They peaked at the late log to early stationary phase of growth for most strains and decreased to much lower or undetectable levels

by 24 hours of growth. The growth phase – dependent presence of extracellular ATP suggests a dynamic process of ATP release and depletion, and the observed H 89 cell line level of ATP in the see more culture supernatant is most likely the combined effect of the two processes. Live E. coli and Salmonella (but not dead bacteria or culture supernatant) appear to actively deplete extracellular ATP and the depletion was not due to uptake (Figure 5). Either α-labeled or γ-labeled phosphate on supplemental ATP remained in the culture medium, suggesting that the extracellular ATP was hydrolysed or degraded at the bacterial surface (Figure 5). There have been a few reports on the extracellular ATP from bacteria [1, 9, 10]. Iwase et al. reported the detection of ATP in the culture supernatant of Enterococcus species, but not strains of E. coli or Staphylococcus aureus KPT330 (Iwase, 2010 #195). A possible reason for the discrepancy between their results and ours is that they used overnight cultures which had very low ATP levels in our study as well, while cultures

at late log and early stationary phases had much higher extracellular ATP levels (Figures 3 and 4). Another report by Ivanova et. al reported the presence of extracellular ATP from cultures of Sulfitobacter, Staleya and Marinobacter at 190 μM to 1.9 mM. These levels approach those reported for intracellular ATP of 1 – 3 mM and are much higher than we observed. If those levels are accurate it would suggest that the total quantity of extracellular ATP

far exceeds that of intracellular ATP since the volume of cell culture medium is at least several hundred times higher than that of bacterial cells. We do not know if the differences between results by Ivanova et al. and our results were due to the different bacterial species used or to technical reasons. After we finished the experiments reported here and were preparing this manuscript, Hironaka et al. reported a follow-up study to their previous Phospholipase D1 report that ATP is secreted by gut commensal bacteria [11]. In the new report, they demonstrated that ATP can be detected in the culture supernatant of log phase cultures of E. coli, Pseudomonas aeruginosa and Staphylococcus aureus but not the stationary cultures, in agreement with our observations reported here [11]. They also reported that glycolysis is essential for ATP secretion which supports our notion that cytochrome bo oxidase and respiration are important for ATP release (Figure 4). Reports in recent years have shown that eukaryotic cells can release ATP without lysis through exocytosis of ATP-containing granules, plasma membrane carriers or large conductance channels [2, 3, 20, 21]. Cells of innate immunity such as dendritic cells and macrophages sense ATP as a danger signal through purinergic receptors of P1 and P2 family and initiate a pro-inflammatory response [2, 3, 20].