The role of ribosome hibernation factor and the stringent response in the survival of Pseudomonas aeruginosa during dormancy

Abstract

Pseudomonas aeruginosa survives in a dormant state in low nutrient environments and is able to resuscitate when favorable conditions are available. In response to stressful environmental conditions including antibiotic stress, osmotic stress, and starvation, P. aeruginosa undergoes the (p)ppGpp-mediated stringent response to induce a variety of genes for entry into the dormant state. One critical mechanism for P. aeruginosa dormancy involves in inactivating translation machinery by converting active ribosomes into inactive 70S and 100S ribosome monomers and dimers. Hibernation promoting factor (HPF~11.6kDa) is a ribosome-associated protein that stabilizes inactive ribosomes. Here, I investigated the relationship between HPF and the stringent response in survival of P. aeruginosa during dormancy. I also investigated role of HPF preserving ribosomes in dormant cells by quantifying the abundance of two ribosomal proteins (L5 of the large ribosomal subunit and S13 of the small ribosomal subunit), during P. aeruginosa starvation. For quantitative analysis during nutrient-limited conditions, I used immunoblotting and image analysis to quantify L5 and S13 abundances in the wild-type strain, PAO1, and in a relA/spoT double mutant strain, which is incapable of producing (p)ppGpp. The results show that the relA/spoT mutant loses HPF proteins after four days of starvation. To explore the role of HPF in preserving ribosomal proteins, I quantified L5 and S13 in wild-type PAO1 and in the Delta hpf deletion mutant and in the relA/spoT mutant. Immunoblots showed that both L5 and S13 rapidly decrease by day 2 of starvation in the Delta hpf mutant strain, but that these proteins are maintained throughout eight days of starvation in the wild-type strain. Notably, L5 and S13 are maintained in the Delta relA/Delta spoT strain throughout starvation. Lastly, I determined if the amount of cellular HPF required for P. aeruginosa ribosome maintenance. Growth in minimal medium (MOPS medium) affects the amount of HPF produced, based on the carbon source. Therefore, I tested ribosome maintenance during starvation of cells first cultured in MOPS-fructose or MOPS-glucose. The results indicate that HPF production during growth in MOPS-fructose is higher than in MOPS-glucose. However, the increased amount of HPF did not affect the amount of L5 and S13 during starvation. Therefore, the amount of HPF is not critical for P. aeruginosa to maintain its ribosomes during starvation. These results demonstrate HPF is essential for maintenance of ribosomal proteins during starvation of P. aeruginosa, and that the ribosomal proteins are likely degraded in the absence of HPF. P. aeruginosa needs a minimum amount of HPF to preserve ribosomes during nutrient-limited condition.