Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair

Abstract

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. DOI: http://dx.doi.org/10.7554/eLife.01322.001.

Keywords: DNA repair; evolution; extremophile; ionizing radiation; mutation.

Figure 1.. Directed evolution scheme for the evolved isolates described in this paper.

Red arrows denote cycles of irradiation and outgrowth. Evolved populations have titles beginning with “IR”. Isolates were derived from each listed population, as indicated by blue arrows. Isolates from each population are listed under the respective blue arrows, and each isolate features a name beginning with CB. Isolates listed in green text are described for the first time in this study. The sequences of the remaining isolates were described previously (Harris et al., 2009), and are listed here since the genomic data was utilized in the current analysis. DOI: http://dx.doi.org/10.7554/eLife.01322.003

Figure 2.. Effects of selected mutations on survival of E. coli.

Mid-logarithmic phase cultures were irradiated to 3000 Gy and plated to measure survival as described in ‘Material and methods’. (A) mutations discovered in CB2000 were moved individually and in combination into the Founder Δe14 background. Mutations present in a given strain are indicated by a + symbol. For reasons not understood, it proved impossible to move the nanE mutation into this background on its own. CB2000 itself is presented in the final lane. (B) the same point mutations (this time including nanE) were mutated back to Founder sequence in the CB2000 background. The first lane is CB2000. Mutations converted to the wild type allele in a given strain are indicated by a–symbol. (C) non-essential genes assayed in Panel A and B were deleted in the Founder Δe14 background, with the deleted gene indicated by a Δ symbol. D, The effects of mutations in genes recA, dnaB, and yfjK are summarized, with symbols as in panels A–C. All results were obtained with a ¹³⁷Cs irradiator (∼7 Gy/min). DOI: http://dx.doi.org/10.7554/eLife.01322.006

Figure 3.. IR survival in direct competition assays.

Mid-logarithmic cultures consisting of a 1:1 ratio of the competiting Ara+ and Ara^–strains were irradiated to 2000 and 3000 Gy and plated on tetrazolium arabinose indicator plates to distinguish the frequency of survival for both strains. (A) CB2000 Ara^– (red, ^), vs CB2000 wtRecA wtDnaB wtYfjK, ☐ (B) Founder Δe14 Ara⁻, (red, △), vs Founder Δe14 RecA D276N DnaB P80H YfjK A151D, ◇ (C) TA plate of 2000 Gy survival competition of Founder Δe14 Ara⁻vs Founder Δe14 RecA D276N DnaB P80H YfjK A151D. The two red Founder Δe14 Ara^–colonies are indicated by red arrows. (D) Founder Δe14 Ara⁻ (red, ◇), vs CB2000 wtRecA wtDnaB wtYfjK, ☐. (E) Log advantage in survival to 2000 Gy of CB2000 Ara⁻over CB2000 wtRecA wtDnaB wt YfjK (in black), Founder Δe14 RecA D276N DnaB P80H YfjK P80H over Founder Δe14 Ara⁻ (in grey), and CB2000 wtRecA wtDnaB wt YfjK over Founder Δe14 Ara⁻ (in white). DOI: http://dx.doi.org/10.7554/eLife.01322.007

Figure 4.. (A), Measurements of metabolites from three representative evolved E. coli strains as compared to Founder.

Metabolites from whole cell pellets collected during logarithmic growth in LB were identified using a two-dimensional ¹H-¹³C Heteronuclear Single Quantum Coherence (HSQC) experiment. Each metabolite is expressed as a ratio of the amount measured in the evolved strain (CB1000, CB1013, or CB2000; black, gray, and white bars, respectively) relative to the Founder. (B) Ratios of manganese to iron are plotted for all isolates for which genomic sequences were obtained. The average increase in Mn/Fe ratio in strains derived from the further evolution of CB1000 is 1.4-fold. DOI: http://dx.doi.org/10.7554/eLife.01322.008

Figure 4—figure supplement 1.. Relative levels of metals are unchanged in evolved E. coli.

Cells sampled during mid-logarithmic growth were subjected to trace metal analysis as described in ‘Materials and methods’. The amount of each metal was plotted as a ratio normalized to the amount of potassium present in each sample, K, which was chosen arbitrarily to correct for variation in metal extraction. DOI: http://dx.doi.org/10.7554/eLife.01322.009