Viruses in ancient ice wedges in the Central Yakutia, Siberia 1 1 1 Elina Karnysheva , Anatoli Brouchkov , Maria Cherbunina , Gennady 3 2 2 2 Griva , Svetlana Filippova , Dmitry Skladnev , Valery Galchenko 1 2 Moscow State University, Russian Federation, Vinogradsky Institute 3 of Microbiology, Russian Federation, ATEMA Lab, Canada ABSTRACT The study of the viral component of ancient microbial communities from permafrost is important for the understanding evolution of microbial communities, possibility of their variations due to climate change, changes in the physical-chemical state of permafrost and practical questions of biosafety. For the first time the virus particles in native samples of ancient ice wedges of the Mammoth Mountain in Siberia have been discovered. Defined morphological diversity of viruses that can be attributed to five main types: miovirus, sifovirus, podovirus, spherical and filamentous. Specific characteristic of these viruses are small size and fever genome. RÉSUMÉ L’étude des virus qui sont présents dans des communautés microbiennes anciennes du pergélisol est cruciale pour la compréhension des questions fondamentales telles que l’évolution des communautés microbiennes, la possibilité de leur changement suite aux changements du climat, de l’état physico-chimique du pergélisol aussi bien que les questions pratiques concernant la sécurité biologique. Les virus ont été découverts pour la première fois dans des glaces éternelles de la montagne Mammouth. La définition de leur diversité morphologique faite, les virus peuvent être classés en cinq types principaux : miovirus, sifovirus, podovirus, virus sphériques et virus filamenteux. Leur spécificité consiste en une petite taille du génome. 1 INTRODUCTION take thousands of years to move a meter. A bacterium of greater size than the thickness of the water layer is likely Permafrost microorganisms in comparison with ancient to move much more slowly than the water. The salt or amber isolates are widely distributed microorganisms are about 0.3 to a few microns in size, (Vishnivetskaya et al., 2006; Steven et al., 2008; Yergeau while the thickness of the water films tends to be less. et al., 2010; Margesin&Miteva, 2011). For more than a One concludes that microorganisms in permafrost have century there have been reports of living organisms in been isolated, certainly from the ground surface, trapped permafrost, some of which are certainly might be millions among the mineral particles and ice. of years old, if they have age which is similar to the age of The longest, continuously frozen permafrost in the permafrost itself. Living (or at least viable) bacteria Northern hemisphere is variously estimated as between apparently occur deep in solid-frozen ground (permafrost) one and three million years old (Foundations of in the cold regions (see the review by Gilichinsky and geocryology, 1998). Abyzov’s investigations at the Vostok Wagener, 1995). Sometimes permafrost as well as station (Abyzov, 1993) revealed bacteria, fungi, diatoms microorganisms in it is dated quite well (Katayama et al., and other microorganisms which were probably carried to 2007). Viruses in permafrost were not broadly reported, Antarctica by winds. The ages of these individuals could however, their presence might be associated with be more than half a million years. Abyzov (1993) has psychrophile bacteria and other organisms (Morita 1997). showed the presence of viable bacteria in the ice which There is a number of questions related in life in ancient was hundreds of thousands of years old and at a depth of permafrost. For example, are isolated bacteria as old as thousand meters which could not have been the permafrost itself or can contamination with more contaminated from the surface or from below in recent recent bacteria have occurred? Do the bacteria grow in time. the permafrost? And to what extent are ‘normal’ metabolic Although most microorganisms do not grow at processes taking place? - or are they inactive and temperatures below 0°C, certain bacteria and fungi can be cryopreserved? An important characteristic of permafrost physiologically active and Friedmann (1994) notes is that some water, held tightly by electrochemical forces metabolic activity in permafrost bacteria at -20°C. Others onto the surfaces of mineral particles or under the reporting evidence concerning bacterial activity in soils influence of capillary forces, occurs in even hard-frozen below 0°C, include Kalinina, Holt and McGrath (1994); permafrost (Williams and Smith, 1991; Brouchkov & and Clein and Schimel (1995). Water is the solvent for the Williams, 2002). The thin liquid layers provide a route for molecules of life, and availability of water is a critical water flow, which is normally from the warmer to the factor affecting the growth of all cells. But the particular colder parts (Derjaguin and Churaev, 1986). The water water which is unfrozen in permafrost, although at less may carry solutes and small particles and thus perhaps, than 0°C and in the presence of ice, differs from ‘ordinary’ bacteria, but its movement is extremely slow (Burt and water. It is attached to the soil mineral particles surfaces. Williams, 1976): at a few degrees below °C it may thus As the temperature falls to -2 or -3°C, the remaining water is in layers so thin that a bacterium could not be fitted in. Figure 1. Variety of microorganisms isolated from ice Metabolic activity and especially the ability of wedge of the Mammoth Mountain (Filippova et al., 2014) microorganisms to grow for a long time are greatly limited in the conditions of the environment within the permafrost. The single bacterial cell is trapped and not even free to move or expand within the unfrozen water layer. Probably some microorganisms grow if only because of the substantial degree of microbial activity at temperatures below 0ºC. But for the most part it appears unlikely. Microscopic pictures of frozen soils show single cells mostly (much less groups of a few cells), not colonies (Figure 1), and that fact is another argument for dormancy microorganisms in permafrost (Melnikov et al., 2011). Studies of viruses are of interest for permafrost, however, they are almost unknown (Allen, 2010). 2 METHOD OF WORK AND ISOLATION 2.1 Overview Samples were collected in at an altitude of 83 m above sea level at the Mammoth mountain exposure (Figure 2) in the Central Yakutia (62°56'N, 133°59'E), exposition north, and at a depth of 1.5 m from the surface of the Neogene formation (Figure 3). A deep hole of approximately 100 cm was horizontally dug into the frozen Neogene horizon. After sterilizing the surface of this sampling hole by flame, pieces of frozen sediment (icy sand) were collected from a horizontal depth of 75–100 cm, cleaved with a sterilized axe, and collected in sterile Figures 2 & 3. Section of Mammoth Mountain 50 mL vials by using sterile spatulas. The mean temperature of the icy sand at the time of sampling was Samples were immediately embedded in frozen −4 °C. natural permafrost material, then stored in a cryogenic mixture of NaCl and water to keep the material constantly frozen. The samples were kept frozen during transport from Yakutia to the laboratory in Moscow where samples were stored at−20 °C. Thus, the collected material was constantly kept frozen and never subjected to thawing. A composite sample was produced under sterile conditions immediately before analysis. At this stage of modern science development, it is possible to determine accurately the age of the amber fossils (Lambert&Poinar, 2002), as well as to determine the age of frozen soils. The age of the permafrost in the Mammoth mountain area exceed 3 million years that was dated by paleoclimatic reconstructions (Bakulina&Spector,2000; Baranova et al., 1976). The exposure is destroyed by the river (more than 1 meter per year); therefore, the sampled sediments were obviously in a state of permafrost. The latter are fine-grained sands, and their age corresponds to the middle Miocenbe, 10–12 million years. The sediments have been intensively studied and did not thaw out because of the cold climate of Yakutia (Markov, 1973; Foundations of Geocryology, 1998; Bakulina and Spector, 2000). Samples of different dilutions in sterile conditions were added to Petri dishes containing liquid ISP1 media for 20-30 days at 20°С. A few isolated strains were described before (Brouchkov et al., 2012; Zhang et al., 2013) from the sample. Observations of the appearance of the negative parts of lysis in the area of active growth of colonies was performed visually using a magnifying glass during the whole period of incubation. Material was collected from the zones of lysis by the bacteriological hook for subsequent electron microscopy analysis. 14,000 rpm for 2 minutes to separate from cell fragments. Colonies with negative portions were separated on an Then equal volume of phenol equilibrated with buffer to agar slant medium and incubated for 2 days at 28°C. pH = 8.0 for 10 seconds was added. Then after a 5 Culturing the isolates was done in liquid medium ISP1. minute centrifugation aqueous (top) fraction was taken to The medium was dispensed into 250 ml flasks at 50 ml, a new tube. Then equal volumes (250mcl / 250mcl) and sterilized in an autoclave at a pressure of 1 atm. for phenol and chloroform mixed for 10 seconds was added 30 minutes. 1 ml cell suspension of 1-2 x overnight culture there. was placed in the flasks with a sterile nutrient medium. After 3 min of centrifugation the resulting mixture the Cultivation was conducted by submerged cultivation on a overhead fraction was taken to a new tube, and rotary shaker while aeration and stirring is carried out chloroform was added in a volume equal to the volume of simultaneously by rotating at a speed of 180 rev / min. obtained the fraction. The solution was mixed for 10 Incubation was carried out at a temperature of 26-28°C for seconds. Then the resulting mixture was again 48 hours. centrifuged for 2 min. The top fraction was separated, and Phage lysate preparation. Liquid submerged lysogenic sodium chloride was added to a final concentration of culture was centrifuged at 9000 g. The resulting 0.5M. Then isopropanol in a volume of 0.7 part of the total supernatant was filtered using a syringe membrane filter, volume of the mixture was added and mixed. After pore size 0.2 µ to release phage lysate from cell centrifugation for 5 minutes the precipitate was separated, fragments of the host bacterium. and 0.5 mL of 70% ethanol solution was added, stirred, The method of phages collection. Phage lysate was then centrifuged again for 5 minutes. The resulting used to accumulate phages in the indicator culture liquid supernatant was removed under vacuum, and then dried or the bacterial culture of phage host. 500 ml of the at 37°C for 10 minutes. The dry material was dissolved in filtered phage lysate was added in the submerged culture 105 µl of ampoule water. To determine the DNA of the indicator strain of lysogenic bacteria or bacterial concentration 5 µl sample was transferred to isolate after 7 hours, then culture was incubated under the spectrophotometer. Spectra were recorded at a same conditions for 20 - 24 hours. wavelength of 260 nm and 280 nm. The resulting culture fluid was centrifuged at 9000 g. The supernatant containing phage particles and cell Electrophoresis in agarose gel: For preparing a substrate fragments were centrifuged at 100000 g for release from 2% agarose was used for gel solution preparation in the the bacterial cell fragments. The result is a phage final TE buffer. A dye (ethidium bromide to a final concentrate. concentration of 2 mg/ml) was added and mixed thoroughly. The sample of DNA and marker fragments of 2.2 Study of lytic properties of phage the phage DNA was applied in an amount of 2 µl in appropriate wells. Electrophoresis was performed for 15 2.2.1 Selection of the indicator culture minutes at 120 V. One day cultures of Bacillus subtilis ATCC 6633, as well 3 RESULTS as strains B.mycoides, B. megatherium and Paenibacillus sp., isolated from the Antarctic Lake Untersee, were used 3.1 Identification of virus-like particles in the sample to study the lysing activity of the phage. from ice wedge by electron microscopy 2.2.2 Study of lysis activity Viral particles of different morphology by the electron microscopy of melted ice samples were found (Figure 4). Concentrated material containing phages in amount of 5µ, -1 -2 -3 and also diluted by 10 , 10 , 10 was applied to freshly 3.2 Identification of lysogenic bacterial forms. prepared bacterial lawns. Thereafter it was incubated at 28°C for a day. Lysing activity was estimated by The number of colony forming viable organisms in the 2 3 appearance of the transparent zones - zones of lysis. samples was an average of 10 -10 CFU / ml. Increasing Methods for microscopic study included phase-contrast the incubation periods has revealed 2-3 colonies of similar protocols by Zetopan microscope with phase-contrast type, in the area with active growth where there is a device. negative sites ranging size 1.5 - 2 mm, whose number is The method of electron-microscopic study. For tests increasing with the aging of the colonies. 10 ml of melted sample was taken. After standing about The appearance of the sterile areas in the peripheral 0.5-2 hours at room temperature, enlightened upper zone of the old colonies suggests that these areas are the portion was selected to produce samples for electron result of the release of the phage from lysogenic bacteria microscopy. Electron microscopic studies were performed cells and subsequent lysis of some of them (Figure 5). on the electron microscope JEM-100CXII (JEOL, Japan). The release of the phage can be due to physiological Samples were viewed with magnification × 40,000. state of the cells, i.e., with aging, there is an accumulation of metabolic products which can induce the phage output. 2.2.3 The method of isolation of phage DNA. It was noted that during the period of normal saline (2 -3 days) appearance of sterile areas were not observed. It is Isolation of DNA from the concentrated lysate: 0.5 ml of known that cells lysogenic cultures of microorganisms the precipitated sample of phage were centrifuged at resistant to contained phage and only a small portion of them can be sensitive and lysed. Aging and death of the hours of immersion indicator culture then the culture was cell population may contribute to the release of the phage continued for another 24 hours. lysogenic cultures. For studying the source of the appearance of bald spots on the colonies electron microscopic examination was carried out. The results revealed filamentous virus particles (Figure 6). Colonies of this bacteria were isolated and maintained on an agar medium ISP1. The study of the morphology of cells lysogenic bacteria showed that their cells are rod-shaped, often grouped into chains in the stationary growth phase, the formation of spores. This can be attributed to the bacteria like Bacillus. Figure 6. Filamentary particles of the negative portions lysogenic bacterial cultures. Scale line 0.12mkm Figure 4. Morphological diversity of viruses attributed to Figure 7. Area of lytic action five main types: miovirus (a,c,e), sifovirus(g,h), podovirus(d), spherical(b) and filamentous(f). Scale line 3.4 Isolation of phage DNA 0,05 mkm After culturing the resulting fagolizat (culture liquid containing cellular material and phage particles) was placed in a refrigerator to 4 ° C and held up to 14 days in order to optimize lysis (Figure 8). Then fagolizat was centrifuged for separating cellular material and concentration of phage particles. Thereafter, DNA was isolated and the electrophoretic separation of virual DNA from impurities bacterial DNA was made. It has been found that the size of the test filamentous phage not greater than 10000 bp (base pairs). Figure 5. The negative (sterile) zone in the region of 4 CONCLUSIONS active growth of bacterial colonies (after 20-30 days of incubation at 200C) The oldest permafrost in Eurasia is likely to be in the Yakutia, where glaciers were not formed and whose age 3.3 The accumulation of phage and identifying its lytic can reach 3 million years, when the surface temperature action was perhaps similar to modern as it follows from paleoclimatic studies (Ershov, 1998; Lisiecki & Raymo, Lytic activities zones were found at the site with initial 2005; Hansen et al., 2010). The upper part of the filtrate, which may indicate its small litic activity or lack of Mammoth mountain section is so-called "ice complex", sensitivity indicator culture (Figure 7). which is a syngenetic ice wedges located in the icy alluvial For getting a concentrated viral material performed its sediments. These deposits are younger, they are late accumulation in a submerged indicator culture conditions. Pleistocene (Vasil'chuk, 1991), but still represent a kind of For this obtained viral material was used to inoculate 7 "time capsule", which have ancient microorganisms which have penetrated into the cracks during its formation with of Russian Academy of Sciences.Nauka: Moscow, surface waters. It gives a unique opportunity to study 1976; 284 p. (in Russian) microbial communities, their ability to survive, various cell- Belda, E., Moya, A., Silva, F.J. 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