Updated project metadata. Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of a number of phylogenetically diverse pathogenic bacteria with different in lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after the surgical injury in humans and animals. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity nor the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA) as a model. However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factor categories, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling and the secretion of proteases, phospholipases and phosphatases.