Most bacterial pathogens are lysogens; yet, if the prophage doesn’t carry identifiable toxins, little credit is given to any role the prophage may play in the biology of the bacterial host. It is not uncommon to find conservation of all or part of a phage genome across a bacterial species, a phenomenon usually associated with powerful selection of beneficial traits. However, this is rarely attributed to prophages lacking identifiable selective markers/traits, with the majority of prophages carrying genes that are still annotated as hypothetical and consigned to the realm of “phage dark matter”. Transcriptomics offers a powerful tool to examine the impact of carrying a prophage or prophages. This has been done before using Shiga toxin encoding prophages, but in this project, we examined the impact of 3 distinct phages of polylysogenic clinical strain of Pseudomonas aeruginosa Liverpool Epidemic Strain (LES) that carry no identifiable virulence genes.

LES is a polylysogenic clinical strain harboring five inducible prophages, a probable prophage remnant and a few genomic islands. LES is associated with increased morbidity of chronic Pseudomonas infections in the patients with cystic fibrosis. We are investigating the influence of a set of three co-habiting prophages in “multiple combinations”, in the model Pseudomonas aeruginosa host strain PAO1. Despite strong evidence that their presence in the wild-type Pseudomonas aeruginosa host strain PAO1 is associated with a gross increase in virulence of their host, the specific molecular detail is not known. We have optimized growth conditions to favor the lysogenic state and used that condition to further study the gene expression landscape of lysogen under conditions in which prophage is induced and where the prophage is held as stably as possible in the lysogen culture. We validated the induction profile by qRT-PCR of the key phage marker genes from crucial stages of LES phage replication in PAO1 in both conditions. Using the information derived from the q-PCR profiling, we prepared RNA samples for RNA seq library production. We have identified 24 clusters of genes that are differentially regulated by these prophages and combinations of these prophages in the PAO1 background. Our RNA seq data demonstrates that the phage carriage in the lysogeny regulates the expression of many host genes, depending on the type of the phage. Moreover, we show the possible epistatic genetic interactions between two different phages using differentially regulated clusters of genes. Work is ongoing to understand how these genes are regulated and what this means for the biology of the lysogen.