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The alarmone pGpp inhibits translation through GTP depletion II

ABSTRACT: Bacteria produce the alarmone nucleotides ppGpp and pppGpp during stress to affect replication, transcription, and metabolism. ppGpp and pppGpp also attenuate translation by competitively binding translational GTPases to conserve resources during stress. Recently, pGpp was identified as a third alarmone, and important pathogens like Clostridioides difficile exclusively produce pGpp in response to stress. Despite its abundance as an alarmone, the precise role of pGpp in mediating stress responses is poorly understood. Here, we show that, while pGpp is a weaker inhibitor of protein synthesis than ppGpp and pppGpp in vitro, pGpp production in the model Gram-positive bacterium Bacillus subtilis leads to faster translation inhibition in vivo. pGpp production leads to fewer ribosomes engaged in translation and more hibernating ribosome dimers than (p)ppGpp production, suggesting that translation initiation is strongly inhibited. Additionally, pGpp production depletes cellular GTP more rapidly than (p)ppGpp production, which we show is sufficient for translation inhibition. Faster GTP depletion during pGpp production is also accompanied by more robust transcriptome remodeling. This work expands the model by which alarmones inhibit translation to include GTP depletion and demonstrates how production of different alarmone species exert varying effects on physiology.

ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168

PROVIDER: GSE319727 | GEO | 2026/02/18

REPOSITORIES: GEO

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Project description:This work investigates the molecular mechanisms that determine the requirement of (p)ppGpp-mediated GTP homeostasis for viability during prolonged nutritional stress. Initially, we observed that the absence of (p)ppGpp in S. aureus resulted in reduced culturability and elevated levels of guanosine nucleotides (GMP, GDP, and GTP) when cells experienced prolonged starvation during stationary phase. Analysis of metabolic activity and cell membrane function revealed, that (p)ppGpp-deficiency does not immediately lead to cell death but rather to a VBNC state where cell become division-incompetent and display reduced metabolic activity. The cell membrane stayed intact, however (p)ppGpp-deficient cells displayed reduced membrane potential and increased membrane fluidity. Since missing translation inhibition and elevated oxidative stress levels could not be identified as the cause for the VBNC state of the (p)ppGpp-deficient cells, we conducted transcriptomic analysis on S. aureus wildtype and a (p)ppGpp0 mutant, as well as isogenic guaBA-deficient strains, to gain a more thorough understanding of the genetic basis through which (p)ppGpp maintains cell homeostasis during nutritional stress in the stationary phase and the importance of cellular GTP homeostasis to ensure viability. RNAseq analysis revealed that the TCA cycle and electron transport chain, particularly complex IV/the terminal oxidase qoxABCD, were strongly downregulated in the (p)ppGpp-deficient starved cells. By mutating the initiation nucleotide (TSS +1) of the qoxABCD operon, we were able to increase qoxA expression and membrane potential in a (p)ppGpp0 strain during stationary phase starvation. This resulted in increased culturability during prolonged nutrient starvation, demonstrating the necessity of PMF maintenance. Furthermore , we could show that the decreased membrane potential in starved (p)ppGpp-deficient cells contributes to decreased antibiotic tolerance towards oxacillin and ciprofloxacin. In summary, our data suggests, that (p)ppGpp-dependent regulation of cellular GTP homeostasis during nutritional stress is crucial for maintenance of culturability and antibiotic tolerance.

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The alarmone pGpp inhibits translation through GTP depletion II

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