Changes in the activity of inorganic pyrophosphatase in a temperature-sensitive DNA-synthesis mutant of Escherichia coli.

Abstract

CRT 46, a derivative of Escherichia coli K12, has temperature‐sensitive initiation of DNA synthesis. The specific activity of inorganic pyrophosphatase in the cells of CRT46 decreased during 30 min to a new constant level (approximately 2/3 of the initial value), when DNA synthesis was arrested after 1 h at restrictive temperature 42 °C. When the culture was shifted back to permissive temperature 30 °C, the normal specific activity of the enzyme was restored in 2.5 h, while DNA was synthesized with abnormally high rate. Both the decrease and the increase in the activity of the enzyme were prevented, if protein synthesis was arrested by chloramphenical or by amino acid starvation. The activity of inorganic pyrophosphatase was stable at all temperatures up to 80 °C in the extracts of the mutant strain. Therefore the observed changes in the activity of the enzyme were evidently due to the corresponding changes in the differential rate of synthesis of the enzyme.In the cultures of the wild‐type parent strain Escherichia coli CR34 the differential rate of synthesis of inorganic pyrophosphatase was similar both at 30 °C and at 42 °C. Valyl‐tRNA synthetase was produced normally by both strains at both temperatures.The synthesis of inorganic pyrophosphatase continued normally, if DNA synthesis was prevented by nalidixic acid or thymine starvation at 30 °C in the cultures of CRT46 or both at 30 °C and at 42 °C in the cultures of CR34.By inhibiting the initiation of new rounds of DNA replication by amino acid starvation or the proceeding of DNA replication by nalidixic acid at 42 °C it was observed that the defect in the synthesis of inorganic pyrophosphatase appeared only when the rounds of DNA replication had reached the end and the temperature was restrictive.In the cultures of spontaneous revertants the differential rate of synthesis of the enzyme slowed down immediately after the shift to 42 °C, although DNA synthesis continued normally. Another derivative of CR34, BT313, which is temperature‐sensitive in the DNA synthesis, produced the enzyme with the lower differential rate both at 30 °C and at 42 °C.The results show that, although the defect in the synthesis of inorganic pyrophosphatase is somehow dependent on the defect in DNA synthesis those two phenomena are separable. Possible divA mutation, which causes the formation of DNA‐less minicells in the cultures of CRT46 at 42 °C is also responsible for the abnormal synthesis of the enzyme.