Radiation-Resistant Thermophiles: From High Temperature and Radiation to Engineered Bioremediation
PREETI RANAWAT1 and SEEMA RAWAT2
'Department of Botany and Microbiology, Hemvati Nandan Bahuguna Carhwal University, Srinagar (Garhwal)-246174, Uttarakhand, India
2School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat-382030, India
Microorganisms are ubiquitous and competent enough to inhabit hostile niches on Earth-like subsurface, deep oceans, extreme acid, and extreme alkaline conditions, frozen glaciers, seawater, and hydrothermal systems (Rampellotto, 2010). The existence of microbial life in inhospitable environments, viz., geothermal regions, hydrothermal vents, acidic, and hot geysers have unveiled the presence of special categoiy of microorganisms known as thermophiles or hyperthennophiles. The habitat of thermo (or hypeithermophilic) microorganisms is versatile and they have been reported from freshly fallen snow to pasteurized milk to geothermal springs (Ranawat and Rawat, 2017a). The famous abode of thermophiles include hot springs, viz., Yellowstone National Park, USA (Reysenbach et al., 1994, 2000; Slobodkin et al., 1997; Schaffer et al., 2004), in India from Bakreshwar hot spring, West Bengal (Ghosh et al., 2003), Tulasi Shyam hot spring, Gujarat (Gehlani et ah, 2015), Unkeshwar hot spring of Maharashtra (Mehetre et ah, 2015), Soldhar hot spring of Uttrakhand (Shanna et ah, 2015), Deulajhari hot spring, Odhisha (Singh and Subudhi,
2016), Grensdalur hot spring, Iceland, Garga hot spring, Russia (Nazina et al., 2004), Nalychevskie, Oksinskie, Apapelskie, and Dachnye hot springs in Kamchatka Peninsula, Russia (Belkova et al., 2007), El Biban hot spring in Northeast of Algeria (Kecha et al., 2007), Rotoura hot spring, New Zealand (Niederberger et al., 2008), Tengchong hot spring, China (Hou et al., 2013). The continental and submarine volcanic areas, viz., solfatara fields, geothermal power plants, hydrothermal vents, and geothennally heated sea sediments also provide suitable conditions for the growth of heat-loving microorganisms and mainly comprised of a population of anaerobic thennophiles and hyperthennophiles (Erauso et al., 1993; Uemori et al., 1993). The variations in environmental parameters, viz., temperature, pH, oxygen, nutrients, and light intensity in the habitat of thennophiles leads to stress conditions for the inhabitants, which results in slow growth or cell death. In stress conditions, the thermophilic life forms exhibit a variety of physiological changes, which include the production of DNA binding proteins, activation of reactive oxygen species (ROS) detoxification system, accumulation of compatible solute, expression of heat shock proteins and alterations in morphology (Ranawat and Rawat, 2017a). Thennophiles are the center of curiosity amongst the scientific community from the viewpoint of the mechanisms, which provide the ability to cope up simultaneously with variety of stresses, viz., desiccation, radiation, and pressure along with elevated temperature (DiRuggiero et al., 1997; Beblo et al., 2011; Caviccholi et al., 2011). The radiation stress at high temperatures is the most interesting phenomenon to explore as it reveals the adaptation mechanisms exhibited by thennophiles to radiations along with adaptations in the temperature range.
The exposure to ionizing radiations (IR) like a, p, у adversely affects DNA, lipids, and proteins, along with the production of oxidative stress (Webb and DiRuggiero, 2012). IR can either cause direct damage or indirect damage. In ‘indirect damage,' the ROS fonned by the radiolysis of water and generate hydroxyl radicals (OH-), superoxide (O,-), and hydrogen peroxide (H,02). The water molecules associated with DNA undergo radiolysis become detrimental for DNA molecules and as a result, generate oxidized DNA bases and sugar moieties, single-strand breaks (SSBs), abasic sites, and cross-links in proteins (Imlay, 2006). The increased intensity of IR leads to damage in the linear density of DNA base and induces SSBs on both strands, which in turn results in doublestrand breaks (DSBs). The proteins are the main target of ROS, which inactivates and denature proteins by introducing amino acid radical chain reactions and cross-linking of proteins (Stadtman and Levine, 2003; Imlay, 2006). O,- cannot react with DNA as well as with the majority of proteins and unable to cross membranes, but it can cause inactivation of enzymes with exposed 2Fe-2S or 4Fe-4S clusters. Fe2+ reacts with H,02 and catalyzes the Fenton reaction (Imlay, 2006). It is now widely accepted that radiations specifically target proteins, and in order to survive damage from IR protection against oxidation is required (Du and Gebicki, 2004; Daly et al., 2007). IR also induces ‘direct damage’ when macromolecules absorb X-ray and y-ray photon (Von Sontaag, 1987). A dose of IR typically causes 40 times more SSBs than DSBs (Von Sontaag, 1987; Daly et al., 1994). The radiation-resistant thermophiles counteract the damaging effects of IR by preventing the formation of ROS via superoxide dismutase and peroxidase. Resistant bacteria also accumulate Mil2- ions that protect Fe-S cluster containing proteins from O,-. Thus, the release of Fe2+from Fe-S cluster containing proteins can be prevented, minimizing the effects of Fenton chemistry and help enzymes to function efficiently (Ghosal et al., 2005; Imlay, 2006; Daly, 2009). The effects of IR on the cell are summarized in Figure 10.1. Thermophiles deal with the damaging effects of IR by using a detoxification system that scavenges ROS, mechanisms that repair DNA damage and accumulation of Mn2+ and trehalose (Makarova et al., 2007; Liedert et al., 2012; Webb and Di Ruggiero, 2012).
Radiation resistant thermophiles possess genes for detoxification and removal of toxic elements, which extends their application in the field of metal bioremediation also (Unnania, 2005). In this process, the radiation- resistant thermophiles not only perform degradation of the toxic metal waste but also oxidize a number of organic and inorganic substances along with metal ions, which results in the production of non-toxic metal ions from toxic ones (Sar et al., 2013). Therefore, these microorganisms are biotechnological assets that can be applied for bioremediation of heavy metals and radionuclides expelled out from nuclear power plants. The thermophilic Clostridium thermosuccinogenes can survive 6mM concentration of U(VI) (Wright et al., 2012) while mesophilic, Serratio marces- ceus has a minimum inhibitory concentration (MIC) of 4 mM (Kumar et al., 2011). Biotechnologically engineered thermophilic radiation-resistant strains like Deinococccus geothermalis can reduce Fe(III)-nitrilotriacetic acid, U(VI) and Cr(VI). This suggests that thermophiles are better and efficient candidates than mesophilic microorganisms for bioremediation of radionuclide wastes (Brim et al., 2003). The present chapter deals with the mechanisms governing radiation-resistant in thermophiles and their potential biotechnological applications ranging from exploitation of engineered strains for treatment of radionuclides contaminated environments to astrobiology.
FIGURE 10.1 Effects of ionizing radiations on bacteria (SSBs: single-stranded DNA breaks; DSBs: double-strand DNA breaks; SODs: superoxide dismutases; bold arrows represent effects of ionizing radiations, and dotted arrows represent responses of thermo- pliiles to radiation stress).
Source: Modified from Ranawat and Rawat, 2017b.
CO-EXISTENCE OF DESICCATION AND RADIATION RESISTANCE IN THERMOPHILES
The surviving conditions of early Earth resemble that of the present-day habitat of thermophiles, which supports the hypothesis that thennophiles were the first life forms on Earth (Di Giulio, 2000; Nisbet and Sleep, 2001). However, the hypothesis of lithopanspennia, suggests that the organisms which were carried by meteorites and survived the harsh environment of space, originated life on Earth (Nicholson et al., 2000; Homeck et ah,
- 2001). It has been observed that there is striking similarity in consequences of heat and water loss hi the cell, which include DNA double-strand breaks and protein denaturation (Prestrelski et ah, 1993; Mattimore and Battista,
- 1996). This suggests that microorganisms are able to survive in elevated temperature, and dehydration develops resistance for desiccation, and it leads to resistance for radiation as well. It has been speculated that the mechanisms that help Pyrococcus furiosus and Thermococcus gammatol- erous to overcome DNA damage caused by high temperature are possibly involved in repair of damage caused by desiccation and radiation (Grogan, 2000; Williams et ah, 2007; Beblo et ah, 2009; Zivanovic et ah, 2009).
In moderate thermophilic microorganism, Halobacterium salinarum, fast recovery from irradiation and desiccation has been reported by regeneration of intact chromosome from scattered fragments due to DSBs (Kotte- mann et ah, 2005; Soppa, 2013). The presence of overlapping genome fragment is necessary for this repair mechanism, and thus, it works only in oligoploid, such as D. radiodurans, or polyploid species, such as Haloar- chaea (Soppa, 2014). In Deinococcus spp. both stresses, i.e., desiccation, and radiation produce a similar type of DNA damage. It has been observed that radiation-sensitive mutants are desiccation sensitive also. Mattimore and Battista (1996), reported that the combination of UV and desiccation resistance in these microorganisms assist in their dispersal to hospitable environments. Proteins play an important role in the repair of damaged DNA, especially double-strand DNA breaks (DSBs). This protein repair system has been reported in Halobacterium salinarum NRC-1 where, Ral (tucHOG0456) helps in double-strand break repair and increases tolerance against radiations (Capes et ah, 2012).
The other mechanisms that enhance radiation resistance in microorganisms include the usage of advanced antioxidant chemical and enzymatic defense systems which mitigate DNA and protein oxidation, protection of DNA from radical damage by using enhanced DNA repair enzymes and nucleoid condensation (Pavlopoulou et ah, 2016). The plausible reason for radiation resistance in thermophiles is that these microorganisms continuously come across radioactive substances in their habitat, which may lead to radiation resistance. Elderfield and Schultz (1996) reported that some hydrothermal chimneys which exist in heavy metal-rich environments are naturally exposed to radioactivity doses, which are expected to be a hundred times higher than those observed in Earth’s atmosphere. The radiotolerance in mesophilic microorganisms like E. coli can be developed comparable to that of D. radiodurans by genetic alterations due to exposure of cells to repetitive irradiation cycles (Harris et al., 2009; Byrne et al., 2014). The concept of genetic alterations possibly holds true for ther- mophiles also as in their natural habitats, they often encounter radioactive elements like Paralana hot springs (PHS), rich in uranium. The phyloge- netically unrelatedness of radiation-resistant strains could be possibly due to the acquisition of radiotolerance during the evolution process (Cox and Battista, 2005).