Sunday, April 29, 2018

Deinococcus radiodurans: The Conqueror of Oxidative Stress?

By: Nyema Harris, Aiza Malik, and Grace Wheeler

In 1998, the bacterium Deinococcus radiodurans claimed the title “Most Radiation Resistant Life-form” from the Guinness Book of World Records. Care to hazard a guess as to how much gamma radiation this red bacterium can withstand? The answer is 1.5 million rads of gamma radiation, which is almost 3000 times the amount that would kill a human! It is thought that Deinococcus radiodurans may be able to survive extreme conditions because of its unusual ability to repair damaged chromosomal DNA. But how does it manage this? Read on to find out!

Image result for deinococcus radiodurans
Source.
This bacterium is hailed as a “polyextremophile,” which means it can survive many different types of extreme conditions, including vacuum, acid, cold environments and radiation. As described by Wikipedia: “The name Deinococcus radiodurans derives from the Ancient Greek words δεινός (deinos) and κόκκος (kokkos) meaning ‘terrible grain/berry’ as well as the Latin words radius and durare, meaning ‘radiation surviving.’” A radiation surviving terrible berry is a pretty accurate description of this bacterium! These polyextremophile traits are unusual in a bacterium, especially to this extent! Naturally, superpowers of this magnitude have peaked scientists’ curiosities all around the world. How do they do it? If we can understand their survival mechanisms, could we apply this technique to other organisms?

One of the types of stressors that Deinococcus radiodurans is able to resist is called oxidative stress. Oxidative stress is basically when a molecule containing oxygen builds up within an organism’s cells -- acting as a toxin -- so that the organism is not able to neutralize the toxins or counteract the damage caused. Oxidative stress is caused by a reactive oxygen species (in this case, H2O2). This type of stress causes all sorts of damage inside the cell, including damage to the DNA strands. So the question is, how does Deinococcus radiodurans repair this damage?

While the answer to this question is not necessarily well understood yet, Lin et al. have begun to formulate an answer that implicates an interesting bacterial process: quorum sensing. Quorum sensing can be thought of as bacteria’s way of ‘talking’ to each other. However, instead of words, bacteria use various signalling molecules such as N-acyl-L-homoserine lactones (AHLs) to communicate with one another. These molecules are able to tell cells to change the genes they are expressing so that bacteria can participate in beneficial processes such as symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. One can imagine that sometimes too much talking may be disruptive to the microbe and quorum sensing can be turned off by quorum quenching. Quorum sensing is fascinating because it challenges the traditional view of microbes as lone agents. Studies have shown that bacteria can communicate both with their own species and with other species, and the system of quorum sensing could have been an evolutionary precursor to multicellular life such as ourselves!
Microbial communities ‘talk’ with each other to control multiple group behaviors

So, you’re asking, what does quorum sensing have to do with D. radiodurans? As mentioned previously, it has been suggested that D. radiodurans doesn’t just use quorum sensing to communicate, the bacterium also uses it to combat oxidative stress! So how does this little fighter, this terrible berry, use quorum sensing to battle this stressor? The study by Lin et. al. reveals possible AHL-mediated quorum sensing pathways that D. radiodurans could use to combat oxidative stress.

The study first set out to show that D. radiodurans indeed has AHLs and AHL synthases, which are enzymes that catalyze the production of AHLs, a signaling molecule. If D. radiodurans has these molecules, then it is possible for D. radiodurans quorum sensing to be AHL-mediated. To detect AHLs in our terrible berry, the researchers used a strain of bacteria called Agrobacterium tumefaciens KYC55 to sense AHL and  determine its levels. It was revealed that AHLs are present in D. radiodurans and that more AHLs are present during the early growth phase of the cells vs. the exponential growth phase. To support that AHLs have a role in D. radiodurans oxidative stress response, D. radiodurans was paired with exogenous AHLs: AHLs that are not produced within the cell itself. D. radiodurans paired with exogenous AHLs had higher resistance to H2O2 stress than D. radiodurans without the exogenous AHLs. This indicates that AHLs take part in D. radiodurans oxidative stress response. To find AHL synthases (enzymes that synthesize AHLs), a BLAST search was conducted using a query sequence from known AHL synthases. Two potential AHL synthases were identified: DqsI-1 and DqsI-2. To test their function, researchers compared normal DqsI-1 and DqsI-2 synthases with those that contained one or two mutations. Because intentional mutations are a common method of making an enzyme lose its function, researchers often use them to create a disruption in a cellular process, making the cellular process dysfunctional. Transformed E. coli BL21 cells were made to overexpress the enzymes, and it was revealed that while mutated versions of DqsI-1 and DqsI-2 still catalyzed the production of AHLs, the levels of AHL were lower than those of cells using non-mutated enzymes. These results support the proposal that DqsI-1 and DqsI-2 are involved in the normal production of AHLs.

Considering that AHLs have been implicated in the oxidative stress response, researchers tried to determine how AHLs are regulated. D. radiodurans’ genome was sequenced for genes that could possibly regulate quorum sensing. Several genes were identified, but one was discovered to be part of AHL-mediated quorum sensing. The product of this gene (dr_0987), DqsR, was found to be part of AHL-mediated quorum-sensing because a mutation in DqsR resulted in decreased expression of DqsI-1 and DqsI-2. To further implicate its involvement in the oxidative stress response, D. radiodurans with mutated DqsR was found to accumulate three times more ROS (reactive oxygen species) than the D. radiodurans with the standard DqsR following treatment with H2O2.

Gel filtration chromatography clues us in to how DqsR regulates DqsI-1 and DqsI-2. It confirms an AHL-DqsR interaction that provides support for the hypothesis that AHL-DqsR interactions play a part in the ability of DqsR to function as a regulatory molecule under oxidative stress. In summary, in the presence of H2O2 , DqsI catalyzes the synthesis of AHLs which play a role in oxidative stress response. This relationship is demonstrated well in the figure below. DqsI is regulated by DqsR, which in turn requires the binding of AHL in order to regulate DqsI production (Figure 6). This means that oxidative stress causes production of AHLs. The AHLs counteract the negative effects of oxidative stress and so this terrible berry becomes the mighty conqueror of oxidative stress and lives another day!



While the paper may be said to raise more questions than it answers, it begins to lay out some details of the processes involved in surviving oxidative stress. We can now claim that AHLs and the quorum sensing/quenching system are important components in withstanding an oxidative assault. The authors do mention that the quorum sensing system is also present in other extremophilic Deinococcus bacteria. It would be interesting to see whether this toolbox is equally as useful for other Deinococcus strains in responding to oxidative stress. The paper dwells on quorum sensing and quenching molecules as a strategy to deal with oxidative stress. However, it does not explicitly address how quorum sensing is being used by D. radiodurans. Is it using the system to talk to other bacteria? Perhaps it has repurposed a system already at its disposal for a new function? Either explanation could provide interesting insights into the life of the fascinating and durable D. radiodurans.

We are provided with a tentative mechanism for the details of how this works: AHLs are involved in transcriptional regulation. This means that they are essential in producing useful substances that counteract oxidative species. This is a good foundation for understanding other extremophiles and gives a more definite direction to unravelling the mystery fully. This research is important because if we can learn more about how these extremophiles handle oxidative stress, we may learn more about extremophiles and bacteria in general. We may be able to use this information in the future for possible medical treatments (oxidative stress is one cause of skin cancer). Having a better understanding of cellular processes will help in future research as well. This type of exploratory research is useful because we never know what we could find!


About the Authors:
Grace Wheeler '18
Grace is a Biology Major with Art Studio and Chemistry Minors. She is studying to become a veterinarian specifically for working dogs! Her favorite particles are Diatoms, because they look like art.
Aiza Malik '18
Aiza is a Biochemistry major with a minor in Cultural Anthropology. She hopes to work in a public health related field. Her favorite biological rotary motor is ATP synthase (sorry flagellum!)
Nyema Harris ‘18
Nyema is a Neuroscience major with a minor in Chinese. Her favorite microbes are the Streptomyces for their earthy smell.


Works Cited:

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Lin, L., Dai, S., Tian, B., Li, T., Yu, J., Liu, C., . . . Hua, Y. (2016). DqsIR quorum sensing-mediated gene regulation of the extremophilic bacterium Deinococcus radiodurans in response to oxidative stress. Molecular Microbiology, 100(3), 527-541. doi:10.1111/mmi.13331

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Narendhirakannan, R. T., & Hannah, M. A. C. (2013). Oxidative Stress and Skin Cancer: An Overview. Indian Journal of Clinical Biochemistry, 28(2), 110–115. http://doi.org/10.1007/s12291-012-0278-8

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Wheeler D, Bhagwat M. BLAST QuickStart: Example-Driven Web-Based BLAST Tutorial. In: Bergman NH, editor. Comparative Genomics: Volumes 1 and 2. Totowa (NJ): Humana Press; 2007. Chapter 9. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1734/
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