The DNA uptake competence system of the intracellular bacterial pathogen Listeria monocytogenes was considered non-functional. There are no known conditions for DNA transformation and the competence master activator gene, comK, is interrupted by a temperate (lysogenic) prophage. We have shown recently that the L. monocytogenes competence system is required during infection to promote bacterial escape from macrophage phagosomes, in a manner that is independent of DNA uptake. Remarkably, we found that regulation of the competence system relies on the formation of a functional comK gene via a controlled process of prophage excision. Prophage excision was specifically induced during intracellular growth, primarily within phagosomes, yet, unlike classic prophage induction, progeny virions were not produced and bacterial lysis did not occur. This study revealed a unique adaptation of a prophage to the intracellular life style of its host, whereby the prophage serves as a genetic switch to modulate the virulence of its host. In the proposed project we aim to investigate this phenomenon and study the give-and-take interactions between the L. monocytogenes 10403S strain and its ϕ10403S-prophage during mammalian infection. We will study the prophage determinants and mechanisms that control intracellular excision and maintenance as well as the mechanisms that prevent its virions production and bacterial lysis. We will explore the crosstalk between phage and bacterial regulatory factors and characterize the mammalian host signals/conditions that trigger this unique prophage response. Lastly, we will investigate the unexpected function of the competence system in phagosomal escape. In particular, we will explore the possibility that the competence system serves as an auxiliary secretion system, which secretes proteins that promote phagosomal escape.