Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Abstract

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 ,g/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 ug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 ug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 ug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 ' This paper is derived from a dissertation submitted by Charles E. Deutch to the Graduate Dvision, University of California, Riverside, in partial satisfaction of the requirements for the degree of Doctor of Philosophy. A portion of this work was

Chemical Evolution of a Bacterium’s Genome

“…Thymine starvation in E. coli and other bacteria results in the rapid loss of viable cell titer, a well‐studied response known as thymineless death 12. 13 Exogenous thymine and thymidine can be utilized for the synthesis of deoxythymidine triphosphate (dTTP) and to rescue thyA mutants by a salvage pathway which converts thymine into dTMP through the action of pyrimidine nucleoside phosphorylases ( udp and deoA gene products) and thymidine kinase ( tdk gene product; see Figure S1a).…”

Thymineless Mutagenesis in Escherichia coli

“…DNA synthesis under these conditions was measured by the incorporation of [3H ]uracil into alkaline-resistant, acid-precipitable material. Both the rate and total amount of incorporation decreased in direct proportion to the thymine concentration (8). A significant amount of DNA synthesis still occurred at thymine concentrations which yielded the maximum number of induced mutations, e.g., 0.1 sg/ml.…”

“…Figure 5 shows that, with decreasing concentrations of thymine, there was a progressive increase in the rate of loss of viability. The effects of limiting thymine on viability were discussed in detail in an earlier paper (8). With thymine concentrations below 0.2 ,ug/ml, the frequency of Arg+ revertants began to increase immediately upon suspension in limiting thymine.…”

“…DEUTCH AND PAULING required. Viable cell counts were performed by plating on enriched medium as previously described (8), and revertants to arginine prototrophy were determined by plating on glucose-salts selective media. Plates were incubated at 37 C and scored after 3 days.…”

Protein Synthesis and Deoxyribonucleic Acid-Membrane Attachment During Thymineless Death in Escherichia coli

“…However, Cummings and Kusy (6) found, using synchronously growing cultures of E. coli B and B/r, that there was no position in the cell cycle at which the bacterial population was more or less immune to TLD. Others have found that immunity to TLD may be detected in the presence of RNA and protein synthesis by varying the growth media and the thymine concentration during growth (9).…”