Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans.
ABSTRACT: Deinococcus radiodurans is among a small number of bacterial species that are extremely resistant to ionizing radiation, UV light, toxic chemicals, and desiccation. We measured proteome oxidation (i.e., protein carbonylation, PC) in D. radiodurans as well as in standard and evolved resistant strains of Escherichia coli exposed to ionizing radiation or UVC light and found a consistent correlation with cell killing. The unique quantitative relationship between incurred PC and cell death holds over the entire range of killing for all tested bacteria and for both lethal agents, meaning that both bacterial species are equally sensitive to PC. We show that the extraordinary robustness of D. radiodurans depends on efficient proteome protection (but not DNA protection) against constitutive and radiation-induced PC consisting of low molecular weight cytosolic compounds. Remarkably, experimental evolution of resistance to ionizing radiation in E. coli coevolves with protection against PC. The decline in biosynthetic efficacy of the cellular proteome, as measured by the loss of reproduction of undamaged bacteriophage lambda in irradiated standard and evolved ionizing radiation-resistant E. coli, correlates with radiation-induced oxidative damage to host cells and their sensitivity to ionizing radiation. This correlation suggests that cell death by radiation is caused primarily by oxidative damage with consequential loss of maintenance activities including DNA repair.
Project description:For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn(2+) and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn(2+)-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.
Project description:Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation-resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by ?-irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein-intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties.
Project description:Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.
Project description:The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.
Project description:Understanding chronic ionizing radiation (CIR) effects is of utmost importance to protecting human health and the environment. Diverse bacteria and fungi inhabiting extremely radioactive waste and disaster sites (e.g. Hanford, Chernobyl, Fukushima) represent new targets of CIR research. We show that many microorganisms can grow under intense gamma-CIR dose rates of 13-126 Gy/h, with fungi identified as a particularly CIR-resistant group of eukaryotes: among 145 phylogenetically diverse strains tested, 78 grew under 36 Gy/h. Importantly, we demonstrate that CIR resistance can depend on cell concentration and that certain resistant microbial cells protect their neighbors (not only conspecifics, but even radiosensitive species from a different phylum), from high-level CIR. We apply a mechanistically-motivated mathematical model of CIR effects, based on accumulation/removal kinetics of reactive oxygen species (ROS) and antioxidants, in bacteria (3 Escherichia coli strains and Deinococcus radiodurans) and in fungi (Candida parapsilosis, Kazachstania exigua, Pichia kudriavzevii, Rhodotorula lysinophila, Saccharomyces cerevisiae, and Trichosporon mucoides). We also show that correlations between responses to CIR and acute ionizing radiation (AIR) among studied microorganisms are weak. For example, in D. radiodurans, the best molecular correlate for CIR resistance is the antioxidant enzyme catalase, which is dispensable for AIR resistance; and numerous CIR-resistant fungi are not AIR-resistant. Our experimental findings and quantitative modeling thus demonstrate the importance of investigating CIR responses directly, rather than extrapolating from AIR. Protection of radiosensitive cell-types by radioresistant ones under high-level CIR is a potentially important new tool for bioremediation of radioactive sites and development of CIR-resistant microbiota as radioprotectors.
Project description:The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3' ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.
Project description: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.
Project description:In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.
Project description:Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.
Project description:Deinococcus radiodurans R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (katA) and superoxide dismutase (sodA) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.