Rate of DNA synthesis during the division cycle of Escherichia coli B/r

Abstract

The rate of DNA synthesis during the division cycle of Escherichia coli B/r has been measured in exponentially growing cultures. This was accomplished by pulse-labeling exponential phase cultures with [ 14C]thymidine and measuring the amount of label incorporated into cells of different ages in the cultures. The amount of label in cells of different ages was found by binding the bacteria to the surface of a membrane filter at the end of the labeling period and counting the radioactivity in the cells which were eluted continuously from the membrane. Since primarily new-born cells are eluted from a membrane-bound culture of E. coli B/r, the radioactivity per effluent cell during each generation of elution was a measure of the amount of [ 14C]thymidine incorporated into the oldest through the youngest cells in the exponential phase culture. This experimental approach was considered to be superior to those employing synchronized cells for these studies, because all manipulations were carried out after, rather than before, the incorporation took place. It was found that there is an abrupt increase in the rate of DNA synthesis during the division cycle of cells growing in glucose, glycerol, succinate or acetate minimal medium which occurs at a cell age, in fractions of a generation, of 0.4, 0.8, 0.9 and 0.9, respectively. In suecinate- and acetate-grown cells, this increase is preceded by a pronounced minimum in the rate of [ 14C]thymidine incorporation during the last half of the division cycle. It is suggested that the period of reduced thymidine incorporation corresponds to a gap between rounds of DNA replication, and that the abrupt increase corresponds to the start of a new round of DNA replication.