By directed evolution in the laboratory, we previously generated populations of Escherichia coli that exhibit a complex new phenotype, extreme resistance to ionizing radiation (IR). The molecular basis of this extremophile phenotype, involving strain isolates with a 3-4 order of magnitude increase in IR resistance at 3000 Gy, is now addressed. Of 69 mutations identified in one of our most highly adapted isolates, functional experiments demonstrate that the IR resistance phenotype is almost entirely accounted for by only three of these nucleotide changes, in the DNA metabolism genes recA, dnaB, and yfjK. Four additional genetic changes make small but measurable contributions. Whereas multiple contributions to IR resistance are evident in this study, our results highlight a particular adaptation mechanism not adequately considered in studies to date: Genetic innovations involving pre-existing DNA repair functions can play a predominant role in the acquisition of an IR resistance phenotype.
X-rays and other forms of ionizing radiation can damage DNA and proteins inside cells. The radiation interacts with aqueous solutions to produce reactive forms of oxygen, which then cause the damage. A range of mechanisms exist to moderate and/or repair this damage, with certain species being able to tolerate extraordinary levels of radiation. The bacterium D. radiodurans, for example, can survive radiation levels that are over 1000 times higher than the levels that can kill human cells.
The molecular basis of high-level resistance to ionizing radiation is not well understood, and several mechanisms have been proposed. Recent work has focused on passive mechanisms that are based on changes in cellular levels of certain small molecules that prevent damage by reactive forms of oxygen molecules.
Now, based on experiments on E. coli, Byrne et al. demonstrate that active mechanisms, involving adaptations in the cellular DNA repair systems, can bring about dramatic increases in radiation resistance. The experiments were performed on populations of E. coli cells that had been subjected to an evolutionary selection for extremely high resistance to ionizing radiation. This involved exposing the E. coli cells to ionizing radiation that killed most of the population, and then growing up the survivors. Many repetitions of this process led to a population of cells with a resistance that was comparable to that of the bacterium D. radiodurans. The same evolution experiment was carried out four times, generating four separate populations of bacteria that were resistant to ionizing radiation.
Byrne et al. sequenced the genomes of the E. coli after 20, 40 or 50 rounds of the selection process, and compared mutations found in the four separate evolved populations. This showed that nine genes were particularly prone to mutations. Together, these genes had roles in repairing and copying DNA sequences, in decreasing damage caused by reactive forms of oxygen, and in manufacturing the molecular wall that shields cells.
To assess the importance of the mutations in the nine genes, Byrne et al. took Founder cells from the initial population of E. coli cells–which were not resistant to ionizing radiation–and introduced the very same mutations, one at a time. Then the mutations that had the largest positive effects on resistance to ionizing radiation were combined. Introducing particular mutations into three DNA repair genes resulted in the highest aggregate levels of resistance. Finally, evolved E. coli cells that were already resistant were made more sensitive to radiation by repairing the same individual mutations. Again, the biggest change was observed with the DNA repair genes. Indeed, repairing the mutations in just the three DNA repair genes completely removed the radiation resistance.
The next step is to determine how the properties of the mutated proteins change, and how those changes lead to radiation resistance. Also, there are clues in the work that suggest the presence of additional ways for cells to become radiation resistant, and these remain to be explored.