ABSTRACTIn 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 thatEscherichia colicould 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 generateE. colipopulations that are as resistant to IR asDeinococcus 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 toD. 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.IMPORTANCESome bacterial species exhibit astonishing resistance to ionizing radiation, withDeinococcus radioduransbeing the archetype. As natural IR sources rarely exceed mGy levels, the capacity ofDeinococcusto 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 ofEscherichia coliwith IR resistance levels comparable toDeinococcus. 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.
Experimental Evolution of Extreme Resistance to Ionizing Radiation inEscherichia coliafter 50 Cycles of Selection