Relationship between Growth Rate and ATP Concentration in Escherichia coli

Abstract

Previous studies showed that adenosine triphosphate (ATP) concentrations in Escherichia coli changed during certain growth transitions and directly controlled the rate of rRNA transcription initiation at those times. The relationship between ATP concentration and rRNA transcription during steady-state growth is less clear, however. This is because two commonly employed methods for measuring ATP concentrations in bacteria, both of which rely on physical extraction followed by chromatographic separation of small molecules, resulted in dramatically different conclusions about whether ATP concentration changed with steady-state growth rate. Extraction with formic acid indicated that ATP concentration did not change with growth rate, whereas formaldehyde treatment followed by extraction with alkali indicated that ATP concentration increased proportionally to the growth rate. To resolve this discrepancy, we developed a bioassay for ATP based on the expression of a variant of the firefly luciferase enzyme in vivo and measurement of luminescence in cells growing in different conditions. We found that the available ATP concentration did not vary with growth rate, either in wildtype cells or in cells lacking guanosine 5-diphosphate, 3-diphosphate, providing insight into the regulation of rRNA transcription. More broadly, the luciferase bioassay described here provides a general method for evaluating the ATP concentration available for biochemical processes in E. coli and potentially in other organisms.ATP is the energy currency of the cell, providing energy for enzymatic reactions. ATP also serves as a substrate for RNA synthesis, and it regulates a variety of biological processes. Because of its potential to affect many aspects of cellular regulation, whether cellular ATP concentrations change when growth conditions change is a key question in molecular biology.The concentration of the initiating nucleoside triphosphate (iNTP) 1 plays a direct role in the regulation of rRNA transcription initiation in Escherichia coli in response to changes in growth conditions (1-4). For example, when cells outgrow from stationary phase, the levels of ATP (the iNTP for 6 rrn P1 promoters) and GTP (the iNTP for the seventh rrn P1 promoter) increase dramatically, resulting in a direct and rapid increase in rRNA synthesis (4). Conversely, when cells enter stationary phase, a decrease in ATP and GTP levels (in conjunction with an increase in the concentration of guanosine 5Ј-diphosphate, 3Ј-diphosphate, ppGpp) directly inhibits rRNA synthesis. Following nutrient shifts during exponential growth, changes in ppGpp concentration are sufficient to account for regulation of rRNA promoters (4).It has long been known that when E. coli cells are grown on different nutrient sources leading to different steady-state growth rates rRNA synthesis is proportional to the square of the culture's growth rate (5). The molecular basis for this phenomenon, called "growth rate-dependent control", still remains unresolved, however. Based on an observed correlati...

The uses and abuses of rapid bioluminescence-based ATP assays

“…Having grown the cells under what they termed 'moisture stress', they found that cell ATP content could be increased 2.2 fold by rewetting dry soils and that furthermore, the addition of glucose and ammonium salts increased ATP levels 5.9 and 2.8 fold respectively. Schneider and Gourse (2004) found that ATP levels in laboratory-grown E. coli remained constant with growth rate, and claimed that earlier work that had presented evidence of growth rate-related differences were in fact at fault due to the method used to extract ATP from the cells.…”

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

“…A standing controversy regarding the reliability of methods of NTP extraction was resolved in favor of the formic acid extraction method on the basis of a newly developed luciferasebased bioassay to measure available ATP pools (60). Using the formic acid extraction method, we observed a substantial increase in all four NTPs during outgrowth of E. coli from sta- lanes 1 and 2), 90 (lanes 3 and 4), 120 (lanes 5 and 6), 150 (lanes 7 and 8), and 390 min (lanes 9 -12).…”

Glyceraldehyde-3-phosphate dehydrogenase of Xanthomonas campestris pv. campestris is required for extracellular polysaccharide production and full virulence

“…The ATP levels of the complemented strain C0972 and the wild-type strain 8004 were almost identical ( Table 2), indicating that the ATP production of the mutant could be restored completely by the wild-type gapA gene in trans. The intracellular ATP levels of the Xcc strains were also determined by the method described by Schneider & Gourse (2004). The result also displayed that the gapA mutant produced significantly less intracellular ATP than the wild-type (Table 2).…”

“…Standard ATP curves were generated with standards containing known amounts of ATP in Tris/EDTA buffer. Intracellular ATP levels were also measured by the modified method developed by Schneider & Gourse (2004).…”

“…Data presented are the means±SD of triplicate experiments. Assays A and B used the methods described by Kimmich et al (1975) and Schneider & Gourse (2004) Different letters a and b in each column indicate significant differences at P¡0.05 (t-test).…”