Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA
Center for Biofilm Engineering, Montana State University, Bozeman, Montana, USA.
J Bacteriol. 2020 Sep 8;202(19). doi: 10.1128/JB.00280-20.
Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In , 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 by performing mutagenesis of the conserved residues and then assaying mutant HPF alleles for their ability to protect ribosome integrity of starved 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 Δ mutant, 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. 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 , an opportunistic pathogen that causes persistent infections, and analyzed the role of HPF in ribosome protection and bacterial survival during dormancy.
休眠促进因子 (HPF) 是一种核糖体辅助蛋白，可在细菌饥饿时使核糖体失活。在 中，HPF 可在细胞休眠时保护核糖体的完整性。HPF 的序列在细菌中已经分化，但在其两个α螺旋中含有与 rRNA 相互作用的保守带电氨基酸。在这里，我们通过对保守残基进行诱变来表征 中的 HPF 功能，然后检测饥饿 细胞中突变 HPF 等位基因保护核糖体完整性的能力。结果表明，HPF 在带电残基和保守 Y71 残基以及 C 末端截短处可耐受点突变。在与 Y71F 取代相结合的情况下，α 螺旋 1 中带电残基的双突变和三突变降低了 HPF 的活性。筛选导致 HPF 活性受损的单点突变，鉴定出两个 HPFα螺旋中的其他取代。然而，在等效位置用丙氨酸取代恢复了 HPF 活性，表明 HPF 可以耐受不破坏蛋白质结构的突变。令人惊讶的是，革兰氏阳性菌的异源 HPF，具有长 C 末端结构域，在功能上可以补充 Δ突变体，这表明 HPF 在其他细菌物种的核糖体保护中可能发挥类似的作用。总之，结果表明，HPF 在细菌中已经分化，并且可以耐受大多数单个氨基酸取代。Y71 残基与螺旋 1 结合对于 HPF 在细菌饥饿期间保护核糖体以及细菌从休眠中复苏的功能作用很重要。在大多数环境中，细菌会遇到营养物质丰富或有限的情况。在营养有限的条件下，即使是非孢子形成的细菌也可能进入休眠状态。休眠伴随着各种细胞生理变化，这些变化是细胞在休眠期间保持存活并在营养物质可用时复苏所必需的。在休眠细菌中发生的生理变化之一是休眠蛋白（休眠促进因子，HPF）使核糖体失活和保存。在这项研究中，我们表征了机会性病原体 的 HPF 活性，该病原体可引起持续性感染，并分析了 HPF 在核糖体保护和细菌休眠期间存活中的作用。
