Oxidative Stress Response in Deinococcus Radiodurans

Understanding Oxidative Stress Response in Deinococcus Radiodurans    

Deinococcus Radiodurans

Also referred to as “Conan the Bacterium” D. Radiodurans belongs to a small family of extremophiles that share an uncanny ability to survive extreme amounts of radiation. Not surprisingly, D. Radiodurans was first isolated in 1956 from a canned meat product that had been treated with gamma radiation. At the time, such levels of radiation where thought to be lethal to all forms of life, but shortly after and to much amazement, the meat began to spoil.

In subsequent years, it was discovered that D. Radiodurans could survive not only gamma radiation but particle radiation as well, and not only a lot of it but an unprecedented 500,000 rad or 5000Gy of acute dosage with no loss of viability. With an LD50 3000 fold greater than in humans and roughly 30 times greater than E coli, D. Radiodurans has been a hot topic for radiation studies for many years.

 

figure 1 radiodurans Picture1

What makes D. Radiodurans so resistant to radiation?

The answer is many-fold and still the subject of ongoing research. Early on, many believed the answer was the methods used by Radiodurans to correct DNA double stranded breaks. More resent research suggests that D. Radiodurans employs robust mechanisms to protect its proteome and that such mechanisms are more likely to account for its extraordinary ability. In reality, there are multiple layers of protection available for D. Radiodurans to implement. Following a fair amount of introductory material, this web-article will summarize the study “Genome-Wide Transcriptome and Antioxidant Analysis on Gamma-irradiated Phases of Deinococcus radiodurans R1”, which illuminates the intricate developments that occur within D. radiodurans.

General Features

  • Deinococcus Radiodurans is a Gram-positive bacteria
  • Cells form diads or tetrads
  • It is nonpathogenic
  • It is nonsporulating

Ecology

  • Though it was first named micrococcus Radiodurans, 16S rRNA analysis confirmed that Radiodurans was its own phylogenetic group of bacteria
  • As of 2009, forty three species of Deinococcus have been isolated
  • Species of Deinococcus can be found in a variety of environments including deserts, intestines, hot springs, Antarctica, soil, clean-rooms and just about everywhere else. D. Radiodurans specifically is however sensitive to heat and especially when accompanied by fluctuations from dry to moist.

Genetics

  • Has a 3.28 megabase genome
  • Its genome is split between two chromosomes and two plasmids
  • Each cell may contain up to ten copies of its genome but never less than two copies
  • D. Radiodurans has a notably large capacity for heterologous DNA that can extend 3Mb by duplication insertion making the bacteria, with selective pressure, good for storing foreign DNA.

Metabolism

  • D. Radiodurans is a organotrophic bacterium with a proteolytic metabolism
  •  Several features of its metabolism work in its favor during periods of environmental stress including irradiation
    • Proteolysis and uptake of exogenous peptide materials: The uptake of external materials and the recycling of damaged proteins is critical for restoration of cellular functions, especially during periods of stress. Indeed, following periods of ionizing radiation intracellular proteolysis is proven to be accelerated.
  • D. Radiodurans encodes relatively few proteins that require iron or iron sulfur cofactors. Extreme oxidative stress follows periods of irradiation brought on by generation of reactive oxygen species, ROS. Enzymes that require iron are easily disturbed by ROS or by iron depletion, resulting in free iron ions. Free iron can present further problems when it is oxidized with hydrogen peroxide to generate the dreaded hydroxyl radical!
  • D. Radiodurans keeps carbohydrate granules that can be broken down to feed directly into glucose metabolism. Following DNA damage, precursors to deoxynucleoside triphosphates are made from glucose released from granular stores.
  • Interestingly, Radiodurans isn’t able to synthesize NAD nor is it able to use the nucleosides uridine and adenosine or the TCA products succinate, malate, fumarate and alpha-ketoglutarate as carbon sources. This metabolic “defect” leads to an accumulation of nucleosides and NAD precursors that, based on current research, may account for D. Radiodurans ability to tolerate radiation more so than any of its DNA repair mechanisms, but more on that later.

Radiation and Cell Damage

Before proceeding, it should be noted that there are several kinds of harmful radiation, and radiation can induce all kinds of reactions in biological systems and otherwise. For the purpose if this summary, know that energy from ionizing radiation causes direct and indirect damage (not to be confused with direct and indirect radiation) most notably single and double stranded breaks in DNA (SSB and DSB), production of ROS, and functionally crippling oxidation of enzymatic proteins.

  • Indirect Damage and Reactive Oxygen Species
  • Indirect radiation can cause severe damage to organisms by supplying energy for the production of ROS. These chemicals are then free to oxidize other, potentially important, molecules.
  • This type of radiation, shown in the figure below() is gamma and X-ray radiation
  • The radiolysis of H2O is the most common source for ROS.

H2O —> HO*+ H+

2HO* —> H2O2

The radical hydroxyl group is extremely destructive, but it has a relatively short lifespan. The peroxide however, is free to diffuse throughout the cell. As mentioned previously, peroxide can still cause damage, especially when in the presence of iron ions. Iron can act as a fenton catalyst, breaking peroxide into yet another radicle.

Fe3+ + H2O2 —>  HO + HO*

  • Dissolved oxygen is also a problem. By taking on charge, oxygen can become destructive for ionic bonds and polar associations. Fe-S bonds in proteins are particularly susceptible to cleavage, releasing iron ions as a result.

O2 + e —> O2

  • Direct Damage by Radiation
  • The same energy used to generate ROS can also affect molecules like DNA, RNA, or proteins by direct bond breaking.
  • The mechanism can be kinetic as with direct particle radiation or photochemical with indirect radiation.

 

figure 2 oxidative table picture

Oxidation and Radiation Resistance

Initial studies of D. radiodurans’s DNA repair pathway suggested that it resembled other common mechanisms found in bacteria. Scientists began to question if radiodurans’s ability was due to DNA repair and began looking at other explanations. Though it has since been found that there are indeed several novel mechanisms and proteins controlling radiodurans DNA repair, according to () the mortality rate of a cell is more correlated with damage to its enzymes rather than its DNA. This notion seems paradoxical. In order to make proteins, a cell must have DNA, but to fix DNA a cell must have functioning repair proteins. It turns out that as long as a cell’s proteins remain intact, especially in the case of D. Radiodurans, DNA can be repaired and the cell can continue to function. Not surprisingly, radiodurans has several mechanisms in place for protection and for relieving the oxidative stress that follows radiation.

 

resistance diagram

DNA Damage Repair       

Early speculation suggested that this organism had the potential for extraordinary DNA repair, and while D. Radiodurans does have a very robust system of repair, new research is focusing on different systems as well. But so summary of the magnificent organism can be complete without at least mentioning a few of the ways DNA is repaired.

  • D. radiodurans uses four types of repair mechanisms to fix DNA breaks, which further increases the efficiency of repair and allows complete chromosomal reconstruction within a few hours.
    • homologous recombination: uses an intact copy of DNA (from another chromosome) as a template to fill in or fix the break in DNA.
    • synthesis-dependent strand annealing: repairs broken DNA with existing copy of DNA and rebuilds missing sections from fragments.
    •  single-strand annealing:  synthesizes missing sections on a strand
    •  non homologous end joining:  broken ends are recognized and rejoined. This process doesn’t require another copy of DNA.
  • Following damage these repair mechanisms are initiated by a whole host of signals and gene factors and proteins.

In addition to DNA repair D. Radiodurans has mechanisms to deal with radiation and oxidative stress.

Cell Cleaning

  • Cell Cleaning: damaged macromolecules are either broken into constituent pieces or are eliminated from the cell. Timely removal of oxidized molecules and oxidizing agents prevents further damage.
    • The Nudix family of hydrolases and nucleotidases recycle damaged nucleotides.
    • Radiodurans has an extensive 23 nudix enzymes, 5 of which are induced by radiation.
    • Directed protein degradation by Lon and Clp families of ATP-depedent proteasis is triggered by Aconitase. Aconitase is an oxidative stress sensor that begins this degradation process. Tagets for degradation include TCA cycle enzymes and several chaperones including GroEL and DnaK.
    • Selective Protein Protection: some proteins are not rapidly replaced even during major oxidative stress.
      • The protected list includes one serine protease, EF-Tu and both of the RNA polymerase subunits beta and beta prime. Protection of the RNA subunits is, intuitively, essential for making a recovery.

Antioxidant Defense

  • Large ROS Savengers
    • Catalases: Catalases remove free hydrogen peroxide. Radiodurans encodes three katE catalases, DR1998, DRA0259 and DRA0146) katE catalases are upregulated by the DrRRA transcriptional regulator and down-regulated by OxyR
    • Peroxidases: The two encoded peroxidases in Radiodurans also remove hydrogen peroxide. DRA0301 is cytochrome C peroxidase and DRA0145 is iron dependent peroxidase.
    • Superoxide Dismutases: While catalases and peroxidases can neutralize oxidative species other than hydrogen peroxide, they are much less efficient with other species. Superoxide dismutases are specialized for dealing with the highly destructive oxygen radicals. Radiodurans has four. DR1279 is manganese dependent, while DR 1546, DRA0202, and DR0644 are Cu/Zn dependent. (Note there are no Fe dependent species) The metal ion cofactors are very important as they become reduced and their valences dictate the redox product, as shown below.

M(n+1)+-SOD + O2− → Mn+-SOD + O2

Mn+-SOD + O2− + 2H+ → M(n+1)+-SOD + H2O2.

  • Dps Proteins: As mentioned earlier, ferrous ions can undergo fenton chemistry. Dps proteins are a multifunctional protein that can chelate iron ions and reduce hydrogen peroxide into water. Radiodurans encodes two proteins from this family, DR2263 and DRB0092, called Dps1 and Dps2, which are induced by ionizing radiation.
  • Carotenoids: Interestingly, even as pigments, carotenoids don’t play a role in direct radiation resistance in radiodurans. However, these small molecules do act as scavengers for each type of ROS.
  • Manganese Complexes: Deinococcus species all have a high availability of manganese, especially radiodurans. These species form several complexes that scavenge ROSs. The short circuit metabolism mentioned earlier ensures an abundance of substrates for these complexes that are shown below.

complexMGfinal

Article Study

In the article, “Genome-Wide Transcriptome and Antioxidant Analysis on Gamma-irradiated Phases of Deinococcus radiodurans R1 , a dynamic transcriptome analysis is performed in order to detect the many transcriptional responses that occur in D. radiodurans following irradiation. I chose this study because I found it gave a unique look at stress response and how cellular systems change. Dynamic transcriptome analysis (DTA) is also I fairly new technique that can really look at expression comprehensively over the course of development. DTA measures the relative amounts of specific RNAs present in a system at a given time and so DTA is particularly suited to developmental response studies. Amounts of specific sequences of RNA will change based on the cells programming. The effects of particular perturbations, like gene knockouts, can also be characterized. Generally, this method works by incorporating labels into growth substrate, doing so produces three fractions of RNA, total RNA, labeled therefor newly synthesized RNA, and old unlabeled RNA. Stand specific RNA-seq analysis along with RT PCR, validates the specific changes happening with the cell’s transcriptional behavior.

Method:

The R1 D. radiodurans strain was grown and split into three groups. One group was a control that received no radiation treatment. One group was the experimental and did receive gamma radiation. The final group was used to create a crtB knockout mutant. This gene is known to code for proteins involved in carotenoid synthesis. LC-MS was used to measure differences in the carotenoid metabolite levels between the mutant and control to measure effects on survival. The radiation experimental group was measured at three time periods to see the changing levels of expression at different stages of radiation and recovery. RT PCR was used to validate the sRNA-seq data by measuring nine pre-selected genes for comparison.

Results:

Once specific RNA sequences are attributed to intended proteins, the data shows what kinds of proteins are called for and when. This study found that:

  • 618 genes (19.2%) were expressed at different levels from the control group.
  • Between 1hr and 3 hrs of radiation 63 genes were deferentially expressed
  • Between 3 hrs and the recovery period 261 genes were deferentially expressed

What kinds of functions these sequences were coding for was determined using the Gene Ontology Database.

  • Genes involving cell component and transporter activity were called for throughout the radiation and recovery process. these were possible to restore membrane structure and function including ATP synthesis
  • Three genes coding for antioxidant activity were induced in the 1hr trial vs the control. DR1014 (a chloroperoxidase protein), DRA0202 (Cu/Zn superoxydase dismutase protein) and DRA0301 (a methylamine utilization protein).
  • The 3hr and recovery groups showed upregulation of DNA repair genes
  • DNA replication genes appeared in the recovery group along with the ATP-dependent Lon protease (DR0349).

Genes Involving DNA Repair

  • Two of eleven known base excision repair genes were expressed LakA and RecJ
  • Only during later stages were the nucleotide excision genes uvrA and uvrB induced
  • 7 genes for homologous recombination were detected in late and recovery stages, especially the recA gene.

It was also found that the carotenoid defficient mutant crtB was 30% more sensitive to radiation than wildtype, implying that the ROS scavenging roles of the carotenoid metabolites are critical to oxidative resistance.

Weaknesses and Future Prospects:

The main problem with this study and with DTA experiments in general is that though there is a correlation with RNA concentration and expression, there is no one-to-one relation between a given concentration of RNA and the number of proteins actually being synthesized. In fact, a single RNA can code for many different proteins depending upon post-translational modifications, and so this data gives no information with what is happening there. The strength of this study lies in its ability to provide a time dependent sequence with what happens as a bigger picture. In the future, there are still many known proteins and pathways involved in Deinococcus radiodurans.

References:

1) Krisko, A., & Radman, M. (2013). Biology of Extreme Radiation Resistance: The Way of   Deinococcus radiodurans. Cold Spring Harbor Perspective Biology, .

2) Englander, J., Klein, E., Brumfeld, V., Sharma, A. K., & Doherty, A. (2004). Dna toroids: Framework for dna pepair in deinococcus radiodurans and in germinating bacterial spores. Journal of Bacteriology186(18), 5973-5977.

3) Cox, M., & Battista, J. (2005). Deinococcus radiodurans-the consummate survivor. Microbiology Nature Reviews,3, 882-892.

4) Slade, D., & Radman, M. (2011). Oxidative stress resistance in deinococcus radiodurans.Microbiology and Molecular Biology Reviews,75(1), 133-191.

5) Krisko, A., & Radman, M. (2010). Protein damage and death by radiation in escherichia coli and deinococcus radiodurans . PNAS, 107(32), 14373-14377.

6) Daly, M. (2009). A new perspective on radiation resistance based on deinococcus radiodurans. Microbiology Nature Reveiws, 7, 237-245.

7) Daly MJ, Gaidamakova EK, Matrosova VY, Kiang JG, Fukumoto R, et al. (2010) Small-Molecule Antioxidant Proteome-Shields inDeinococcus radiodurans. PLoS ONE 5(9): e12570.

8) Luan H, Meng N, Fu J, Chen X, Xu X, et al. (2014) Genome-Wide Transcriptome and Antioxidant Analyses on Gamma-Irradiated Phases of Deinococcus radiodurans R1. PLoS ONE 9(1): e85649.

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