By: Mayang Hasibuan
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Figure 1. Damage by ionizing radiation to a double stranded DNA, causing detachment of electrons that disrupts the double-helix structure. Source
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Microbes are tough fighters when it comes to surviving against unfavorable conditions such as drought, drastic temperature, nutrient scarcity, and even vacuum space. Some microbes, such as Eschericia coli, could survive a nuclear disaster because they have evolved to have an intricate DNA damage response that repairs damaged DNA due to ionizing radiation. On the other hand, if humans were to be exposed to ionizing radiation the results could be fatal depending on the intensity of it. Ionizing radiation carries high energy and therefore it has the ability to ionize a molecule by removing one or more electrons from the molecule itself. This could result in a double-stranded DNA breakage that would then lead to other adverse health effects. In the microbial world, E. coli is not the only microbe that have the skills to repair DNA. An amazing extremophile called Deinococcus radiodurans is considered to be the toughest bacterium by the Guinness World Records and could survive a dose of 5,000 grays (Gy) of ionizing radiation! For comparison, a chest x-ray radiates about 1mGy and 5Gy could kill a human.
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| Figure 2. A very arbitrarily-illustrated graph of humans and D. radiodurans resistance to radiation. It is apparent that humans are complete weaklings when it comes to surviving a radiation apocalypse. Source |
D. radiodurans was first discovered when a can of ground meat became spoiled even after it was sterilized by radiation. It is a gram-positive bacterium that have an unusually thick cell wall and inner layer. The inner layers form four chambers of cells, separated by septa into a tetrad of cells. D. radiodurans have evolved to carry a set of genes responsible to repair damaged DNA after exposure to radiation. This set of genes is regulated as a unit called the radiation-desiccation response (RDR) regulon; RDR regulon is only activated when cells are exposed to radiation. Upstream of the RDR regulon is a conserved motif of nucleotide base-pairs, abbreviated as RDRM, that serve as binding sites for regulatory proteins to bind. To hone you guys on how important gene regulation is, I would like to use the analogy of a traffic road to talk about the RDR regulon. In a traffic system, there are rules and regulations to exercise discipline for the sake of everyone’s safety. During a normal road condition, the red traffic light is arguably the most essential regulator by which cars are expected to stop when they see it turn on. A protein called DdrO in D. radiodurans is the equivalent of a red light; DdrO represses the RDR regulon by binding to the RDRM sequence and repress its transcription. In this sense, the RDR regulon is the equivalent of the cars on the road. In the case of an accident, however, a police officer is expected to help regulate the traffic and instruct the cars to ignore the red light and move away from the site of accident. In other words, the police officer represses the activity of the red light. For D. radiodurans, exposure to radiation is the equivalent to a horrible accident that also requires other regulatory mechanism, such as the police officer, to help regulate the RDR regulon. This is where the protein called IrrE comes in. IrrE is the designated police officer that represses the activity of DdrO by cleaving the protein so that it can no longer repress the RDR regulon. Therefore, DNA-damage response can now be activated. Overall, this means that DdrO is the repressor protein and IrrE is indirectly the activator protein of the RDR regulon.
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Figure 3. Gene regulation in microbes are just as complex, and important, as any traffic regulators that assure the safety of the people on the road.
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Now what happens to the cells if and when these regulatory proteins are depleted? In terms of a road system, it is obvious that proper function of traffic regulators are necessary to maintain the flow of the cars. When it comes to D. radiodurans, the necessity of gene regulators might not seem as obvious. A paper published by Devigne et al. (2015) shows exactly why regulatory protein, especially DdrO, is critically important for the organism to maintain proper function of the genes involved in DNA repair. In the interest of trying to understand the role of these regulatory proteins, the authors took microscopy images of normal and mutant D.radiodurans cells. They compared and contrasted the morphology of wild type cells, that have functioning DdrO, and mutant cells that are DdrO-depleted. The images below show nuclei staining, also known as DAPI, of the cells taken from different time points during the bacterium’s exponential growth. Notice that the images from both rows are quite distinct. We can see that the nuclei on the second row, pointed out by the arrowheads, are displaced throughout each cell. Also shown in the middle image on the second row, are highly condensed nuclei staining, indicating DNA condensation.
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| Figure 4. DAPI staining of D. radiodurans taken from 16 hours, 18 hours, and 24 hours of growth, consecutively. The top row shows normal, wild type cells and the bottom row show cells that are DdrO-depleted. |
Not only does DdrO depletion effect proper nucleus segregation, it also plays a big role in septum formation. The images below show membrane staining of D.radiodurans in which the wild-type cells, depicted on the first row, form two septa, creating a tetrad of cells. However, cells that are DdrO-depleted show no sign of the second septum that forms the tetrad; This is pointed out by the arrowheads on the first image of the second row. The third image on the second row show the formation of vesicles also known as membrane ‘blebbing’, indicating a protrusion of the plasma membrane. This is indicative of an apoptotic cell response, also known as programmed cell death. In addition to membrane blebbing, another morphology of apoptosis is also exhibited through DNA condensation shown in Figure 4.
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| Figure 5. Images of D. radiodurans membrane staining. The arrowheads indicate imperfect formation of the septum and the white arrows indicate formation of vesicle that is pertinent to an apoptosis-like cell response. |
Lastly, but not least, shown below are images of unstained cells using high contrast imaging. Cells that are DdrO-depleted, shown on the second row, exhibit a larger cell size as if the cells have become swollen. From previous images, we have seen that nuclei organization and septum formation are both deformed in cells that are DdrO-depleted. All these morphological changes, which include: membrane blebbing, DNA condensation and DNA degradation, are characteristics of an apoptosis cell response.
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| Figure 6. High contrast microscopy image of D. radiodurans cell, exhibiting larger cell size and membrane ‘blebbing.’ |
The DdrO protein has been the main focus of this post so far, so let us refresh our minds and talk about what it actually does. Remember? Figure 7. will help us visualize the job of DdrO, represented as the grey squares. As you can see, the grey squares are resting on top of the black square labelled “RDRM”, which is the promoter region of the RDR regulon, indicating that transcription of the regulon is inhibited. However, when cells are exposed to radiation, IrrE is activated and cleaves DdrO so it can no longer inhibit transcription, illustrated in figure 8. Now, you must be wondering: why is it that when DdrO is depleted, cells exhibit apoptotic-like death morphology? Here is the thing, when D.radiodurans are exposed to radiation, it automatically activates its RDR regulon, which is a DNA repair mechanism. This gene can only be activated if and when DdrO is no longer bound to RDRM. If damaged DNA cannot be repaired soon enough, cells will activate pro-apoptotic genes that are also controlled by DdrO that will lead to cell death. Logically, if DdrO is depleted, then RDR regulon is always activated since there is nothing to repress transcription. If RDR regulon is always activated, cells will think that DNA damage is too severe and will activate programmed cell death. Hence, the apoptosis-like morphology will be exhibited.
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| Figure 7. The role of DdrO as a repressor protein of the RDR regulon by binding to RDRM, which inhibits transcription. |
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Figure 8. DdrO is cleaved by IrrE, disabling DdrO to bind to RDRM. Thus, transcription of RDR regulon is activated.
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There are some really cool stuff to learn from microbes such as these. I would like to suggest some future studies that, in my opinion, could and should be done. Due to the bacterium’s resistance to radiation, it might be interesting to see how they can use their expertise to consume and digest solvents and heavy metals especially in highly radioactive sites as a way of bioremediation. D. radiodurans could potentially help humans clean up the mess that they have made over time to the environment. This bacterium is smarter than us in the sense that they have evolved to unlock the key to survival, without damaging and taking advantage of other forms of life. One last thing about what these amazing microbes teach us: The next time you think that you are the strongest beings on earth, think again!
Learn more: Devigne et al. (2015) DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic-like cell death in the radioresistant Deinococcus radiodurans bacterium. Mol Microbiol 96: 1069. https://doi.org/10.1111/mmi.12991
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| Meet the author: Mayang Hasibuan |
My love for microbes have increased tenfold after writing this blog post. You can call me Mayang and I was born and raised in a city called Bogor, which is one of the most populated cities in Indonesia. I am a non-premed, Biology major and a Gender Studies Minor in Mount Holyoke College. I will be graduating in 2018 (yeay) and I hope to one day be able to go home and work in the areas of bioethics. I love working in a research lab as well, but I do not think I would want to do that my whole life. My goal in life is to be as awesome as the many millions of microbes, which without, us humans would not be able to thrive and survive.
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