Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

Abstract

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D 37 values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

Oxidative Stress Resistance inDeinococcus radiodurans

“…Diaz and Schulze-Makuch (141) showed that D. radiodurans could survive Martian-like conditions of low temperatures (Ϫ35°C for 10 days), low pressure (83.3 kPa for 10 days), and high UV radiation (37 W/m 2 for 24 h), which occur near the surface of Mars. The "training" of radiation resistance was demonstrated experimentally on different bacteria (E. coli, Bacillus pumilus, and Salmonella enterica serovar Typhimurium) exposed to multiple cycles of high radiation doses that yielded a permanently acquired increase in radiation resistance (132,238,358,481).…”

Parallel evolution of transcriptome architecture during genome reorganization

“…Experimental evolution studies combined with genome resequencing have correlated specific mutations to phenotypic changes to demonstrate that these dynamic genome reorganizational events are tightly coupled to adaptive evolution (Herring et al 2006;Barrick et al 2009). Interestingly, several studies have reported that, along with metabolic and structural genes, mutations within regulatory genes are also adaptive (Harris et al 2009;Wang et al 2010), which suggests that altering gene regulatory programs is an effective strategy for adaptation over short time intervals. Despite benefits of efficient coordinate regulation due to operonization, there is a surprisingly low level of conservation in operon structures across organisms.…”

Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans

“…To explore the existence and nature of such putative protective mechanisms, we quantitatively examined oxidation (carbonylation) of the proteomes of sensitive and resistant strains of D. radiodurans and E. coli (including two radiation-resistant variants of E. coli evolved experimentally by exposure of surviving cells to successively increasing doses of ionizing radiation (20 cycles) (7). We show here that killing of D. radiodurans and E. coli strongly correlates with proteome carbonylation (PC) and that this correlation is independent of the extent of cellular resistance or the type of lethal agent used.…”