Impact of the (p)ppGpp synthesis and maintenance of GTP homeostasis during stationary phase starvation on the transcriptome
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. Overall design: Samples of S. aureus HG001 wildtype, a (p)ppGpp0 mutant, ?guaBA mutant and a (p)ppGpp0 ?guaBA mutant were grown under aerobic conditions (37°C, 200 rpm) to late stationary growth phase (24h)in chemically-defined medium. The final optical densitiy of the cultures was app 4.6 - 5.0. Each strain and condition was performed in biological triplicates.