Role of Hibernation Promoting Factor in Ribosomal Protein Stability during Pseudomonas aeruginosa Dormancy

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes biofilm-associated infections. P. aeruginosa can survive in a dormant state with reduced metabolic activity in nutrient-limited environments, including the interiors of biofilms. When entering dormancy, the bacteria undergo metabolic remodeling, which includes reduced translation and degradation of cellular proteins. However, a supply of essential macromolecules, such as ribosomes, are protected from degradation during dormancy. The small ribosome-binding proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), inhibit translation by inducing formation of inactive 70S and 100S ribosome monomers and dimers. The inactivated ribosomes are protected from the initial steps in ribosome degradation, including endonuclease cleavage of the ribosomal RNA (rRNA). Here, we characterized the role of HPF in ribosomal protein (rProtein) stability and degradation during P. aeruginosa nutrient limitation. We determined the effect of the physiological status of P. aeruginosa prior to starvation on its ability to recover from starvation, and on its rRNA and rProtein stability during cell starvation. The results show that the wild-type strain and a stringent response mutant (∆relA∆spoT strain) maintain high cellular abundances of the rProteins L5 and S13 over the course of eight days of starvation. In contrast, the abundances of L5 and S13 reduce in the ∆hpf mutant cells. The loss of rProteins in the ∆hpf strain is dependent on the physiology of the cells prior to starvation. The greatest rProtein loss occurs when cells are first cultured to stationary phase prior to starvation, with less rProtein loss in the ∆hpf cells that are first cultured to exponential phase or in balanced minimal medium. Regardless of the pre-growth conditions, P. aeruginosa recovery from starvation and the integrity of its rRNA are impaired in the absence of HPF. The results indicate that protein remodeling during P. aeruginosa starvation includes the degradation of rProteins, and that HPF is essential to prevent rProtein loss in starved P. aeruginosa. The results also indicate that HPF is produced throughout cell growth, and that regardless of the cellular physiological status, HPF is required to protect against ribosome loss when the cells subsequently enter starvation phase.