Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans

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.

Download Full-text

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

DNA Repair ◽

10.1016/j.dnarep.2014.12.001 ◽

2015 ◽

Vol 26 ◽

pp. 30-43 ◽

Cited By ~ 16

Author(s):

Joseph R. Piechura ◽

Tzu-Ling Tseng ◽

Hsin-Fang Hsu ◽

Rose T. Byrne ◽

Tricia A. Windgassen ◽

...

Keyword(s):

Ionizing Radiation ◽

Biochemical Characterization ◽

Extreme Resistance ◽

Resistance To Ionizing Radiation

Download Full-text

Effects of Mn and Fe Levels onBacillus subtilisSpore Resistance and Effects of Mn2+, Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation

Applied and Environmental Microbiology ◽

10.1128/aem.01965-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 32-40 ◽

Cited By ~ 49

Author(s):

Amanda C. Granger ◽

Elena K. Gaidamakova ◽

Vera Y. Matrosova ◽

Michael J. Daly ◽

Peter Setlow

Keyword(s):

Ionizing Radiation ◽

Deinococcus Radiodurans ◽

Dipicolinic Acid ◽

Divalent Cations ◽

Wild Type ◽

Divalent Metal Ions ◽

Protein Resistance ◽

Wet Heat ◽

Resistance To Ionizing Radiation

ABSTRACTSpores ofBacillus subtilisstrains with (wild type) or without (α−β−) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl2concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H2O2). However, α−β−spores were more sensitive to H2O2when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α−β−spores to H2O2, probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn2+complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiationin vitro, and Ca2+complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca2+, much like high levels of Mn2+complexed with small molecules protect the bacteriumDeinococcus radioduransagainst ionizing radiation.

Download Full-text

Author response: Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair

10.7554/elife.01322.012 ◽

2013 ◽

Author(s):

Rose T Byrne ◽

Audrey J Klingele ◽

Eric L Cabot ◽

Wendy S Schackwitz ◽

Jeffrey A Martin ◽

...

Keyword(s):

Dna Repair ◽

Ionizing Radiation ◽

Author Response ◽

Genetic Adaptation ◽

Extreme Resistance ◽

Resistance To Ionizing Radiation

Download Full-text

Ultraviolet-B Radiation Stress-Induced Toxicity and Alterations in Proteome of Deinococcus radiodurans

Microbial Physiology ◽

10.1159/000512018 ◽

2020 ◽

pp. 1-15

Author(s):

Jay Kumar ◽

Paushali Ghosh ◽

Ashok Kumar

Keyword(s):

Gene Expression ◽

Ionizing Radiation ◽

Stress Responses ◽

Deinococcus Radiodurans ◽

In Silico Analysis ◽

Ultraviolet B ◽

Percentage Survival ◽

High Doses ◽

Separation Of Proteins ◽

Resistance To Ionizing Radiation

Deinococcus radiodurans is a polyextremophilic bacterium capable to survive and grow at high doses of ionizing radiation. Besides resistance to ionizing radiation, the bacterium is also resistant to toxic chemicals and desiccation. This study deals with the effects of non-ionizing radiation (ultraviolet-B) on survival, alterations in proteomic profile, and gene expression in D. radiodurans. Exposure of culture to UV-B caused decrease in the percentage survival with increasing duration, complete killing occurred after 16 h. D. radiodurans also showed enhancement in the generation of reactive oxygen species and activities of antioxidative enzymes. Separation of proteins by 2-dimensional gel electrophoresis revealed major changes in number and abundance of different proteins. Twenty-eight differentially abundant protein spots were identified by MALDI-TOF MS/MS analysis and divided into 8 groups including unknown proteins. Gene expression of a few identified proteins was also analyzed employing qRT-PCR, which showed differential expression corresponding to the respective proteins. In silico analysis of certain hypothetical proteins (HPs) suggested that these are novel and as yet not reported from D. radiodurans subjected to UV-B stress. These HPs may prove useful in future studies especially for assessing their significance in the adaptation and management of stress responses against UV-B stress.

Download Full-text

Decision letter: Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair

10.7554/elife.01322.011 ◽

2013 ◽

Keyword(s):

Dna Repair ◽

Ionizing Radiation ◽

Genetic Adaptation ◽

Extreme Resistance ◽

Resistance To Ionizing Radiation

Download Full-text

Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair

eLife ◽

10.7554/elife.01322 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 38

Author(s):

Rose T Byrne ◽

Audrey J Klingele ◽

Eric L Cabot ◽

Wendy S Schackwitz ◽

Jeffrey A Martin ◽

...

Keyword(s):

Dna Repair ◽

Ionizing Radiation ◽

Predominant Role ◽

Extreme Resistance ◽

Adaptation Mechanism ◽

Genetic Changes ◽

Resistance Phenotype ◽

Order Of Magnitude ◽

Resistance To Ionizing Radiation ◽

Magnitude Increase

By directed evolution in the laboratory, we previously generated populations of Escherichia coli that exhibit a complex new phenotype, extreme resistance to ionizing radiation (IR). The molecular basis of this extremophile phenotype, involving strain isolates with a 3-4 order of magnitude increase in IR resistance at 3000 Gy, is now addressed. Of 69 mutations identified in one of our most highly adapted isolates, functional experiments demonstrate that the IR resistance phenotype is almost entirely accounted for by only three of these nucleotide changes, in the DNA metabolism genes recA, dnaB, and yfjK. Four additional genetic changes make small but measurable contributions. Whereas multiple contributions to IR resistance are evident in this study, our results highlight a particular adaptation mechanism not adequately considered in studies to date: Genetic innovations involving pre-existing DNA repair functions can play a predominant role in the acquisition of an IR resistance phenotype.

Download Full-text

A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene's 5′ Untranslated Region in the Radiation and Desiccation Response

Applied and Environmental Microbiology ◽

10.1128/aem.00039-17 ◽

2017 ◽

Vol 83 (12) ◽

Cited By ~ 5

Author(s):

Jordan K. Villa ◽

Paul Amador ◽

Justin Janovsky ◽

Arijit Bhuyan ◽

Roland Saldanha ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Ionizing Radiation ◽

Untranslated Region ◽

Deinococcus Radiodurans ◽

Dna Gyrase ◽

Regulatory Motif ◽

Subunit A ◽

Content Type ◽

Rna Elements

ABSTRACT Tight regulation of gene expression is important for the survival of Deinococcus radiodurans, a model bacterium of extreme stress resistance. Few studies have examined the use of regulatory RNAs as a possible contributing mechanism to ionizing radiation (IR) resistance, despite their proffered efficient and dynamic gene expression regulation under IR stress. This work presents a transcriptome-based approach for the identification of stress-responsive regulatory 5′ untranslated region (5′-UTR) elements in D. radiodurans R1 that can be broadly applied to other bacteria. Using this platform and an in vivo fluorescence screen, we uncovered the presence of a radiation-responsive regulatory motif in the 5′ UTR of the DNA gyrase subunit A gene. Additional screens under H2O2-induced oxidative stress revealed the specificity of the response of this element to IR stress. Further examination of the sequence revealed a regulatory motif of the radiation and desiccation response (RDR) in the 5′ UTR that is necessary for the recovery of D. radiodurans from high doses of IR. Furthermore, we suggest that it is the preservation of predicted RNA structure, in addition to DNA sequence consensus of the motif, that permits this important regulatory ability. IMPORTANCE Deinococcus radiodurans is an extremely stress-resistant bacterium capable of tolerating up to 3,000 times more ionizing radiation than human cells. As an integral part of the stress response mechanism of this organism, we suspect that it maintains stringent control of gene expression. However, understanding of its regulatory pathways remains incomplete to date. Untranslated RNA elements have been demonstrated to play crucial roles in gene regulation throughout bacteria. In this work, we focus on searching for and characterizing responsive RNA elements under radiation stress and propose that multiple levels of gene regulation work simultaneously to enable this organism to efficiently recover from exposure to ionizing radiation. The model we propose serves as a generic template to investigate similar mechanisms of gene regulation under stress that have likely evolved in other bacterial species.

Download Full-text