Bidirectional sequestration between a bacterial hibernation factor and a glutamate metabolizing protein

Abstract

Bacterial hibernating 100S ribosomes (the 70S dimers) are excluded from translation and are protected from ribonucleolytic degradation, thereby promoting long-term viability and increased regrowth. No extraribosomal target of any hibernation factor has been reported. Here, we discovered a previously unrecognized binding partner (YwlG) of hibernation-promoting factor (HPF) in the human pathogen Staphylococcus aureus . YwlG is an uncharacterized virulence factor in S. aureus . We show that the HPF–YwlG interaction is direct, independent of ribosome binding, and functionally linked to cold adaptation and glucose metabolism. Consistent with the distant resemblance of YwlG to the hexameric structures of nicotinamide adenine dinucleotide (NAD)–specific glutamate dehydrogenases (GDHs), YwlG overexpression can compensate for a loss of cellular GDH activity. The reduced abundance of 100S complexes and the suppression of YwlG-dependent GDH activity provide evidence for a two-way sequestration between YwlG and HPF. These findings reveal an unexpected layer of regulation linking the biogenesis of 100S ribosomes to glutamate metabolism.

Epitranscriptional m6A modification of rRNA negatively impacts translation and host colonization in Staphylococcus aureus

“…A total of 0.1–0.2 Abs 280 units of cell lysate were analyzed on 4–20% TGX SDS-PAGE gels (BioRad), or 4–12% Bis-Tris NuPAGE minigels (Invitrogen) and the proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo system (BioRad). The membrane was stained with Ponceau red (Amresco #K793-500mL) to ensure equal loading, followed by immunoblotting using anti-ErmB (1/1,000 dilutions, a gift from Julian Rood [ 115 ]), anti-SdrE (1/1,000 dilutions, a gift from Dominique Missiakas), anti-SigB and anti-Asp23 (1/2,000 dilutions; 1/100 dilutions, gifts from Bischoff Markus), anti-YwlG (1/1,000 dilutions, [ 116 ]) anti-SasG (1/200, Abnova PAB16066), anti-mCherry (1/1,000 dilutions, Novus Biologicals NBP196752X), anti-YFP (1/500 dilutions, Roche 11814460001), anti-Nuc2 (1/500 dilutions, OriGene AP05314SU-N), anti-Goat Anti-SpA (1/2,000, OriGene AP05314SU-N), Donkey anti-Goat Alexa Fluor Plus 800 (1/10,000 dilutions, Invitrogen A32930), and HRP-conjugated anti-IgG secondary antibody (1/15,000 dilutions, Cytiva #NA9120). SuperSignal West Dura chemiluminescence substrate was used (Thermo Scientific #34075).…”

Rippling life on a dormant planet: hibernation of ribosomes, RNA polymerases, and other essential enzymes

“…Recent studies suggest that dedicated mechanisms exist to constrain hibernation factors in an inactive state in actively growing cells to prevent them from interfering with protein synthesis. For example, S. aureus produces YwlG under normal growth conditions, which acts as an inhibitor that sequesters the hibernation factor HPF ( Ranava et al, 2022 ). Similarly, Saccharomyces cerevisiae has been shown to inactivate the hibernation factor Serbp1/Stm1 via phosphorylation by the TOR kinase ( Shetty et al, 2023 ).…”