Anraku N, Landman O E
J Bacteriol. 1968 May;95(5):1813-27. doi: 10.1128/jb.95.5.1813-1827.1968.
Studies of Maaløe, Lark, and others with amino acid- and thymine-starved cultures revealed successive steps in the biosynthesis of Escherichia coli chromosomes. In this study, the corresponding mechanisms in Bacillus subtilis 168 were examined. Using a strain requiring both thymine and tryptophan, we found that, 3 hr after the start of amino acid starvation, when the deoxyribonucleic acid (DNA) content of the culture had increased 40 to 50%, DNA synthesis ceased. After 4 to 5 hr, 100% of the cells were immune to thymineless death; their chromosomes had presumably been completed. Immune cultures slowly incorporated (3)H-thymine. Thymine incorporation increased 20-fold 30 min after readdition of amino acids, indicating reinitiation of chromosome synthesis. Simultaneous presence of amino acids and thymine was required for reinitiation. If 5-bromouracil (5-BU) was added instead of thymine, newly replicated DNA segments could be separated by centrifugation in CsCl. Analysis of the CsCl fractions by a transformation assay showed that the order in which the markers were synthesized was ade-16, thr-5, leu-8, metB5. Less than half the chromosomes started resynthesis synchronously in 5-BU. Nevertheless, chromosome alignment in the amino acid-starved culture is probably very good: marker frequency analysis of its DNA gives the same normalized frequencies as DNA from "perfectly" aligned spores. Full viability is maintained in the chromosome-arrested culture for 10 hr in thymine-free medium in the absence or presence of amino acids. In the latter condition, protein synthesis proceeds, and the cells filament and become more lysozyme-sensitive. Such cells must be incubated and plated on hypertonic or on slow-growth media; otherwise, they undergo "quasiosmotic" thymineless death. This death is thus apparently not directly attributable to any damage of chromosomal DNA. Further, weakening of the teichoic acid portion of the cell wall is not involved, since (32)P incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits (32)P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue.
马勒、拉克及其他一些人对氨基酸和胸腺嘧啶饥饿培养物的研究揭示了大肠杆菌染色体生物合成的连续步骤。在本研究中，对枯草芽孢杆菌168中的相应机制进行了研究。使用一株同时需要胸腺嘧啶和色氨酸的菌株，我们发现，在氨基酸饥饿开始3小时后，当培养物的脱氧核糖核酸（DNA）含量增加40%至50%时，DNA合成停止。4至5小时后，100%的细胞对无胸腺嘧啶死亡具有抗性；它们的染色体大概已经完成。具有抗性的培养物缓慢掺入（3）H-胸腺嘧啶。重新添加氨基酸30分钟后，胸腺嘧啶掺入增加了20倍，表明染色体合成重新启动。重新启动需要同时存在氨基酸和胸腺嘧啶。如果添加5-溴尿嘧啶（5-BU）代替胸腺嘧啶，新复制的DNA片段可通过在CsCl中离心分离。通过转化试验对CsCl级分进行分析表明，标记物合成的顺序是ade-16、thr-5、leu-8、metB5。在5-BU中，不到一半的染色体同步开始重新合成。然而，氨基酸饥饿培养物中的染色体排列可能非常好：对其DNA进行标记频率分析得到的归一化频率与来自“完美”排列孢子的DNA相同。在无胸腺嘧啶培养基中，无论有无氨基酸，染色体停滞的培养物在10小时内都能保持完全活力。在后一种情况下，蛋白质合成继续进行，细胞形成丝状体并对溶菌酶更敏感。此类细胞必须在高渗或缓慢生长培养基上培养并接种；否则，它们会发生“准渗透”无胸腺嘧啶死亡。因此，这种死亡显然并非直接归因于染色体DNA的任何损伤。此外，细胞壁磷壁酸部分的弱化也不涉及，因为（32）P掺入磷壁酸是正常的。氯霉素可防止准渗透无胸腺嘧啶死亡，也抑制（32）P掺入磷壁酸。进行染色体合成的培养物会遭受两种类型的无胸腺嘧啶死亡：准渗透死亡和对渗透拯救不敏感的死亡。
