Temperate phages influence the density, diversity and function of bacterial populations and thus are recognised as direct modulators of the gut microbiome, and indirectly of host health and disease.
More...Temperate phages influence the density, diversity and function of bacterial populations and thus are 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. Here, we present methods for using high-throughput sequencing data to localise 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 accurately quantifying prophage activity and precisely 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 model bacteria from the murine gut microbiome, we located five novel inducible prophages in three gut bacterial strains, quantified their activity and showed signatures of lateral transduction for two of the prophages. Moreover, by read alignment analysis, we were able to differentiate between inducible prophages and linear mobile genetic elements in an Escherichia coli HS strain. Finally, we demonstrate that experimental validation of prophage prediction is essential since none of three tested bioinformatic prophage prediction tools could accurately and unambiguously locate prophages in the reference genome. Methods to locate prophages in sequence data and quantify their activity can provide insight into how prophage-bacteria interactions influence our microbiomes and impact human health.
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