scholarly journals Experimental evolution of extremophile levels of radiation resistance in Escherichia coli

2021 ◽  
Author(s):  
Steven T Bruckbauer ◽  
Benjamin B Minkoff ◽  
Takeshi Shinohara ◽  
Anna Lipzen ◽  
Jie Guo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Deinococcus Radiodurans ◽  
Dna Packaging ◽  
Cellular Systems ◽  
Cell Physiology ◽  
Oxygen Species ◽  
Iron Sulfur ◽  
Selection For ◽  
High Level

Recent human development of high-level sources of ionizing radiation (IR) prompts a corresponding need to understand the effects of IR on living systems. One approach has focused on the capacity of some organisms to survive astonishing levels of IR exposure. Using experimental evolution, we have generated populations of Escherichia coli with IR resistance comparable to the extremophile Deinococcus radiodurans. Every aspect of cell physiology is affected. Cellular isolates exhibit approximately 1,000 base pair changes plus major genomic and proteomic alterations. The IR resistance phenotype is stable without selection for at least 100 generations. Defined and probable contributions include alterations in cellular systems involved in DNA repair, amelioration of reactive oxygen species, Fe metabolism and repair of iron-sulfur centers, DNA packaging, and intermediary metabolism. A path to new mechanistic discoveries, exemplified by an exploration of rssB function, is evident. Most important, there is no single molecular mechanism underlying extreme IR resistance.

scholarly journals Experimental Evolution of Extreme Resistance to Ionizing Radiation inEscherichia coliafter 50 Cycles of Selection

2019 ◽  
Vol 201 (8) ◽  
Author(s):  
Steven T. Bruckbauer ◽  
Joseph D. Trimarco ◽  
Joel Martin ◽  
Brian Bushnell ◽  
Katherine A. Senn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Ionizing Radiation ◽  
Experimental Evolution ◽  
Deinococcus Radiodurans ◽  
Bacterial Species ◽  
Genomic Sequencing ◽  
Novel Mutations ◽  
Content Type ◽  
Resistance To Ionizing Radiation

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.

scholarly journals Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in Escherichia coli

2002 ◽  
Vol 184 (24) ◽  
pp. 6866-6872 ◽  
Author(s):  
Joanna Klapacz ◽  
Ashok S. Bhagwat
Keyword(s):  
Escherichia Coli ◽  
Genetic System ◽  
Base Substitution ◽  
E Coli ◽  
Base Substitutions ◽  
Selection For ◽  
Substitution Mutations ◽  
High Level ◽  
C To T Mutations ◽  
Genetic Assay

ABSTRACT We showed previously that transcription in Escherichia coli promotes C · G-to-T · A transitions due to increased deamination of cytosines to uracils in the nontranscribed but not the transcribed strand (A. Beletskii and A. S. Bhagwat, Proc. Natl. Acad. Sci. USA 93:13919-13924, 1996). To study mutations other than that of C to T, we developed a new genetic assay that selects only base substitution mutations and additionally excludes C · G to T · A transitions. This novel genetic reversion system is based on mutations in a termination codon and involves positive selection for resistance to bleomycin or kanamycin. Using this genetic system, we show here that transcription from a strong promoter increases the level of non-C-to-T as well as C-to-T mutations. We find that high-level transcription increases the level of non-C-to-T mutations in DNA repair-proficient cells in three different sequence contexts in two genes and that the rate of mutation is higher by a factor of 2 to 4 under these conditions. These increases are not caused by a growth advantage for the revertants and are restricted to genes that are induced for transcription. In particular, high levels of transcription do not create a general mutator phenotype in E. coli. Sequence analysis of the revertants revealed that the frequency of several different base substitutions increased upon transcription of the bleomycin resistance gene and that G · C-to-T · A transversions dominated the spectrum in cells transcribing the gene. These results suggest that high levels of transcription promote many different spontaneous base substitutions in E. coli.

scholarly journals Global Change of Gene Expression and Cell Physiology in YidC-Depleted Escherichia coli

2010 ◽  
Vol 192 (8) ◽  
pp. 2193-2209 ◽  
Author(s):  
Peng Wang ◽  
Andreas Kuhn ◽  
Ross E. Dalbey
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Protein ◽  
Gene Chip ◽  
Cell Physiology ◽  
Global Changes ◽  
Protein Insertion ◽  
Genes Encoding ◽  
Membrane Protein Insertion ◽  
High Level

ABSTRACT YidC depletion affects membrane protein insertion and leads to a defect in the growth of the Escherichia coli cell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131 (yidC depletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregulated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism; various transporters, proteases and chaperones; stress response; and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation of the genes encoding the energy transduction proteins F1Fo ATP synthase and cytochrome bo 3 oxidase due to perturbation in assembly when YidC was depleted. We also determined that the high-level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which dissipates the proton motive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion. The data show the broad physiological contribution of YidC to the bacterial cell and the considerable ramification to the cell when it is depleted.

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

2020 ◽  
Vol 11 ◽  
Author(s):  
Steven T. Bruckbauer ◽  
Joel Martin ◽  
Benjamin B. Minkoff ◽  
Mike T. Veling ◽  
Illissa Lancaster ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Deinococcus Radiodurans ◽  
Atp Synthesis ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Trade Offs ◽  
Radiation Resistant ◽  
Replication Terminus ◽  
High Doses

Ionizing radiation (IR) is lethal to most organisms at high doses, damaging every cellular macromolecule via induction of reactive oxygen species (ROS). Utilizing experimental evolution and continuing previous work, we have generated the most IR-resistant Escherichia coli populations developed to date. After 100 cycles of selection, the dose required to kill 99% the four replicate populations (IR9-100, IR10-100, IR11-100, and IR12-100) has increased from 750 Gy to approximately 3,000 Gy. Fitness trade-offs, specialization, and clonal interference are evident. Long-lived competing sub-populations are present in three of the four lineages. In IR9, one lineage accumulates the heme precursor, porphyrin, leading to generation of yellow-brown colonies. Major genomic alterations are present. IR9 and IR10 exhibit major deletions and/or duplications proximal to the chromosome replication terminus. Contributions to IR resistance have expanded beyond the alterations in DNA repair systems documented previously. Variants of proteins involved in ATP synthesis (AtpA), iron-sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA) each contribute to IR resistance in IR9-100. Major genomic and physiological changes are emerging. An isolate from IR10 exhibits protein protection from ROS similar to the extremely radiation resistant bacterium Deinococcus radiodurans, without evident changes in cellular metal homeostasis. Selection is continuing with no limit to IR resistance in evidence as our E. coli populations approach levels of IR resistance typical of D. radiodurans.

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