Many systems are proposed to be involved in the AR of both logarithmic- and stationary-phase cells (5
). The well-studied systems are amino acid-dependent systems. It has been reported that the amino acid-dependent systems enhance survival under acidic conditions via the consumption of cytoplasmic protons by amino acid decarboxylation (29
). Why is such pHi
regulation required for the survival of nongrowing cells under acidic stress? Some metabolic processes may work under such nongrowing conditions. In addition to amino acid-dependent induction, many studies have demonstrated that various genes participate in AR (5
). Carbon dioxide, a substrate for nucleic acid and amino acid biosynthesis, induces AR (38
), leading us to assume that nucleotide biosynthesis induces AR, but there has been no report to show the role of nucleotides in AR. We therefore examined the participation of purine nucleotide biosynthesis in survival under acidic conditions in the present study.
The present study with cells growing exponentially revealed that the survival of the cells under acidic stress required ATP in both the presence and absence of amino acids such as glutamate, arginine, and lysine. The ATP level increased during the adaptation of E. coli cells at pH 5.5 and decreased during acidic challenge at pH 2.5 (). The ATP level was low at pH 5.5 and ATP was lost rapidly at pH 2.5 in the purA and purB mutants, and these mutants showed a low survival rate at pH 2.5. The defect of the adenylate kinase activity decreased the ATP level and the AR. In contrast, the deletion of genes for IMP biosynthesis did not decrease the AR. The ATP levels of these mutants were higher than those of the purA and purB mutants but lower than that of the wild type. Furthermore, the requirement of genes for GMP or purine deoxyribonucleotide biosynthesis was suggested to be less significant for AR. These data implied that the ATP level is more important for survival under acidic conditions than the levels of other purine nucleotides and deoxynucleotides.
All pur mutants tested were unable to grow without the addition of adenine at pH 7.5, hence 0.1 mM adenine was added to EG medium at pH 7.5. The experimental conditions for the acidic adaptation and challenge were the same in all pur mutants, but the ATP level of the purA and purB mutants was lower than the level of the purC, purK, and purL mutants (). It may be possible that the level of IMP produced from adenine at pH 7.5 is enough for the maintenance of the ATP level required for survival at pH 2.5 in the purC, purK, and purL mutants. The other possibility is that E. coli has alternative enzymes or pathways functioning at acidic pH instead of PurC, PurK, and PurL. The functions of more than 2,000 genes in E. coli still are unknown.
It remains unclear why the ATP level of cells grown at pH 5.5 is higher than that at pH 7.5. The greater pH gradient at pH 5.5 might account for the high level of ATP. However, the membrane potential that drives ATP synthesis, together with the pH gradient, was low or inside positive at low pH (29
). An alternative explanation is that many metabolic processes consuming ATP, such as the biosynthesis of macromolecules, decline at low pH because of the decrease in enzyme activities. In fact, the growth rate was low at pH 5.5.
ATP is a substrate to supply energy for various metabolic processes. Which ATP-dependent metabolic process supports the survival at pH 2.5? Jeong et al. (12
) showed that mutants deficient in the genes required for DNA repair had low survival rates at low pH and that mutation caused more DNA damage. We found in the present study that the single deletions of recA
, and urvB
had no significant effect on the AR (data not shown), but the AR of the recB
mutant was lower than that of the parent strain. Furthermore, multiple mutations (recB
, and sbcB
) brought about low survival at pH 2.5 (). These data confirmed the suggestion that the DNA repair system is indispensable for survival in acidic conditions. The DNA damage analysis showed that the deletion of purA
caused more DNA damage in acidic conditions, suggesting that ATP keeps the DNA repair systems active.
E. coli has many other ATP-requiring systems, such as ion transport systems and macromolecule biosynthesis. We found that the intracellular levels of Na+ and K+ decreased at acidic pH, but the levels of these ions in the purA and purB mutants were almost the same as those of their parent strain (data not shown), suggesting that the low ATP level shown in the mutants is sufficient for the maintenance of the cytoplasmic levels of these cations in acidic conditions. The biosynthesis of macromolecules may not occur under nongrowing conditions at pH 2.5.
It was reported that the pHi
of the wild type was 3.6 to 3.7 in medium of pH 2.3 to 2.4 without the addition of amino acid (29
). In contrast, the pHi
was 3.7 to 4.0 under our experimental conditions (). In the previous study, the cells grown to stationary phase were transferred to pH 2.5 medium and the pHi
was measured. We adapted the cells in the logarithmic phase at pH 5.5, and the pHi
was measured after the cells had been transferred to pH 2.5 medium. Therefore, cells growing logarithmically at pH 5.5 may have an increased ability to regulate pHi
in the absence of amino acids.
No significant decrease in pHi
was observed in the purA
mutants (). The mechanism for the maintenance of the pH gradient remains unclear. It has been clarified that the pH gradient is generated by the F-type H+
ATPase in enterococci (14
). The same mechanism might work in E. coli
, as proposed by Richard and Foster (29
). However, the ATP hydrolysis activity of the H+
ATPase was negligible at pH of less than 5, and the Km
for ATP was 0.6 mM in E. coli
). ATP at 0.6 mM corresponds to approximately 1.8 nmol ATP per mg protein. Therefore, the ATPase may have difficulty extruding protons at acidic pH. It is possible that E. coli
has an unidentified system for pH homeostasis. In any case, the pHi
regulation is essential for survival under acidic stress. In addition, our present results suggested that the ATP-dependent repair system has an essential role in the AR.