Polyhydroxyalkanoate carbonosomes impact the activity and viability of growth-arrested Pseudomonas aeruginosa during nutrient fluctuations and tobramycin exposure

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.