Role of phoU in P. aeruginosa virulence and the emergence of compensatory mutations in the ΔphoU mutant

Bacteria respond to phosphate (Pi) availability by modulating the PHO regulon, which encodes proteins for Pi scavenging. The phoU gene is part of the pst operon, a high-affinity phosphate transport system that also represses PHO regulon expression under Pi excess. In the absence of phoU , Pi transport continues but repression fails, resulting in constitutive PHO gene transcription. In Pseudomonas aeruginosa and other Proteobacteria, phoU knockouts display pleiotropic effects, including stress and antibiotic sensitivity and polyphosphate (polyP) accumulation. Here, using cryo-STEM imaging and EDX analysis, we show that the \(Δ\) phoU mutant of P. aeruginosa accumulates large polyP granules containing over seven-fold more phosphorus than the wild type. Moreover, \(Δ\) phoU was markedly less virulent in vivo in the Galleria mellonella model. We further isolated two suppressor classes from \(Δ\) phoU : low-constitutive alkaline phosphatase (LCAP) and not-constitutive alkaline phosphatase (NCAP). LCAP mutants remain PHO-constitutive, less virulent than wild type, and accumulate polyP similarly to \(Δ\) phoU . In contrast, NCAP mutants are not PHO-constitutive, do not accumulate polyP, and retain wild-type virulence. We also tested swarming motility, pyocyanin, and pyoverdine production. Significant differences were limited to swarming, where \(Δ\) phoU was substantially less motile than the wild type, with LCAP showing partial recovery and NCAP full restoration.