Franklin, Michael J.Sandvik, ElizabethYanardag, SilaWilliamson, Kerry S.2022-05-092022-05-092020-07Franklin MJ, Sandvik E, Yanardag S, Williamson KS. 2020. Functional characterization of the Pseudomonas aeruginosa ribosome hibernation-promoting factor. J Bacteriol 202:e00280-20.0021-9193https://scholarworks.montana.edu/handle/1/16764Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In Pseudomonas 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 and then as-saying 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 trunca-tion. 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 struc-ture. Surprisingly, heterologous HPFs from Gram-positive bacteria that have long C-terminal domains functionally complement the P. aeruginosa Δhpf mutant, sug-gesting 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 dur-ing bacterial starvation and resuscitation of the bacteria from dormancy.en-USArticle