On the mechanism of thymineless death in Bacillus subtilis.

Thymineless death (TD), the loss by a thymine-requiring auxotroph of the ability to multiply after a period of thymine starvation, was first described1 in Escherichia coli 15T-, and was attributed to "unbalanced growth," the synthesis of RNA and protein in the absence of DNA synthesis. This phenomenon has been investigated in thymineless auxotrophs of a number of bacterial species: E. coli 15Tand various derivatives;2' I E. coli B3;4 E. coli K12 thy-;' B. megaterium thy-;6 and B. subtilis thy-.7 8 A number of different hypotheses have been advanced to explain TD, including "unbalanced growth,"' "nuclear damage,"4 single-stranded nucleolytic scissions in DNA,9 10 colicin induction,"' 11, 12 and prophage induction., 13 The involvement of macromolecular synthesis in TD has been examined in E. coli 15Tand other strains with conflicting results. Although several investigators have claimed an absolute requirement for protein synthesis,14 3. 5. 6 others have found that TD proceeds in the apparent absence of protein synthesist. 15 It has been suggested that the synthesis of RNA rather than protein is essential for TD,4 and there have been reports suggesting that messenger-RNA synthesis may be involved.'5 16 Perhaps complicating the interpretation of many of these studies is the fact that the course of TD may be influenced by conditions prevailing before or after the period of thymine starvation. Full immunity to TD in E. coli 15 TAU is attained by chromosome completion prior to thymine starvation.2 Also, the level of survival of a thymine-starved culture of this strain depends upon the conditions in which thymine is restored to the cells: in cells starved for thymine, arginine, and uravil, there is a higher proportion of survivors if starvation for arginine and uracil persists for a time after thymine is restored.17 We have utilized exponentially growing cultures of thymineless strains of Bacillus subtilis to show that actinomycin blocks TD. This effect ensues at concentrations of actinomycin just sufficient to inhibit RNA synthesis, with little inhibition of DNA synthesis. It is known that actinomycin blocks protein synthesis by preventing the synthesis of messenger RNA,18 and we have found that other treatments which inhibit protein synthesis also stop TD in B. subtilis. Studies in which actinomycin is administered at different times after removal of thymine suggest that TD involves two distinct steps, only the first of which is blocked by actinomycin. Materials and Methods.-Strains: B. subtilis S (S22, thy-i, arg-) derived from ATCC 6633 was obtained from R. Romig.7 B. subtilis strains R (R168, thy-, tryp-) and W (W23, thy-, his-) were obtained from F. Rothman.'9 Media: Luria broth20 (LB) or tris-salts, minimal medium21 (TS), were used with the following supplements: amino acids, 20 ,jg/ml; thymine, 2 ,g/ml (S), or 20 Ag/rnl (R, W); glucose, 5 mg/ml; P as phosphate, 10 Ag/ml. Glutamate (10 mg/ ml) reduces the lag before growth of strains R and W in TS, and was used in a number of experiments.