementation of polyethylene glycol (PEG)/NaCl to concentrate bacteriophages from fecal and rumen fluid phage filtrates resulted in the observation of plaques which were further purified to create

Summary

Introduction

Despite the abundance of viruses in the rumen, little is known about the bacteriophage (phage) population in the rumen microbiome compared to other predominant microbes in this environment (Gilbert et al, 2020). Studies based on electron microscopy observations of the viral fractions of rumen fluid (Hoogenraad et al, 1967; Paynter et al, 1969; Klieve and Bauchop, 1988) established that a highly diverse phage population exists in high abundance in ruminal fluid and could influence bacterial populations (Orpin and Munn, 1973) It is only with the application of metagenome sequencing to rumen associated viruses that the full extent of the phage community in this environment is beginning to be uncovered and better understood (Berg Miller et al, 2012; Gilbert et al, 2017). There is still much to be learned about phages in this environment and the opportunity exists to further investigate their presence in samples taken directly from rumen fluid and fresh feces, as well as exploring phages that target other rumen bacterial hosts

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Conclusion