Listeria monocytogenes prophage induction is activated by ppGpp and inhibited by c-di-AMP

Bacteriophages, particularly temperate prophages that integrate directly into the host genome, are crucial drivers of bacterial evolution and act as fundamental architects of microbial communities. Listeria monocytogenes 10403S has two phage elements - prophage {Phi}10403S and monocin element. In this study, we found that c-di-AMP, a crucial second messenger in L. monocytogenes, regulates phage production. C-di-AMP accumulation down-regulates the gene expression of prophage and monocin gene loci and inhibits phage production, both spontaneous phage production as well as under phage induction through mitomycin-C treatment. We found that in genetically heterogenous cultures, super-infection of non-lysogenic strains with phage-containing strains can significantly amplify spontaneous prophage production. Using these cultures as an induction system, we found other inducers of spontaneous phage production. We found that ppGpp accumulation and nutrient starvation acts as an inducer of the spontaneous prophage production in L. monocytogenes. H2O2 can also play a role in inducing spontaneous phage production. Moreover, {Phi}10403S prophage production is suppressed in co-cultures of L. monocytogenes 10403S with L. monocytogenes F2365 and L. innocua CLIP11262. ImportanceMost Listeria and L. monocytogenes strains are lysogens, although the impact of phage genes on bacterial host metabolism or host fitness during bacterial competition is still unexplored. Bacteriophages have been shown to influence the evolution of their host and, in several cases, have a major effect on environmental fitness, pathogenicity and/or virulence of bacterial pathogens, either by regulating expression of critical genes or encoding beneficial genes. Prophages provide their hosts with a competitive edge through lysogenic conversion-- introducing novel toxins and defense systems--while simultaneously maintaining a molecular trigger, in the form of lysogenic-to-lytic switch, capable of initiating population-wide lysis in response to environmental stress. In the human pathogen Listeria monocytogenes, these prophages are near-ubiquitous and studies by other groups have described them as critical regulators of the bacteriums SOS pathways and its pathogenic lifestyle. This study identifies important novel regulators of phage induction in Listeria monocytogenes - c-di-AMP, ppGpp and H2O2, and how phage induction and production can shape the dynamics of bacterial competition of its host.