Abstract
BackgroundTemperate phages influence the density, diversity and function of bacterial populations. Historically, they have been described as carriers of toxins. More recently, they have also been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. Despite recent advances in studying prophages using non-targeted sequencing approaches, methodological challenges in identifying inducible prophages in bacterial genomes and quantifying their activity have limited our understanding of prophage-host interactions.ResultsWe present methods for using high-throughput sequencing data to locate inducible prophages, including those previously undiscovered, to quantify prophage activity and to investigate their replication. We first used the well-established Salmonella enterica serovar Typhimurium/p22 system to validate our methods for (i) quantifying phage-to-host ratios and (ii) accurately locating inducible prophages in the reference genome based on phage-to-host ratio differences and read alignment alterations between induced and non-induced prophages. Investigating prophages in bacterial strains from a murine gut model microbiota known as Oligo-MM12 or sDMDMm2, we located five novel inducible prophages in three strains, quantified their activity and showed signatures of lateral transduction potential for two of them. Furthermore, we show that the methods were also applicable to metagenomes of induced faecal samples from Oligo-MM12 mice, including for strains with a relative abundance below 1%, illustrating its potential for the discovery of inducible prophages also in more complex metagenomes. Finally, we show that predictions of prophage locations in reference genomes of the strains we studied were variable and inconsistent for four bioinformatic tools we tested, which highlights the importance of their experimental validation.ConclusionsThis study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity. The ability to generate such quantitative information will be critical in helping us to gain better insights into the factors that determine phage activity and how prophage-bacteria interactions influence our microbiome and impact human health.3cD-fDFs4PLzgiMyqjRmsZVideo abstract.
Highlights
Temperate phages influence the density, diversity and function of bacterial populations
This study demonstrates that the integration of experimental induction and bioinformatic analysis presented here is a powerful approach to accurately locate inducible prophages using high-throughput sequencing data and to quantify their activity
Gut prophages are inducible in ex vivo-cultured murine gut bacterial community samples Having experimentally validated inducible prophages in pure cultures of Oligo-MM12 strains, we investigated the inducibility of prophages from the intestinal community of gnotobiotic Oligo-MM12 mice
Summary
Temperate phages influence the density, diversity and function of bacterial populations. They have been described as carriers of toxins. They have been recognised as direct modulators of the gut microbiome, and indirectly of host health and disease. The widespread manifestation of lysogeny amongst phages found in the gut microbiome suggests an important role for prophages in controlling the density, diversity and function of gut bacterial populations [8,9,10]. The activity and abundance of phages may cause substantial alterations to gut microbial community compositions [17] and in turn, impact host-microbe interactions. Alteration in the abundance and composition of viral populations have been detected, for example, in the context of gastrointestinal disorders [18,19,20]; after dietary perturbation in humans [21] and mice [22, 23], as well as in response to antibiotic administration in pigs [14]
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