A useful tool for answering those questions is the thermo-sensitive CV2 strain [20, 21]. This strain contains

a heat-sensitive AK that is rapidly inactivated when the bacteria are grown at temperatures higher than 30°C. At 37°C, the cellular energy charge drops within two hours from 0.9 to 0.2, GDC-0449 molecular weight the intracellular ATP Smad pathway concentration being around 0.2-0.3 mM. When an energy substrate is present, ATP is produced at a normal rate, but its hydrolysis coupled to nucleic acid synthesis results in an accumulation of AMP that cannot be converted to ADP because of lack of AK activity. Therefore, the energy charge remains low despite the presence of an energy substrate. Here, we observe that at 37°C, CV2 cells accumulate AThTP in the absence of carbon sources as expected, but not when D-glucose or L-lactate are present (Table 2). This is surprising, as the

presence of those substrates does not induce any substantial increase in intracellular ATP concentration. Thus, AThTP production does not occur in the presence of substrates, even when the energy charge remains very low. However, under these conditions ThTP levels are very high [21] and it is therefore possible that AThTP accumulation is inhibited by ThTP (see below). The effects of the uncoupler CCCP were also investigated in CV2 cells. The cells were transferred to a minimal medium supplemented with L-lactate (10 mM) either at 25°C (Figure 6A) or at 37°C (Figure 6B) and CCCP was

added after 1 BI 2536 supplier hour. At 25°C addition of CCCP induced a rapid decrease of the energy charge (from 0.9 ± 0.1 to 0.3 ± check details 0.1 after 20 min). In contrast, at 37°C, addition of CCCP only slightly decreased the energy charge as it was already very low (from 0.29 ± 0.04 to 0.26 ± 0.02 after 20 min and less than 0.2 after 1 h). However, at both temperatures, CCCP induced a rapid increase in AThTP content. This change occurred even more rapidly at 37°C than at 25°C. At 37°C, ATP content was less than 1 nmol per mg protein (corresponding to an intracellular concentration of 0.3 mM) 1 h after addition of CCCP. Thus AThTP accumulation occurred when the Δp was abolished and did not appear to be significantly influenced by variations in the ATP pool. Figure 6 Effect of CCCP on AThTP levels in the E. coli CV2 strain incubated in minimal medium containing L-lactate at 25 and 37°C. The bacteria were grown overnight in LB medium and transferred to minimal M9 medium containing 10 mM L-lactate either at 25 or at 37°C. CCCP (50 μM) was added after 60 min (arrow). (Means ± SD, n = 3) At both temperatures, CCCP increased the respiratory rate by a factor of approximately 2 with glucose (from 21 ± 7 to 41 ± 9 nmol.mg-1.min-1, n = 3) and L-lactate (from 19 ± 8 to 38 ± 1 nmol.mg-1.min-1, n = 3) as substrates. These results suggest that the CV2 strain retains a significant Δp even at 37°C, when the energy charge is very low.