Prophage host interactions: pulling back the curtains on Pseudomonas puppet masters

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Prophages are viruses (bacteriophages) that have integrated into bacterial genomes, but their contribution to the success of their hosts has been grossly under-estimated. We aim to provide a more detailed view of prophage influence on the bacterial host, which will greatly expand our understanding of a system where they are known to be important, but not how they are important.Pseudomonas aeruginosa, a pathogen of plants, animals and humans will be our model prophage/bacterial host system. We will specifically focus on the Liverpool Epidemic Strain (LES) and, a less pathogenic model strain, PAO1. The LES strain is known to carry multiple prophages that have been proven to enhance its competitiveness. The LES prophages do not encode any known toxins or virulence factors but have been associated with altered metabolic pathways and other biological traits of P. aeruginosa. Prophage sequences are found in almost all bacterial strains. However, very little is actually known about the genetic information that these prophages carry. In fact 75% or more of most prophage genes are annotated as hypothetical (often referred to as phage dark matter). These hypothetical sequences may be shared across many different phages but, unless they have been identified as virulence-related factors, the relevance of these sequences to the biology of the host is generally ignored. Recently, the importance of some of this dark matter has been elucidated and shown to regulate bacterial host genes or to promote bacterial survival. At this point in time when we are losing the battle in controlling the spread of multidrug resistant bacterial infections, there is an urgent need to better understand all of the genetic elements that are controlling bacterial biology. These data will help to produce better informed strategies for the control of bacterial infection and also aid the understanding of lytic bacteriophages that are currently being used in the development of phage therapy. Our objectives will uncover the hidden mechanisms by which prophage "puppet masters" affect the biology and fitness of P. aeruginosa: We have purified multiple inducible prophages from the LES strain of P. aeruginosa. We have constructed a precise set of tools and strains to investigate the direct effect of each prophage (separately and in combination) on a well-characterised model host strain, compared to a strain where the prophages are absent. We will use cutting edge techniques, combining knowledge of genome architecture, changes in gene expression and putative regulators to reveal the different ways that prophages impact their bacterial hosts. Cloning of identified regulators and mutant construction will enable association of prophage genes to functional pathways. We will monitor the impact of identified prophage-encoded elements under many varied environmental parameters that reflect the niches of P. aeruginosa. This combined approach will elucidate the interactions between three cohabiting LES prophages and their host to understand better their control of the bacterial behaviour.Applications and benefits: Not only will these studies inform a better understanding of P. aeruginosa biology, but the techniques can be applied to other phage-host systems. Bioinformatic tools have been much improved in recent years, for identifying prophages in bacterial genomes. But without functional studies such as ours, the relevance of the phage genes remains part of the dark matter. Identifying function of unknown genes (that are conserved across phage databases) will transform the field of phage biology and our fundamental understanding of microbiology.