The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness.
The consequences of growth of a mutator strain of Escherichia coli as measured by loss of function among multiple gene targets and loss of fitness.
复制标题
通过多个基因靶标的功能丧失和适应性丧失来衡量大肠杆菌突变株生长的后果。
DOI：
10.1093/genetics/154.3.959
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发表时间：
2000
期刊：
影响因子：
3.3
通讯作者：
Miller,JH
中科院分区：
文献类型：
--
作者：
Funchain,P;Yeung,A;Stewart,JL;Lin,R;Slupska,MM;Miller,JH
我们已经通过采用一组广泛的表型筛选来检查大肠杆菌变构子群体成员的组成，这些表型筛选使我们能够监测超过700个基因的功能，这些基因占基因组的约15%。我们观察了在丰富培养基上重复循环的单菌落分离以产生被迫通过严重瓶颈的谱系后的错配修复缺陷细胞，并将结果与野生型菌株的结果进行了比较。随着菌落分离周期的增加，增变基因谱系继续快速积累突变。到第40个周期结束时，经过约1000代，大多数谱系的菌落大小减少，4%已经灭绝，55%具有营养缺陷型需求（60个周期后增加到80%），70%在至少一种糖或分解代谢途径中存在缺陷。此外，33%的细胞运动有缺陷，26%的细胞对温度敏感或对冷敏感。另一方面，在40个周期后，只有3%的野生型谱系具有可检测的任何类型的突变。到第60个周期，典型的突变体细胞在被监测的15%的基因组中携带4-5个失活基因，表明平均细胞携带至少24-30个失活基因，分布在整个基因组中。值得注意的是，30%的谱系已经失去了利用木糖作为碳源的能力。DNA测序显示，大多数Xyl−突变体在xylB基因中有一个8个G（GGGGGGGG）的移码，或者增加或删除一个-G-。进一步的分析表明，渲染E.大肠杆菌错配修复缺陷揭示了某些基因或基因间区域的超变位点。生长曲线和竞争测试的血统，通过90个周期的单菌落隔离表明，所有的血统遭受降低健身。我们讨论了这些结果在细胞进化的增变基因的价值。
We have examined the composition of members of mutator populations of Escherichia coli by employing an extensive set of phenotypic screens that allow us to monitor the function of >700 genes, constituting ~15% of the genome. We looked at mismatch repair deficient cells after repeated cycles of single colony isolation on rich medium to generate lineages that are forced through severe bottlenecks, and compared the results to those for wild-type strains. The mutator lineages continued to accumulate mutations rapidly with each increasing cycle of colony isolation. By the end of the 40th cycle, after ~1000 generations, most of the lineages had reduced colony size, 4% had died out, 55% had auxotrophic requirements (increasing to 80% after 60 cycles), and 70% had defects in at least one sugar or catabolic pathway. In addition, 33% had a defect in cell motility, and 26% were either temperature-sensitive or cold-sensitive lethals. On the other hand, only 3% of the wild-type lineages had detectable mutations of any type after 40 cycles. By the 60th cycle, the typical mutator cell carried 4–5 inactive genes among the 15% of the genome being monitored, indicating that the average cell carried at least 24–30 inactivated genes distributed throughout the genome. Remarkably, 30% of the lineages had lost the ability to utilize xylose as a carbon source. DNA sequencing revealed that most of the Xyl−mutants had a frameshift in a run of eight G's (GGGGGGGG) in the xylB gene, either adding or deleting one -G-. Further analysis indicated that rendering E. coli deficient in mismatch repair unmasks hypermutable sites in certain genes or intergenic regions. Growth curves and competition tests on lineages that passed through 90 cycles of single colony isolation showed that all lineages suffered reduced fitness. We discuss these results in terms of the value of mutators in cellular evolution.