Bridges B A, Ereira S
MRC Cell Mutation Unit, University of Sussex, Brighton, United Kingdom.
J Bacteriol. 1998 Jun;180(11):2906-10. doi: 10.1128/JB.180.11.2906-2910.1998.
Escherichia coli WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10(8) on plates lacking the required amino acid. They lose viability for some days, and then a subpopulation recovers and there is cell turnover. At very low plating densities (around 10(2) per plate), almost every cell will eventually form a small but visible colony. At intermediate plating densities (10(6) to 10(7) per plate), there is an immediate increase in the number of viable bacteria. The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species. Mutations in mutT bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10(7) per plate). We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10(8) scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid. The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a polA derivative. We cannot exclude the possibility that this residual DNA synthesis is peculiar to mutT bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain. If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10(7) per plate) and reduced the rate of appearance of mutants at plating densities above 10(8) per plate.
具有赭石色氨基酸营养缺陷型的大肠杆菌WP2细菌，在缺乏所需氨基酸的平板上以高于10⁸的密度接种后的最初几天内，没有生长迹象。它们会在几天内丧失活力，然后一个亚群恢复生长，并且存在细胞更替。在非常低的接种密度（每平板约10²个）下，几乎每个细胞最终都会形成一个小但可见的菌落。在中等接种密度（每平板10⁶至10⁷个）下，存活细菌的数量会立即增加。这些结果与一个模型一致，该模型假设生长依赖于微量的色氨酸或色氨酸互补物质，而死亡是由于培养基中的细胞外有毒物质，包括活性氧物种。在这些条件下，mutT细菌中的突变是由于7,8 - 二氢 - 8 - 氧代 - dGTP掺入DNA所致，因此在很大程度上反映了与所用初始密度（每平板10⁷个）下存活细胞数量增加相关的DNA合成。我们表明，通过存在10⁸个清除细菌以及去除早期出现的释放所需氨基酸的突变菌落，可以消除细胞数量的增加和大部分这种DNA合成。剩余的合成量相当于每天不到四分之一个基因组，并且在polA衍生物中即使有减少也非常微小。我们不能排除这种残余DNA合成是mutT细菌特有的可能性，原因是转录渗漏，尽管没有证据表明这是该菌株中的一个主要问题。如果这种DNA合成也发生在野生型细菌中，那么它对于适应性突变可能很重要，因为在选择性平板中使用更精细的琼脂既消除了低密度（每平板10⁷个）下观察到的细胞数量的初始增加，又降低了接种密度高于每平板10⁸个时突变体出现的速率。
