Team:Botchan Lab Tokyo/Description

Botchan Lab. Tokyo

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Project Inspiration and Description

　　What our team has been working on

We have been searching for effective genetic combinations which make E. coli resistant to radiation. This time, We focused on oxidative stress resistance and DNA repair ability. This is because when an organism is exposed to radiation, it not only damages DNA directly, but leads to generation of reactive oxygen species(ROS), and ROS cause severe damages to DNA, protein and lipid. Then, we spotlighted Deinococcus radiodurans which has rapid DNA repair mechanisms and also displays many protective features against ROS⁽¹⁾. It’s one of the most radiation-resistant organisms ever known, and it can survive after exposure to a high dose of ionizing radiation (IR) －up to 5 kGy, and 5 - 10 Gy are lethal to most vertebrates including humans, even a few hundred Gy are lethal to most known bacteria⁽²⁾.－, or under cold, dehydration, vacuum⁽³⁾, and acid conditions. As a first step in searching effective genetic combinations, we chose 3 genes (See below “The genes we introduce”) that are related to high DNA repair ability and oxidative stress tolerance from D. radiorurans and tried to transform E. coli with plasmids including 1 gene or some genes. We’re trying to evaluate the efficacy of each gene against radiation by comparing postradiation survival rate.

What we hope to accomplish

As a future task, we will evaluate the efficacy of other various genes from D. radiorurans that we didn’t choose this time, and discover effective genetic combinations for radioresistance. Thereby, we expect to be able to create radiation-resistant bacteria.

　　Background

In 2011, Great East Japan Earthquake causes the nuclear accident in Fukushima Daiichi Nuclear Power Plants(NPP). This is why a large amount of radiation was emitted. People have tried to tackle the contamination problems since then. However, we still have a lot of problems that need to be solved for example, tons of contaminated water(See Figure 1), soils(See Figure 2) and biomass⁽⁴⁾. These are the main approaches that are taken now to deal with contaminants. For contaminated water, Advanced Liquid Processing System(ALPS) is used. However, not all contaminants are removed by just processing it once, so the water from the ALPS needs to be stored in tanks which predicted to be full in 2022⁽⁵⁾. For contaminated soils and biomass, thermal treatment is used⁽⁶⁾. However, there is too much contaminants, and processing cannot keep up. So, they are kept temporary in Fukushima. Both of the treatments require a big specialized facility. Then, Is it better to construct more treatment facilities? In this case, it must be difficult to gain public acceptance due to the high risk. Therefore, we thought it is better to develop a new approach that does not depend on the current technology and to use a different approach depending on the situation.

Figure 1. A lot of tanks for contaminated water (Photo credit: Tokyo Electric Power Company Holdings)

Figure 2. contaminated soils that are kept temporary (Photo Credit: Fukushima Prefecture)

　　Inspiration for our project and why we chose this project

So, we considered a biological approach, that is, bioremediation. If we accomplished this goal, decontamination would be possible even in areas where there are no big specialized facilities. When we visited National Institute of Genetics in last December (See Human&Practice Page), Mr. Andachi told us that we need to consider the effect of uptake radioactive materials on bacteria. If bacteria take up cesium, cell death or mutation may occur. Thus, for bioremediation approach, we need not only the technology that can take up radioactive materials but also radiation-resistance. In addition, he told that cesium uptake was recent trend, and Many researchers have already been searching for cesium recovery method. In fact, the 2011 iGEM team SYSU-China searched about cesium absorption⁽⁷⁾. So, we decided to focus on radiation resistance to make use of cesium recovery technology that has already been actively researched and finally to be able to achieve bioremediation.

　　Application（How we will achieve our goal）

To accomplish bioremediation, we are thinking of combining our radioresistant E. coli and Cesium uptake technology invented by the 2011 iGEM team SYSU-China. We would also like to expand bioremediation potential by combining our bacteria and other technologies that take up radioactive materials. The reason why we decided to search radiation resistance was to solve radioactive contamination by bioremediation. However, radioresistant bacteria could work not only under high radiation but in environments where mutations are likely to occur. Radiation-resistant bacteria have the unlimited possibilities by combining them with a variety of technologies, and we hope that various iGEM teams will make use of our project in the future. As another use, our radioresistant bacteria have high DNA repair ability, and that contribute to improve cloning technology (in Vivo E.coli Cloning).

　　The genes we introduce

RecA

recA is a DNA-dependent ATPase recognized in many organisms. E. coli also has it. recA is a multifunctional protein that plays an important role in homologous recombination⁽⁸⁾, recombinant DNA repair⁽⁹⁾, and SOS response to DNA damage and DNA replication arrest⁽¹⁰⁾. recA from Deinococcus radiodurans we use in our experiment has the feature that binding to dsDNA more often than ssDNA⁽¹¹⁾. One the other hand, recA that E. coli and other Deinococcus genera such as Deinococcus geothermalis and Deinococcus murrayi have are binding to ssDNA more often than dsDNA⁽¹²⁾.
That is to say, the features of D. rad's recA are unique, and recA of Escherichia coli and Deinococcus murrayi are considered to have partially lost the function of D. rad's recA. In addition, D. rad's recA plays an important role in the mechanism that repair DNA when it breaks apart by double-strand breaks⁽¹³⁾. This mechanism consists of two steps: ESDSA and double-strand break repair. recA is essential for reactions in which making single-stranded DNA get under double-stranded DNA in this mechanism⁽¹³⁾.

PprM

pprM is a cold shock protein homologue of D. rad (although it works even at physiological temperatures). pprM is an important modulator of pprA along with recA, in radiation resistance and normally suppresses the expression of pprA. When radiosensitive pprI is expressed, pprM is suppressed, and as a result, pprA is promoted. Thus, pprM is an essential gene in the control of a gene that promotes DNA repair of D. rad, and D. rad lacking this is highly radiosensitive⁽¹⁷⁾. pprM is also essential for the production of KatE1, the major catalase of D. rad⁽¹⁸⁾. Furthermore, when pprM is introduced into E. coli, it is believed that OxyR, a transcription factor that is activated during oxidative stress, is oxidized⁽¹⁴⁾. OxyR regulates about 40 regulons, which are activated when oxidized OxyR binds to the promoter region⁽¹⁶⁾. This OxyR regulon promotes the uptake of Mn^{2 +} via MutH, thereby substituting Fn^{2 +} for Mn^{2 +} and suppressing the Fenton reaction by Fn^{2 +} and active oxygen⁽¹⁵⁾. That is, pprM contributes to suppression of the Fenton reaction in E. coli.

PqqE

pqqE is one of operons consisting of six genes that encode an antioxidant called pyrroloquinoline quinone. PQQ is possessed by various organisms, and Klebsiella pneumoniae has six genes, pqqA-F. On the other hand, E. coli cannot produce PQQ, so pqqA, pqqC, pqqD, and pqqE from Klebsiella pneumoniae are required for production by cloning⁽¹⁹⁾. Of these pqqA-F, D.rad has only the pqqE homolog, and other D genus bacteria do not have pqq-related genes⁽¹⁹⁾. This D.rad pqqE can produce pqq even in the absence of homologues of other pqq genes⁽²¹⁾. Furthermore, pQQ not only acts as an antioxidant in E. coli, but also during DNA damage. pQQ binds to periplasmic lipoprotein (yfgL) and activates yfgL. Activated yfgL activates genes necessary for DNA repair such as recA⁽²²⁾.

　　References

1.Dea Slade, Ariel B. Lindner, Gregory Paul, and Miroslav Radman, (2009), “Recombination and Replication in DNA Repair of Heavily Irradiated Deinococcus radiodurans”, Cell 136, 1044-1055

2.Sangyong Lim, Jong-Hyun Jung, Laurence Blanchard and Arjan de Groot, (2018), “Conservation and diversity of radiation and oxidative stress resistance mechanisms in Deinococcus species”, FEMS Microbiology Reviews, 43 (1), 19 – 52

3. Emanuel Ott, Yuko Kawaguchi, Denise Ko¨lbl, Palak Chaturvedi, Kazumichi Nakagawa, Akihiko Yamagishi, Wolfram Weckwerth, and Tetyana Milojevic, (2017), “Proteometabolomic response of Deinococcus radiodurans exposed to UVC and vacuum conditions: Initial studies prior to the Tanpopo space mission”, PLOS ONE, 12 (12), 1 - 25. https://www.ncbi.nlm.nih.gov/pubmed/7968921

4.Ministry of the Environment, “放射線物質汚染廃棄物処理情報サイト”, http://shiteihaiki.env.go.jp

5.Tokyo Electric Power Company Holdings, “Contaminated Water Treatment,” https://www7.tepco.co.jp/responsibility/decommissioning/action/w_management/treatment-e.html

6.Masahiro Osako, ”Sustainable Management of Radiocesium-contaminated Soil and Waste,” Center for Material Cycles and Waste Management Research National Institute for Environmental Studies https://www.ncbi.nlm.nih.gov/pubmed/12045091

7.the 2011 iGEM team SYSU-China https://2011.igem.org/Team:SYSU-China

8. Kowalczykowski SC, Dixon DA, Eggleston AK, Lauder SD, Rehrauer WM. "Biochemistry of homologous recombination in Escherichia coli" Microbiol Rev. 1994;58:401–465. pubmed

9.Kuzminov A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ Microbiol Mol Biol Rev. 1999;63:751–813. pubmed

10.Lusetti SL, Cox MM. The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu Rev Biochem. 2002;71:71–100. pubmed

11.Wanarska M, Krawczyk B, Hildebrandt P, Kur J (2011) RecA proteins from Deinococcus geothermal is and Deinococcus murrayi—cloning, purification and biochemical characterisation. BMC Mol Bio 12:17. pubmed

12.Kim JI, Sharma AK, Abbott SN, Wood EA, Dwyer DW, Jambura A, Minton KW, Inman RB, Daly MJ, Cox MM. (2002) RecA protein from the extremely radioresistant bacterium Deinococcus radiodurans: Expression, purification, and characterization. JOURNAL OF BACTERIOLOGY;184(6) 1649-1660. pubmed

13.Cox, Michael M.; Keck, James L.; Battista, John R. (2010) “Rising from the Ashes: DNA Repair in Deinococcus radiodurans” PLOS GENETICS ;6(1) e1000815 pubmed

14.Park SH, Singh H, Appukuttan D et al. PprM, a cold shock domain-containing protein from Deinococcus radiodurans, confers oxidative stress tolerance to Escherichia coli. Front Microbiol 2017;7:2124. pubmed

15.Anjem A., Varghese S., Imlay J. A. (2009). Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli. Mol. Microbiol. 72, 844–858. pubmed

16.Chiang S. M., Schellhorn H. E. (2012). Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Arch. Biochem. Biophys. 525, 161–169. pubmed

17.Ohba H., Satoh K., Sghaier H., Yanagisawa T., Narumi I. (2009). Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans. Extremophiles 13, 471–479. pubmed

18.Jeong S.-W., Seo H. S., Kim M.-K., Choi J.-I., Lim H.-M., Lim S. (2016b). PprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions. J. Microbiol. 54, 426–431. pubmed

19.Klinman JP, Bonnot F. Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ. Chem Rev 2014;114:4343–65. pubmed

20.Rajpurohit YS, Gopalakrishnan R, Misra HS. Involvement of a protein kinase activity inducer in DNA double strand break repair and radioresistance of Deinococcus radiodurans. J Bacteriol 2008;190:3948–54. pubmed

21.Khairnar NP, Misra HS, Apte SK. Pyrroloquinoline–quinone synthesized in Escherichia coli by pyrroloquinoline–quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization. Biochem Bioph Res Co 2003;312:303–8.

22.Khairnar, Nivedita P.; Kamble, Vidya A.; Mangoli, Suhas H.; et al.(2007) Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli MOLECULAR MICROBIOLOGY; 65(2): 294-304 pubmed