Induction of the pho regulon and polyphosphate synthesis against spermine stress in Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa on spermine requires a functional γ‐glutamylpolyamine synthetase PauA2. Not only subjected to growth inhibition by spermine, the pauA2 mutant became more sensitive to β‐lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant, which alleviated toxicity, were isolated from selection plates containing spermine. These suppressors share common phenotypic changes including delayed growth rate, retarded swarming motility, and pyocyanin overproduction. Genome resequencing of a representative suppressor revealed a unique C₅₉₉T mutation at the phoU gene that results in Ser₂₀₀Leu substitution and a constitutive expression of the Pho regulon. Identical phenotypes were also observed in a ΔpauA2ΔphoU double knockout mutant and complemented by the wild‐type phoU gene. Accumulation of polyphosphate granules and spermine resistance in the suppressor were reversed concomitantly when expressing exopolyphosphatase PPX from a recombinant plasmid, or by the introduction of deletion alleles in pstS pstC for phosphate uptake, phoB for Pho regulation, and ppk for polyphosphate synthesis. In conclusion, this study identifies polyphosphate accumulation due to an activated Pho regulon and phosphate uptake by the phoU mutation as a potential protection mechanism against spermine toxicity.