Functional Characterization of the Pseudomonas aeruginosa Ribosome Hibernation-Promoting Factor

Abstract

Hibernation promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In P. aeruginosa, HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria, but contains conserved charged amino acids in its two alpha helices that interact with the rRNA. Here, we characterized the function of HPF in P. aeruginosa by performing mutagenesis of the conserved residues, then assaying mutant HPF alleles for their ability to protect ribosome integrity of starved P. aeruginosa cells. The results show that HPF functionally tolerates point mutations in charged residues and in the conserved Y71 residue, as well as a C-terminal truncation. Double and triple mutations of charged residues in Helix 1 in combination with a Y71F substitution reduce HPF activity. Screening for single point mutations that caused impaired HPF activity identified additional substitutions in the two HPF alpha-helices. However, alanine substitutions in equivalent positions restored HPF activity, indicating that HPF is tolerant to mutations that do not disrupt the protein structure. Surprisingly, heterologous HPFs from Gram-positive bacteria, that have long C-terminal domains, functionally complement the P. aeruginosa Δhpf, suggesting that HPF may play a similar role in ribosome protection in other bacterial species. Collectively, the results show that HPF has diverged among bacteria, and is tolerant to most single amino acid substitutions. The Y71 residue in combination with Helix 1 is important for the functional role of HPF in ribosome protection during bacterial starvation and resuscitation of the bacteria from dormancy. SIGNIFICANCE In most environments, bacteria experience conditions where nutrients may be readily abundant or where nutrients are limited. Under nutrient limitation conditions, even non-spore-forming bacteria may enter a dormant state. Dormancy is accompanied by a variety of cellular physiological changes that are required for the cells to remain viable during dormancy and to resuscitate when nutrients become available. Among the physiological changes that occur in dormant bacteria is the inactivation and preservation of ribosomes by the dormancy protein, hibernation promoting factor (HPF). In this study, we characterized the activity of HPF of Pseudomonas aeruginosa, an opportunistic pathogen that causes persistent infections, and analyzed the role of HPF in ribosome protection and bacterial survival during dormancy.

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

“…Plasmid constructs. For purification of ribosomal proteins, the genes for rplE and rpsM (encoding ribosomal proteins L5 and L13) were cloned into the poly-histidine expression vector pET28a to produce C-terminal 6x-poly-histidine tags, and hpf was purified using the 6x-poly-histidine tag as described previously [ 22 , 51 ]. Restriction endonucleases and DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA).…”

“…The IgG fraction of the antisera was purified using NAb TM Protein A Plus Spin Columns (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Polyclonal antisera to HPF was purified as described previously [ 22 ]. The efficacies of the purified antibodies were tested using immunoblots against the purified protein, against P. aeruginosa PAO1 protein extracts, and against extracts of P. aeruginosa containing fusions of the ribosomal proteins to the yellow fluorescent protein.…”

“…As shown for Escherichia coli and many other species of bacteria, ribosomes are inactivated during cell starvation by the small ribosome-binding accessory proteins, ribosome modulation factor (RMF) and ribosome hibernation factor (HPF) [ 16 , 17 , 18 , 19 , 20 ]. In P. aeruginosa , HPF is also critical for maintenance of rRNA integrity during starvation and for optimal resuscitation of the cells from starvation [ 21 , 22 ].…”

“…Therefore, cleavage of rRNA [ 34 ] and loss of rProteins [ 35 ] represent the early stages of ribosome recycling. Since HPF is broadly distributed in bacteria and since heterologous HPFs protect ribosome integrity in P. aeruginosa [ 22 ], HPF likely evolved to protect against these initial steps in ribosome degradation, and ensure a sufficient supply of ribosomes are available when the bacteria emerge from dormancy.…”

“…In previous studies, we demonstrated that HPF, but not RMF, is essential for optimal resuscitation of P. aeruginosa from dormancy, and for preservation of the rRNA component of ribosomes [ 21 , 22 ]. Here, we further characterized ribosome recycling of P. aeruginosa by characterizing preservation and degradation of rProteins during P. aeruginosa starvation.…”