Investigating the non-growing physiology of Pseudomonas aeruginosa: A focus on translation and carbon storage during starvation

The opportunistic pathogen Pseudomonas aeruginosa frequently adopts slow and non-growing phenotypes while occupying human-associated ecological and clinical niches. These phenotypes are characterised by the cessation of growth, extreme lows of physiological activity and increased tolerance to many clinical antibiotics. P. aeruginosa secretes numerous carbon-rich virulence-factors; exhibits impressive central metabolic versatility and can stockpile carbon intracellularly within carbonosomes. These activities can be maintained in the absence of growth and despite extreme energetic and material stress. However, the resource allocation strategies employed by growth-arrested P. aeruginosa, and the molecular mechanisms which regulate them, remain understudied. Here, the fundamental behaviours, activities and priorities of non-growing Pseudomonas aeruginosa UCBPP-PA14 were studied. Complete nitrogen and carbon starvation were used to induce robust non-growing phenotypes, which were defined with classical microbiological methods, fluorescence-based techniques and omics-level approaches. P. aeruginosa exhibited broadly distinct behavioural responses during either starvation. Using bio-orthogonal non- canonical amino acid tagging and fluorescence in situ hybridisation, carbon and nitrogen starved cells showed significant distinctions in ribosome counts and protein synthetic dynamics. Nascent proteomics methods and transposon- insertion sequencing identified conserved and distinct activities which were actively maintained by non-growing P. aeruginosa and which impacted fitness. Carbonosome production was highlighted as a dominant and heavily regulated activity during the starvation of P. aeruginosa. Under conditions which prevented growth but provided excess carbon; carbonosomes were found to significantly impact the flagellar motility, antibiotic susceptibility and protein synthetic capacity of P. aeruginosa. The enigmatic carbonosome-linked proteins PhaF and PhaI were shown to be important regulators of carbonosome dynamics, and inspection of each’s interactome alluded to the deep metabolic wiring and chromosomal- association of carbonosomes in P. aeruginosa. Broadly, these findings show that non-growing P. aeruginosa maintains active, complex, and dynamic behaviours during starvation. Although observed in closed systems, these behaviours may be exhibited by growth-arrested P. aeruginosa in a more relevant contexts, including during the stresses of infection. Ultimately, better definition of the physiology maintained by growth-arrested, antibiotic- tolerant bacteria could highlight prime targets for the development of novel antimicrobials or inform more appropriate, stewarded use of existing drugs.