Sources of the Nucleotide Pools in Escherichia coli after Infection with Bacteriophage T2

Abstract

Abstract It has long been known that infection of Escherichia coli with bacteriophage T2 causes degradation of bacterial deoxyribonucleic acid and that 70 to 80% of the degradation products appear in viral DNA within 30 min after infection. It is reasonable to assume that exonucleases participate in this process and catalyze the production of mononucleotide intermediates. To study this system in more detail, the amounts of free mononucleotides present in the cells were measured at various times after infection with phage T2. The extent of transferal of labeled nucleotides from bacterial nucleic acids to the free mononucleotide pools was also studied. Only a gradual and small increase in the total amounts of deoxyribonucleotides in the pools was observed after infection, in spite of drastic changes both in the rate of nucleic acid synthesis and in the pathways of nucleic acid metabolism that occur at this time. This suggests that the intracellular concentrations of these intermediates are precisely controlled. By labeling the bacterial nucleic acid with 32P-orthophosphate and subsequently infecting the cells with T2 phage, the amount of nucleic acid-derived nucleotides present in the deoxyribonucleotide pools was found to increase rapidly 4 min after infection and decline precipitously 10 to 15 min after infection. At 7 to 8 min after infection, 50 to 75% of the nucleotides entering these pools came from the bacterial nucleic acids. Of the known bacterial and phage-induced exonucleases, only T2 exonuclease A exhibits sufficient activity in vitro to readily account for the rate of nucleotide production that is observed. The time of appearance of this enzymatic activity is correlated with the time of increase of nucleic acid-derived nucleotides in the pools. It is paradoxical that, 10 to 15 min after infection, a rapid decline of labeling of the pools occurs at a time when half of the bacterial DNA remains. Knowledge of the relative specific activities of the deoxycytidylate and thymidylate pools makes it possible to estimate the rate of their interconversion in vivo by the pathway involving the phage-induced enzymes deoxycytidylate deaminase and thymidylate synthetase. The data indicate that, at 9 min after infection, approximately 15% of the deoxycytidylate derived from bacterial DNA is converted to thymidylate via this pathway. The high specific activity of the deoxyadenylate pool suggests that some of this compound may have been derived from a deoxyguanosine nucleotide. A small but reproducibly measurable amount of nucleic acid-derived phosphate was found in the nucleotide pools of uninfected cells. This suggests that a slow turnover of stable ribonucleic acid, or perhaps both RNA and DNA, occurs in rapidly growing E. coli.