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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.

SUBMITTER: Harris DR

PROVIDER: S-EPMC2725583 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Directed evolution of ionizing radiation resistance in Escherichia coli.

Harris Dennis R DR Pollock Steve V SV Wood Elizabeth A EA Goiffon Reece J RJ Klingele Audrey J AJ Cabot Eric L EL Schackwitz Wendy W Martin Joel J Eggington Julie J Durfee Timothy J TJ Middle Christina M CM Norton Jason E JE Popelars Michael C MC Li Hao H Klugman Sarit A SA Hamilton Lindsay L LL Bane Lukas B LB Pennacchio Len A LA Albert Thomas J TJ Perna Nicole T NT Cox Michael M MM Battista John R JR

Journal of bacteriology 20090605 16

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 stra ...[more]

PMID: 19502398

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Project description: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.

Project description:Background and Objectives:Metallic antimicrobial materials are of growing interest due to their potential to control pathogenic and multidrug-resistant bacteria. Yet we do not know if utilizing these materials can lead to genetic adaptations that produce even more dangerous bacterial varieties. Methodology:Here we utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance. Results:By day 10 of evolution, increased gallium resistance was evident in populations cultured in medium containing a sublethal concentration of gallium. Furthermore, these populations showed increased resistance to ionic silver and iron (III), but not iron (II) and no increase in traditional antibiotic resistance compared with controls and the ancestral strain. In contrast, the control populations showed increased resistance to rifampicin relative to the gallium-resistant and ancestral population. Genomic analysis identified hard selective sweeps of mutations in several genes in the gallium (III)-resistant lines including: fecA (iron citrate outer membrane transporter), insl1 (IS30 tranposase) one intergenic mutations arsC ?/? yhiS; (arsenate reductase/pseudogene) and in one pseudogene yedN ?; (iapH/yopM family). Two additional significant intergenic polymorphisms were found at frequencies > 0.500 in fepD ?/? entS (iron-enterobactin transporter subunit/enterobactin exporter, iron-regulated) and yfgF ?/? yfgG (cyclic-di-GMP phosphodiesterase, anaerobic/uncharacterized protein). The control populations displayed mutations in the rpoB gene, a gene associated with rifampicin resistance. Conclusions:This study corroborates recent results observed in experiments utilizing pathogenic Pseudomonas strains that also showed that Gram-negative bacteria can rapidly evolve resistance to an atom that mimics an essential micronutrient and shows the pleiotropic consequences associated with this adaptation. Lay summary:We utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance.

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Directed evolution of ionizing radiation resistance in Escherichia coli.

Publications

Directed evolution of ionizing radiation resistance in Escherichia coli.

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