Analyses of protein expression and genetic fitness determinants reveal dynamic pathways active in starved Pseudomonas aeruginosa

Heterotrophic bacteria rapidly deplete essential macronutrients during growth and must navigate subsequent periods of growth arrest imposed by starvation. Nutrient limitations can be dynamic in nature, requiring ongoing regulatory adjustments involving new protein synthesis despite total biosynthetic activities being dramatically lower than during growth. Here, we have characterized the responses of the opportunistic pathogen Pseudomonas aeruginosa to prolonged starvation for carbon or nitrogen sources, and to transitions between these states. We find that most cells survive both types of starvation for more than a week and maintain low but robustly detectable levels of protein synthesis in the absence of growth. Nitrogen-starved cells are larger, make more proteins and retain fewer ribosomes than carbon-starved cells, indicating that distinct physiological strategies are adopted during the two starvation types. We found that the newly synthesized proteomes of each starvation type are distinct, although many of the most highly synthesized proteins are shared between both conditions. Interestingly, we observed a temporary burst of protein synthesis as cells were transitioned between the two starvation conditions, which may reflect active remodelling of the proteome during growth arrest. We also used transposon insertion sequencing to identify genes impacting fitness in both starvation conditions and during transitions between the two and found that a highly overlapping set of global regulators most strongly influenced survival. Combining these datasets, we highlight proteases and chaperones; flagellar motility; and the nitrogen-related phosphotransferase system as key fitness-impacting functions that are actively maintained by growth arrested Pseudomonas aeruginosa .