Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection

Abstract

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 phenotypic 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 K1172I. 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.

Physiology of Highly Radioresistant Escherichia coli After Experimental Evolution for 100 Cycles of Selection

“…We have noted previously (Bruckbauer et al, 2019b) that all strains tested, including IR-resistant isolates from previous trials using 60 Co and Deinococcus radiodurans itself, are more sensitive to the high dose rate of the high energy electron beam irradiation provided by the Linac than they are to the 60 Co irradiation. This can readily be seen in the reduced survival seen here for Deinococcus radiodurans at 5,000 Gy relative to that reported previously (Cox and Battista, 2005;Daly, 2009;Slade and Radman, 2011).…”

“…Using the Linac, the dose required to kill 99% of each population increased from ∼750 Gy after the first cycle, to 2,300-2,500 Gy after 50 cycles of selection (Bruckbauer et al, 2019b). After 100 total cycles of selection, this further increased to 2,900-3,200 Gy ( Figure 1A).…”

“…Using our previously published selection protocol (Bruckbauer et al, 2019b), we continued experimental evolution of IR resistance in Escherichia coli for 100 total cycles of selection. The lineages are denoted IR9, IR10, IR11, and IR12.…”

“…To carry out a long-term evolution trial with a new radiation source, we initiated a second trial. The IR source of choice was a Varian 21EX clinical linear accelerator (Linac), generating high energy electron beam IR at a dose rate nearly four times higher than that of the previous study (Bruckbauer et al, 2019b). Source decay was no longer a problem.…”

“…Furthermore, recent advances in sequencing technologies have allowed for whole-genome sequencing of entire populations throughout the cycles of selection, illuminating the underlying genetic complexities of experimental evolution. Both in the original evolution trial and in the first 50 selection cycles of the new one, the first prominent genetic alterations conferring IR resistance centered on mutations modifying or eliminating the function of particular DNA repair enzymes (Harris et al, 2009;Byrne et al, 2014;Bruckbauer et al, 2019b).…”