8 to 1.6 nmol per mg of protein. This corresponds to a decrease in intracellular concentration from 1.8 to 0.5 mM, assuming an intracellular volume of 3.2 mL/mg of protein, ). The drop (about 70%) was rapid, occurring in less than 30 min, but the subsequent decrease in ATP levels was slow, the intracellular concentration after several hours remaining ≥ 0.3 mM in spite of PI3K inhibitor the absence of a carbon source. This suggests that the bacteria are able to use endogenous energy sources (such as glycogen for instance) in order to maintain a minimal energy charge, allowing survival, but not growth. When AThTP was allowed to
accumulate for 4 h in the absence of a carbon source, addition of various metabolizable substrates induced a sharp decrease in AThTP content (inset
of Figure 1). As previously shown , glucose addition (10 mM) triggered a drop of 80-90% in AThTP in less than 5 min and nearly 100% after 30 min, while the decrease was slower with other carbon sources (especially succinate and acetate). We also confirmed that virtually no AThTP was produced when a metabolizable carbon source was present at zero time (when bacteria were transferred from LB to M9 medium). As shown in Table 1, glucose was very effective in antagonizing AThTP accumulation, as an external concentration as low as 1 mM reduced the AThTP content (measured after 60 min) by about 80% while a concentration ≥ 5 mM nearly completely prevented the accumulation of AThTP. However, at high ionic strength (1 M NaCl, EPZ5676 KCl or choline chloride), glucose was unable to prevent AThTP accumulation. This is not surprising, as the high ionic strength is known to impair glucose utilization by E. coli cells . Table 1 Effect of various
carbon sources on AThTP production in the BL21 E. coli strain. AThTP(pmol/mg of protein) Control 88 ± 6 D-Glucose (1 mM) 13 ± 4 D-glucose (2.5 mM) 9 ± 2 D-Glucose (5 mM) < 2 D-Glucose (10 mM) < 2 L-Lactate (10 mM) 14 ± 2 Succinate click here (10 mM) 6 ± 1 L-Malate (10 mM) 8 ± 2 D-Glucose (10 mM) + NaCl (1.2 M) 94 ± 13 D-Glucose (10 mM) + KCl (1.2 M) 92 ± 6 D-Glucose (10 mM) + Choline Cl (1.2 M) 131 ± 15 Streptomycina (10 μM) 62 ± 2 Neomycina (10 μM) 68 ± 3 AAb 12 ± 2 AAb + serine hydroxamate (0.5 mg/mL) 18 ± 2 aAll amino acids (40 μg/mL each) with the exception of serine bNo carbon source present The bacteria (A600 > 1) were incubated for 60 min at 37°C in minimal M9 medium containing substrates at the concentrations indicated. Mean ± SD for 3 – 9 experiments. The antibiotics streptomycin and neomycin have little effect on AThTP accumulation in the absence of a carbon source, suggesting that protein synthesis is not required for AThTP accumulation. We also wanted to know whether the appearance of AThTP was specifically linked to carbon starvation or could be triggered by other forms of nutritional downshifts or cellular stress.