Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection
Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection
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DOI：
10.1128/jb.00784-18
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发表时间：
2019-04-01
影响因子：
3.2
通讯作者：
Cox, Michael M.
中科院分区：
文献类型：
--
作者：
Bruckbauer, Steven T.;Trimarco, Joseph D.;Cox, Michael M.
在之前的工作(D.R.Harris等人，J细菌191：5240-5252,2009，https://doi.org/10.1128/JB.00502-09；B.T.Byrne等人，eLife3：E01322,2014，https://doi.org/10.7554/eLife.01322)，)中，我们证明了大肠杆菌可以通过定向进化获得对电离辐射(IR)的大量抗性。主要的表型贡献包括有机系统对DNA修复的适应。我们现在已经进行了一项扩展的努力，以产生与耐辐射球菌一样对IR具有抵抗力的大肠杆菌种群。在使用高能电子束IR进行最初50个周期的选择后，四个重复群体的IR抗性显著增加，但尚未达到与耐辐射金黄色葡萄球菌相当的IR抗性。常规的深度测序揭示了复杂的进化模式，并带有丰富的克隆干扰。突出的IR抗性机制包括对DNA修复系统的新适应和RNA聚合酶的改变。适应是高度专门化的，以抵抗IR暴露，因为来自进化种群的分离株对其他形式的DNA损伤表现出高度不同的抗性模式。从种群中测序的分离株具有184到280个突变。IR9-50-1分离株的IR抗性主要来自四个影响DNA和RNA代谢的新突变：RecD A90E、RecN K429Q和RpoB S72N/RpoC K11721。一些细菌对电离辐射表现出惊人的耐受性，耐辐射葡萄球菌就是其中的典型。由于天然红外源很少超过mGy值，因此耐脱水能力已被认为是耐受5,000 Gy值的能力。为了了解真正的极端红外线抗性的分子基础，我们正在利用实验进化来培育出具有与奇诺球菌相当的红外线抗性水平的大肠杆菌菌株。实验进化之前已经对多种细菌物种产生了中等的辐射抗性。然而，这些努力无法利用现代基因组测序技术。在这份报告中，我们检查了50个选择周期后的四个复制细菌种群。基因组测序使我们能够在整个选择过程中跟踪种群中突变的起源。影响编码DNA修复蛋白和RNA聚合酶的基因的新突变增强了辐射抗性。然而，更多的贡献者是显而易见的。
In previous work (D. R. Harris et al, J Bacteriol 191:5240-5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major pheno-typic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans. After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans. Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K11721. Additional mechanisms of IR resistance are evident.IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus. Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.