Relict microorganisms of cryolithozones as possible objects of gerontology
The original of a very, very scientific article, disassembled by the bones in another article,
not scientific at all, as you can guess from the author's signature – A Healthy Skeptic,
Microbiological analysis of Mammoth Mountain: the experience of investigative journalism
Tyumen Scientific Center SB RAS
The study of cells capable of surviving for many millennia may be of interest to gerontology.
Brushkov A.V. (1), Griva G.I. (2), Melnikov V.P. (2), Sukhovey Yu.G. (2), Repin V.E. (3), Kalenova L.F. (2), Brenner E.V. (3), Subbotin A.M. (2), Trofimova Yu.M. (1), Tanaka M. (4), Katayama T. (4), Utsumi M (4).
1 - Tyumen State Oil and Gas University; 2 - Tyumen Scientific Center SB RAS; 3 - Institute of Chemical Biology and Fundamental Medicine SB RAS; 4 - Hokkaido University; 5 Institute of Agricultural and Forest Engineering, University of Tsukuba
introduction
At a time when the study of the mechanisms of aging is still an urgent fundamental problem, the study of cells capable of surviving for many millennia may well be of interest to gerontology. In itself, the discovery of relict living cells is an exceptional event. It was believed that bacteria, being, in fact, simplified likenesses of eukaryotic cells, live or remain viable for a long time. However, confirmation of this has been found only recently. It has been shown that anthrax spores persist for about 105 years (Puskeppeleit et al., 1992). Colonies of bacteria were grown that were extracted from amber aged 40 million years or more (Greenblatt et al., 1999). However, these findings do not allow us to establish ourselves in the idea of the phenomenal lifespan of bacteria, as the permafrost studies have done. The permafrost area is large (Fig. 1), only in the Russian Federation it occupies about 70% of the area. It has temperatures mainly around -2 - - 8 ° C, and its age is in some places millions of years.
Fig. 1. Permafrost distribution area in the Northern Hemisphere
Evidence of the viability of microorganisms in permafrost appeared in the nineteenth century. In 1979, bacteria, fungi, diatoms and other microorganisms were discovered at the Vostok Antarctic Station (Abyzov et al., 1979). Cyanobacteria were found in Antarctica at a depth of 3600 meters, their age is about 500 thousand years. The metabolism of bacteria in permafrost was observed at temperatures of about -20°C (Friedmann, 1994). A bacterial community was found in the Antarctic permafrost (Hubbard et al., 1968). There are some other facts regarding bacterial growth below 0°C (Forster, 1887; Lozina-Lozinsky, 1972; Flanagan, Veum, 1974; Bunt, Lee, 1970; Morita, 1975; Kalinina et al., 1995; Clein, Schimel, 1995). Microorganisms are resistant to freezing; many of them easily tolerate it (Psenner, Sattler, 1998). It is known that at temperatures below -20 °C, part of the water in the tissues (over 10%) remains unfrozen (Brushkov et al., 1995). Without denying the likelihood of the development of microorganisms in frozen rocks, we note that their growth is difficult. Even in laboratory conditions, aging cultures are known to stop growing. The crystallization of water and the stoppage of external metabolism reduces the ability to grow. The thickness of the interlayers of unfrozen water at temperatures of - 2 and - 4 ° C is approximately 0.01 - 0.1 microns, that is, much smaller than the size of microorganisms, which are 0.3 - 1.4 microns or more. These pathways are practically unsuitable for life support, and there can be no question of noticeable cell transfer in such a material. Therefore, we can be sure that the bacteria in permafrost rocks are fossils, relict organisms. Their age is confirmed by the geological conditions of their location, the history of the formation of frozen strata, radiocarbon dating, the results of the study of optical isomers of amino acids and, indirectly, the biodiversity of the species encountered.
The nature of the long-term viability of microorganisms in the ancient permafrost does not have an exhaustive explanation. There is an opinion based on experiments that no chemical and biological reactions occur in the body in a state of suspended animation (Hinton, 1968). However, most proteins in a living cell are known to be unstable (Alexandrov, 1985) and live for minutes and less often days. Genetic structures are subject to mutations (Cairns et al., 1988), and reparative mechanisms are not so effective as to prevent the accumulation of damage. Free radicals and radiation act on cellular structures. The thermal motion of atoms and molecules in an aqueous solution is an obvious destructive factor - permafrost temperatures are far from absolute zero. The cytoplasm of the cell does not freeze at the same time (Low temperature ..., 1968), probably it does not freeze at all (Kanwisher, 1955). It can be assumed that the body is subject to destruction and decay even in suspended animation. Thus, data on the thermal stability of nucleotides (Levy, Miller, 1998) show that due to the instability of cytosine, the time during which the DNA chain is capable of transmitting information hardly exceeds several hundred years. If we take into account the average rate of mutations, almost every gene in the bacterial chromosome will undergo a change in a thousand years, and within a million years there will be "nothing alive" from the genetic apprata, even if the repair systems function. This is confirmed by studies of ancient DNA of mummies, mammoths, insects in amber and other organisms, which turns out to be fragmented and partially destroyed by Greenblatt et al., 1999). Calculations show that even small fragments of DNA (100-500 nucleotides) can last no more than 10,000 years in normal climates and up to a maximum of 100,000 years in cold areas due to hydrolysis. Thus, it seems unclear how bacteria survive in the thousand-year permafrost. The ability of relict microorganisms to remain viable for a long time implies the existence of mechanisms that prevent the accumulation of damage. It seemed to us expedient to study the influence of these microorganisms on higher organisms, especially since the immune reactions of the latter may be of interest.
This paper describes the bacteria found in the ancient frozen strata of Yakutia, and provides preliminary results of testing the biological activity of their culture on fruit flies and laboratory mice.
MATERIALS AND METHODS OF ISOLATION OF MICROORGANISMS
To study microorganisms in frozen rocks, samples were taken from outcrops and underground structures in several areas. One of them is located on the left bank of the Aldan, 325 kilometers upstream from its confluence with the Lena, on Mammoth Mountain. The samples were taken 0.9-1 m deeper than the seasonal thawing layer. The outcrop is destroyed by the river (more than a meter per year), so that the sediments from which the samples were taken were obviously in a permafrost state. At the same time, there is an annual spring washout of collapses, preventing blockages and mixing of rocks. The latter are fine-grained sands and siltstones; their age corresponds to the Middle Miocene and is 10-12 million years old (Baranova et al., 1976). Cooling and the associated freezing of sediments began here at the end of the Pliocene, about 3 - 3.5 million years ago. In the later stages of geological development, the deposits did not thaw due to the cold climate of Yakutia. According to paleoclimatic reconstructions of the region, the average annual temperatures in the Pleistocene ranged from -12 to -32°C in winter and from +12 to +16°C (Bakulina, Spector, 2000). Thus, the age of permafrost on Mammoth Mountain can reach 3.5 million years. In addition to the described outcrop, samples for microbiological studies were taken from younger re-vein ice of the Yakutia ice complex, from the walls of the dungeon of the P.I.Melnikov Permafrost Institute in Yakutsk, as well as from underground ice in the Fox tunnel and at the gold mine near Fairbanks in Alaska.
Samples of frozen rocks were taken with the greatest possible precautions for field conditions. Metal instruments sterilized with alcohol and burnt in flames (drills, tweezers, scalpels) were used. For surface sterilization of samples, a sample weighing about 50 g was placed in a glass with 96% ethanol solution, then in a burner flame and packed in a sterile tube. In addition, monoliths of frozen rocks weighing 4-5 kg were selected. The selected rocks were stored at a temperature of -5°C, which was close to natural conditions. The samples were transported in thermocontainers with refrigerants in a frozen state.
Samples of various dilutions under sterile conditions were added to Petri dishes containing YPD, MRS and NA media (Manual of enviromental microbiology, 1997). Samples were also added to liquid meat-peptone broth under anaerobic and aerobic conditions.
The ribosomal DNA of the microbial culture was extracted using Fast DNA kit for soil (BIO 101 Inc., Vista, CA), which uses a method based on the destruction of the cell by glass beads. Nucleic acids were precipitated from the solution using a solution consisting of 0.1 parts of 3M sodium acetate (pH 5.2) and 2.5 parts of ethanol, incubated on ice and then centrifuged for 30 minutes at 12,000 rpm. The precipitated nucleic acids were then dissolved in distilled water (free of RN-az and DN-az), and stored at -20 °C. Fragments of 16S rRNA were amplified by polymerase chain reaction (PCR) conducted with bacterial primers (27F; 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R; 5’-TGACTGACTGAGGYTACCTTGTTACGACTT-3’). PCR was performed in 20-µl volume using GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) as follows: 4 min at 94°C, then 30 cycles of 1 min at 94°C, 1 min at 50°C, and 1.5 min at 72°C, then 7 min at 72°C. PCR amplicons were subjected to electrophoresis and purification using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Purified amplicons were cloned using pCR2.1 vector, E. coli culture, and TA cloning kit (Invitrogen) in accordance with the manufacturer's recommendations. From a daily culture, plasmid DNA containing 16S rDNA was obtained using a Mini prep spin kit (Quiagen, Crawley, UK). Purified DNA plasmids were sequenced on the ABI PRISM 3100 Genetic Analyzer using the Big Dye Terminator cycle-sequencing kit (Applied Biosystems). Amplified products 27F-1492R were sequenced in both directions with primers 27F, 357F (5'-CTACGGGAGGCAGCAG -3'), 520R (5'-ACCGCGGGGTGCTGGC-3'), 920F (5'-AAACTCAAAGGAATTGACGG-3'), 1080R (5'-CCCAACATCTCACGAC-3') and 1492R as described (Mori et al., 1997). The sequence length was 1488 bp. The resulting sequence was compared with others using BLAST (Altschul et al., 1997). The phylogenetic tree was constructed using the method of Saitou and Nei (1987), using the CLUSTAL W software package (Thompson et al., 1994). The 16S rRNA nucleotide sequence was deposited in DDBJ/EMBL/GeneBank under the number AB178889, identification number 20040510203204.24251.
RESULTS OF ISOLATION, STUDY OF GROWTH AND IDENTIFICATION OF MICROORGANISMS
A cultured bacterium capable of aerobic and anaerobic growth in YPD, MRS and NA media was found in frozen Miocene sediments on Mammoth Mountain; the optimal growth temperature was determined at +37°C. The microorganism is psychrotolerant, because it is capable of metabolic activity at -5 ° C. The bacillus is a relatively large (1-1.5 x 3-6 microns) rod, which is connected in a chain in culture (Fig. 2) and is capable of forming round-shaped spores. It is immobile and has hemolytic activity, gram-positive. At a temperature of -5 ° C, the bacillus slowly (signs of growth were detected after 2-3 months) grew in both frozen and supercooled environments. The microorganism belongs to the genus Bacillus, but, apparently, is a new species. The greatest species similarity of the isolated bacillus was noted with Bacillus simplex, B. macroides, homology with 16S rRNA of which is 96-97%.
Fig. 2. Isolated strain of Bacillus sp.: a) Gram staining, b) growth at a temperature of -4 ° C for 5 months
The growth of bacilli at low temperatures was observed earlier (Ashcroft, 2000). It is known, for example, that Bacillus anthracis easily tolerates freezing (Luyet, Gehenio, 1940). However, the optimal growth temperature of the found bacillus is quite high. Despite the fact that it was able to grow in an artificial environment and at temperatures below 0 ° C, no visible colonies were observed on frozen samples. Bacillus spores are known to be the most resistant (Nicholson et al., 2000); thus, B. thuringiensis and B. macroides were found in amber with an absolute age of 120 million years (Greenblatt et al., 1999). Therefore, the finding of a living balilla in the ancient permafrost of Mammoth Mountain is not surprising in general. It is of interest that in the described case the greatest similarity is noted with the species B. macroides. How active its life is in the permafrost, however, is unclear; this also applies to microorganisms isolated from the ice of Central Yakutia and Alaska.
From the re-vein ice of Yakutia and Alaska with an age of about 25 - 40 thousand years according to the method described above, T.Katayama et al., 2007) isolated several types of microorganisms. Most of the isolated bacteria are gram-positive and close to Arthrobacter and Micrococcus spp., and fungi - to Geomyces sp. Many isolates were able to grow at -5 °C, but did not grow at +30 ° C, i.e. they were obligate psychrophiles. Interestingly, the DNA of methanogens of several groups was also found and identified in the alluvial frozen deposits of Yakutia. Their research is not finished and it is not yet clear whether we are dealing with live methanogens. However, incubation of these permafrost deposits brought positive results: methane emission was observed at -5 °C. Studies of methanogens seem promising, because they may be responsible for the high content of methane in permafrost deposits.
In the dungeon of the Institute of Permafrost named after P.A.Melnikov, at a depth of about 7 meters, a white mushroom mycelium was found on the walls. A similar mycelium is also observed on the walls of the Fox tunnel in Alaska. The identification of the isolated species (PF strain) performed by M.Tanaka was based on its morphological characteristics and analysis of the nucleotide sequence amplified by 18S rRNA. This fungus is close to Penicillium echinulatum and may represent a new species. Samples from frozen sediments were prepared together with samples of P. echinulatum strains obtained from a culture bank and incubated at temperatures of 25°C, 5°C and -5°C. The characteristics of spore germination and growth of PF strain from frozen sediments and IFO 7760 and IFO 7753 P. echinulatum strains at lower temperatures were different: PF strain grew relatively rapidly at -5 °C. Interestingly, at -5 °C, the isolated strain grew in Petri dishes, both in those where the medium crystallized, and in supercooled medium (potato agar). At the same time, it grew faster on a crystallized medium (Fig.3). The isolated strain of Penicillium echinulatum in the dungeon of the Institute of Permafrost named after P.I. Melnikov in Yakutsk, despite its adaptation to cold and food conditions, may well be modern, brought from the surface. In addition, this fungus grows only in aerobic conditions. Therefore, its ability to grow inside the ancient permafrost is doubtful.
THE NATURE OF LONG-TERM VIABILITY OF MICROORGANISMS
The molecular basis of the thermal stability of biological materials is an unsolved problem (Baker, Agard, 1994; Jaenicke, 1996; Levy, Miller, 1998). In general, protein stability is determined by the amount of free energy, G, for the "folded - unfolded structure" reaction under physiological conditions. Most proteins are characterized by a value of G = 5 - 15 kcal/mol (Alexandrov, 1975). The proportion of destroyed areas of the structure attributed to the proportion of ordered areas (k) is as follows:
where: G is the free energy of the transition from an ordered region to a random one, kcal/mol;
T - temperature, °K;
R is the gas constant, ~0.001989, kcal/mol*°K.
Fig. 3. Results of nucleotide stability studies. The half-life of cytosine at temperatures of 0 - -10 degrees Celsius for about 300 years is about 350 years
(according to Levy, Miller, 1998)
This expression can be used to approximate the lifetime of biological molecules. At the same time, it turns out that for G = 30 kcal / mol, the lifetime of bonds is about 300 years. For the period of temperature fluctuations of molecules of 10-8 - 10-9 seconds and G = 20 kcal / mol, their lifetime is less than a year. The maximum known value of the activation energy is approximately 45 kcal/mol; usually less (Alexandrov, 1975). These estimates are very approximate, but they also show how unstable proteins and DNA are. Data on the thermal instability of cytosine are given in the work of M. Levy and S. Miller (Levy, Miller, 1998, Fig. 3). In this sense, our data on the lifetime of the most resistant among the viruses of the smallpox virus are interesting. It is shown that it coincides with the above calculated data and is several hundred years old (Repin et al., 2000). Unlike viruses, bacteria are living objects. They are capable of metabolic activity both without the presence of nutrients in the medium and at low temperatures (Repin et al., 2007). Evolutionarily significant transformations have been noted for non-dividing cultures (Cairns et al., 1988). Of particular interest, of course, are microorganisms that persist in natural conditions at low temperatures for extended time periods (Repin et al., 2007; Greenblatt et al., 1999). The possibility of combinatorial transformations predicted earlier is traced (Zavarzin, 1979). It is impossible not to mention in this aspect the existence of such biocatalysts as ribozymes, whose activity is associated with the origin of life and the world of RNA (Lutay et al. 2007). In our case, it may be important that the ribozymes are stable and active at temperatures below 0 °C. A large variety of relict microorganisms isolated from an ice vein aged 25-40 thousand years may be explained by the encoding of the long-term viability property by mobile genetic structures such as plasmids.
Thus, the long-term existence of living microorganisms is difficult to explain by the slowing down of vital activity during suspended animation. Apparently, it is due to the presence of unique repair mechanisms that support cellular structures.
TESTING OF BACILLUS CULTURE ON HIGHER ORGANISMS
Testing on Drosophila melanogaster flies
Drosophila melanogaster individuals of the same age (1 day) were selected for the experiment. They were placed in tubes with a nutrient medium (5-7 ml) of 5 pairs. The sample size for each variant was 100 flies. The selection of individuals for the experiment was carried out by etherealization, dead and surviving flies were taken into account every third day. The experiment was carried out with a daily culture of Bacillus sp., strain 3M, grown on meat-peptone broth. A culture in the volume of 20 µl was introduced into the test tube. The experiment included a control variant in which flies were kept on a medium with yeast added; and an experimental variant when the first 5 days of flies were kept on a medium with yeast added, then 1 day on a medium with a bacillus (alternating the entire observation period). To determine fertility, virgin flies were selected on the day of departure. Females and males were placed separately and kept for 5 days after they reached maximum fertility. Then they were placed in pairs in test tubes with removable lids. The bottom of the lids was filled with agar-agar confectionery with added sugar. Fertility was recorded daily for 6 days. The number of eggs laid was taken into account, and the number of undeveloped eggs was determined a day later.
When 75 µl of strain 3M was added to the surface of the nutrient medium in saline solution (1 billion cl/ml), a 5-fold decrease in fertility was observed in relation to the control. The average fertility of the female in the control was 58.1 ± 8.61 pcs., in the experimental version this value was 10.2 ± 3.44 pcs. In another experiment, a liquid culture of strain 3M was tested in a meat-peptone broth, the culture age was 1 day. Pure meat-peptone broth was used as a control. Low fertility was observed in both variants (in the control – 8.1 ± 0.82 pcs., in the experiment - 20.5± 3.30 pcs.).
When adding bacterial culture after keeping flies on yeast, despite the death of flies in the experiment and control after 50 days, there was an excess of the proportion of surviving flies from the 24th to the 42nd day of the experiment compared with the control (Fig. 4).
Rice. 4. The effect of Bacillus sp. culture, strain 3M, on the lifespan of Drosophila melanogaster
Testing on laboratory mice
Preparation of the bacterial culture was carried out similarly to the method of testing on fruit flies; a daily culture of Bacillus sp., strain 3M, was used, however, it was frozen and thawed before administration. The experiments were carried out on F1 CBA/Black-6 mice, with an average of 15 individuals in each group. In the first series of experiments, the effect of culture doses on the parameters of the immune system of young animals (age 3-4 months) was studied. Two groups of animals were used in the control, one of which was intact, and the second group was injected with a saline solution. Bacterial culture was administered to animals once intraperitoneally 5 000 (1); 50 000 (2); 500 000 (3); 5 000 000 (4) and 50,000,000 (5) microbial bodies (mt) per animal. In the second series of experiments, the effect of bacterial culture on the physiological and behavioral reactions of "elderly" mice (age 17 months) was evaluated, while the culture was administered once intraperitoneally at a dose of 5000 m.t. The control group was represented by animals of the same age. Euthanasia of animals was carried out by dislocation of the cervical vertebrae. According to standard methods, morphophysiological activity of the thymus and spleen was assessed by the organ index (the ratio of organ weight to body weight, %), the activity of nonspecific immunoresistance factors by the level of phagocytic (AF, %) and metabolic (HCT test, %) activity of splenic macrophages, cellular immunity in the reaction of HRT in vivo according to Crowle (1975), activity of humoral immunity by the number of antibody-forming cells (AOC) in 1 million nucleated cells in the spleen according to Cunningham (1968), animal muscle strength in the load lifting test (Ostrovsky M.M., 1970), behavioral reactions in the Open Field test (Buresh Ya. et al., 1991), life expectancy, as well as other indicators.
It turned out that the dose of Bacillus Bacillus sp., strain 3M in 5,000 microbial bodies contributes to an increase in the indices of the thymus and spleen (Fig. 5).
Rice. 5. The effect of Bacillus sp. culture, strain 3M with parenteral administration of various numbers of cells on the indices of the thymus and spleen of 3-month-old laboratory mice
Culture of bacilli in a small dose (5000 mt.) stimulate, and in medium doses (500,000 and 5000000 mt.) – suppress phagocytic activity of splenic macrophages. The culture of bacilli in almost all doses increases the activity of humoral immunity, and a dose of 5000 m.t. contributes to an increase in the functional activity of both cellular and humoral immunity.
In this regard, a dose of 5,000 mt was chosen for studies of the influence of culture on life expectancy..The minimum life span of mice from the control group was 589 days, and the maximum was 833 days. The minimum life span of mice from the experimental group was 836 days, and the maximum – 897 (Fig. 6).
Rice. 6. The effect of Bacillus sp. culture, strain 3M with parenteral administration of 5000 cells on the life expectancy of laboratory mice aged 17 months
The body weight of the animals of the experimental group was higher than that of the animals of the control group 2 months after the introduction of the culture. Muscle strength in experimental animals increased (about 60%) relative to peers from the control group. The increase in the ability of animals to orient themselves in space and research activity was evidenced by more frequent visits to the inner sectors of the open field, an increase in the number of vertical racks and visits to minks. Apparently, bacterial culture with parenteral administration stimulates the immune system and improves the emotional stability of laboratory animals. An increase in the lifespan of mice testifies to the possible presence of geroprotectors in the bacterial culture of Bacillus sp.
It should be emphasized that studies of the properties of cultures of relict microorganisms should be considered very preliminary both in terms of their volume and formulation. It is obvious that the ability of microorganisms from permafrost to maintain viability for thousands and millions of years can hardly be transferred or used without understanding its mechanism. It is important to note that such a task is not only fundamental, but also of exceptional practical importance in the future.
conclusions
1. Isolation and identification of relict microorganisms from permafrost is an independent task for both microbial ecology and taxonomy in connection with the content of new species. The nature of their long-term viability for thousands and millions of years in permafrost at temperatures around -2 - - 8 ° C needs to be investigated.
2. Isolated from the permafrost of Mammoth Mountain, which is about 3.5 million years old, and identified by 16S rDNA strain 3M Bacillus sp. it belongs to the group of psychrotolerant microorganisms and has a high resistance to adverse environmental factors. For permafrost of a younger age (25-40 thousand years) the content of a large group of microorganisms, including fungi, is characteristic.
3. The reduction in mortality of Drosophila melanogaster flies in individual experiments, as well as laboratory mice, combined with stimulation of the immune system and improvement of the physical condition of the latter, makes it possible to consider the research of relic microorganisms in gerontology promising.about the prevalence of grooming activity.
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ACKNOWLEDGEMENTS OF THE AUTHORS
We are grateful to Academician V.V.Vlasov (Institute of Chemical Biology and Fundamental Medicine SB RAS), Professor V.N.Anisimov (Institute of Oncology named after P. Williams (Scott Polar Research Institute, Cambridge, UK) for supporting this work and useful discussions, employees of the Ministry of Ecology of the Republic of Sakha-Yakutia for assistance in field work, as well as employees of Hokkaido University and Tsukuba University, Japan for assistance in microbiological research.