Дата публикации: 20 октября 2018
Автор(ы): Mikhail VAINSHTEIN, Natalya SUZINA, Tatyana ABASHINA
Публикатор: Шамолдин Алексей Аркадьевич
Номер публикации: №1540051663

Mikhail VAINSHTEIN, Natalya SUZINA, Tatyana ABASHINA, (c)

by Mikhail VAINSHTEIN, Dr. Sc. (Biol.), Pushchino State University, RAS Institute of Biochemistry and Physiology of Microorganisms (IBPM) named after G. K. Skryabin, Natalya SUZINA, Cand. Sc. (Biol.), Tatyana ABASHINA, Post-graduate, Pushchino State University


Bacteria belong to one of the least studied groups of organisms on the Earth. We know least of all about the minutest of them, with their dimensions on or below the border of the light microscope resolution. Discovery and isolation of nanobacteria is of importance to medicine, for they are supposed to serve as the center of attraction for and the reason behind mineral deposits formed in man's body, including renal sand and calculus.


The number of known species serves as an important indicator of the level of research carried out into various high-ranking taxonomic biological groups. The indicator is in the amount of about 1 mln for insects in the animal kingdom and of 200,000 - for gymnosperms in the vegetable kingdom. And today less than 10,000 species of the bacterial superkingdom are known to us. The fact is that the genetic analyses of samples, made, for instance, for soil populations, have shown that bacterium cultures that are on display in all collections of the world account for less than a half of the existing natural variety. Consequently, most of the species of these microorganisms are so far terra incognita for us.


For a long period of time specialists labored under the delusion that bacteria not isolated in cultures may be revealed by light microscopy methods (the limit of its resolution - 0.2 μm). In other words, if you cannot study them, you may at least see them, for most of the bacterial cells have linear dimensions of about 2 - 5 mkm, and this provides an opportunity for optical monitoring in microbiological works. Small-sized forms were neglected due to the methodic advantages of work with relatively large cells. So, if discovered, small forms were regarded as something out of the ordinary. And those discovered in natural surrounding were described as extremely exotic, as "elementary bodies", "subunits", "ultramicrobacteria", "filterable bacteria forms", "dwarf bacteria" or "nanobacteria" ("nano" in this case means that it would be more suitable to describe cell dimensions in nanometers, not in microns, not that the cells are by three orders smaller than their larger counterparts). Theoretical calculations published by various authors assume the existence of viable cells of 0.14 - 0.20 μm in diameter, i. e., invisible in the light microscope.


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Nanosymbiont from permafrost samples: A - an electron-dense symbiont cell between two ordinary-sized bacterial cells; B - a nanocell with a grown hypha; C - a cell with a hypha and a daughter cell being formed on it.


The term "nanobacteria" was coined at the close of the 1980s (to be more precise, American microbiologist Richard Morita was the first to use it in 1988), and it gained broad currency in the late 1990s, when it was frequently used by Finnish microbiologist Olavi Kajander in his works. He discovered such cells inside small-sized renal grains of sand in urolithiasis patients. American geologist Robert Folk also used the term in describing similar cells in the samples of sedimentary rock. At the same time, judging by biological writing, interest in viable small-sized forms was displayed earlier too, and it periodically increased in connection with some of other discoveries of importance to medicine: in the late 1920s for description of formation of nanocells inside tubercular bacillus, or in the 1930s - 1950s when its was discovered that "elementary bodies" were formed after bacteria had been treated with antibiotics, and later, in discussion of the mechanism of transmission of infectious diseases by the respective filterable forms.


In the classical Russian textbook on microbiology by Vasily Omelyansky (1924) the author mentioned the minutest bacteria known at that time, such as Pseudomonas indigofera - 60 nm, Spirillumparvum - 100 nm, Micrococcus progrediens - 150 nm, Bacillus radicicola - 180 nm (all those names are invalid now). Since the electronic microscope was yet to be designed at the time, the dimensions of the micro-objects under study were to a considerable degree determined on the basis of the diameter of the pores through which they passed in filtration.


Even now there are no pure nanobacterium cultures in the practice of microbiological laboratories. Olavi Kajander with a group of co-authors isolated nanobacteria from blood serum and German researcher Karl Stetter with colleagues obtained a symbiotic (unable to grow independently, without a partner) culture of another nanoprokaryotype - nanoarchea. Neither of them grow in synthetic or simple organic media, so their cells' small size is probably due to the loss of synthesis functions required for certain compounds.


Research into isolation of nanobacteria is successfully carried out at the RAS IBPM laboratory of structural-and-functional adaptation of microorganisms, led by Vitaly Duda, Dr. Sc. (Biol.). We have managed to obtain symbiotic cultures of nanobacteria growing together with satellite bacteria from tests of permafrost sediments. Those symbionts have a budding ability with formation of hyphas, i. e., mycellial filaments, with daughter cells growing next to the mother cell on them. Such hyphas, a rare form as it is, is to be met among bacteria of ordinary size (for instance, Caulobacter). Just as the symbiont discovered by us, some of them may attach themselves to biological objects.


Judging by studies devoted to the subject, the possible loss of a part of the synthesis functions does not rule out nanobacteria's great activity in mineralization processes. For instance, Robert Folk described his finds largely precisely in mineral concentrations. Olavi Kajander and his colleagues have come to the conclusion that renal sand and calculus are formed under the strong effect of nanobacteria serving as mineralization centers. The Finnish researchers' hypothesis was followed by a wave of publications with the idea expressed that it was precisely microorganisms that provoked most varied sediments in man's body - up to atherosclerotic plaques. At the same time, this theory fails to account for the way the biostruc-


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Nanobacteria in placenta calcification: A - a microlacuna in placenta tissues, a black (electron-dense) formation - mineral sediments; nodular cells - nanobacteria; B, C - ultra-thin sections of nanobacteria showing the presence of cell membranes (M) and nucleic acids. Photo from "Frontier Perspectives"(USA), 2003


ture, immured as it is in a grain of sand and isolated from the environment, manages to take part in such processes.


We carried out research into calcification in the placenta of women who had given birth to babies. Such mineral formations represent dangerous pathology that may not only add to the placenta's thickness but also lead to the fetus' death. In the course of our research we discovered nanobacteria in calcification zones and their absence in healthy tissues. In the former case they really contribute to mineral sediment formation in tissue microlacunas probably due to the medium alkalization. It is difficult to diagnose microbiological calcification agents, for they have so far been insufficiently studied.


On the basis of the foregoing the question of nanobacteria status is of great importance: are they a radically new taxonomic group or new species of the known groups, or temporary small forms of known species? The strains, deposited by Olavi Kajander and his colleagues at the German Collection of Microorganisms, have been classified by researchers as members of a new genus in the known large group of proteobacteria. The same is true of the symbiotic nanoarchea described by Karl Stetter et al. The symbiotic nanobacteria we have isolated from permafrost probably also belong to a new species or genus. Since the genetic analysis of the said microorganisms is made with the help of known primers (molecular markers), this testifies to the nanoforms' similarity to known bacteria and to the absence of new features in their phylogenesis. At the same time nanoforms may be represented by new species or genera, and that's only natural, for over half of the existing bacteria have not yet been isolated into cultures or identified.


The possibility of temporary formation of small forms, or nanobacteria, has been proved for many bacterium species known in cultures. Research carried out in Russia back in the 1920s and subsequently in the 20th century proved the reality of heteromorphism and formation of filterable forms in many bacterium species. "Heteromorphism is a


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form of biological reactions by bacteria contributing to the species' survival and preservation under the short-term unfavorable effect of certain physical, chemical or biological factors," wrote Academician of the USSR Academy of Sciences and of the USSR Medical Academy Vladimir Timakov* and Professor Henrietta Kagan in 1973. "Heteromorphic forms may die or decompose into sub-microscopic filterable forms as a result of those factors' durable effect or of a greater dose. Upon the removal of the effect that caused the formation of heteromorphic forms, they may easily revert to the initial bacterium species."


Depending on the nature of the effect, we may speak of the microorganisms' gradual process of growing smaller, formation of smaller cells inside the maternal cell or about multiple division of one cell into numerous small ones with their subsequent growth. The last process is possible owning to the multicopy nature of DNA: one bacterium cell may contain more than one double-chain DNA, i. e., their great number sufficient for a number of independent cells.


Typical is the example of anaerobic polysporous bacterium Anaerobacter polyendosopus, discovered and described by Vitaly Duda and his colleagues. One such cell can form up to eight spores, i. e., contains at least eight DNA sets. Consequently, nanobacteria represent in nature not only new species and genera, but also small forms of those already known ones.


The fundamental significance of research into nanobacteria is only too obvious, for we are speaking about a large group of microorganisms, which have been overlooked by microbiologists making use of light microscopes, and about the mechanism of launching the process of multiple division of prokaryotic cells. At the same time the special features of the above-mentioned biological structures assume the application of new technologies. Let's discuss a few of them by way of example.


Diagnosis of the presence of pathogenic nanobacteria in the body is not yet developed although it is absolutely necessary. It may be developed without isolation of their pure cultures, for instance, on the basis of the antibodies created with fluorescent marking.



* See: N. Bochkov, "Microbiology Opened Up Its Secrets to Him", Science in Russia, No. 4, 2005. - Ed.


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Emergence of iron-based globular magnetic nanoparticles (indicated by arrows) from purple bacteria cells R. PALUSTRIS (scale section - 0.5 μm). Photo from "Biology of the cell" (USA), 2002


Globular magnetic cobalt-based nanoparticles (indicated by arrows) in purple bacteria cells RHODOPSEUDOMONAS PALUSTRIS (scale section - 0.5 μm).


Regulation of the ordinary to nanoforms ratio is of interest. Ordinary forms produce humus keeping moisture in the soil and the latter form grains of sand as mineralization centers. The microbiote of the desert sand, according to Imre Friedman, director of the Desert Research Institute of Florida University (USA), is practically represented solely by nanoforms. Hence the conclusion: the replacement of ordinary bacteria by them is accompanied by land desertification, which should be taken into account by specialists in many countries.


Formation of a great quantity of nanoforms leads to growth of the total area of cells upon their impact with the surface of insoluble substratum. In our experiments transformation of ordinary bacteria into the said forms substantially added to the process of fermentation of insoluble starch, also contributing to the output of soluble intracellular enzymes into the medium. Such fermentation of insoluble substrata is applied in practice, for instance, in alcohol production.


Multiple division of bacterium cells leads to increase in their total quantity or, in microbiological lingo, the number of colony-forming units. They add to the effectiveness of bacterial preparations intended for alimentary tract microflora regulation in cases of such pathology, as well as for animal fodder additives.


Development of biomedical technology with the use of magnetic nanoparticles has good prospects for the future. They are used as vehicles for antitumoral agents without any harm to healthy cells. If such preparations are introduced in the blood, with a magnet located next to the tumor, the medicine-carriers will be concentrated at the affected area without any damage caused to the whole body. This method has not been so far applied on a broad scale due to the high cost of obtaining products of high purity and perfectly safe in biological respect. The fact is that the method was based on man-made micro- and nanoparticles of iron oxide or magnetosomas of bacterial origin, which are obtained with great difficulty due to the low productivity of cultivation of a special group of magnetotactile bacteria. However, in the past few years the associates of the RAS IBPM discovered jointly with the Puschino State University researchers a radically new type of magnetic nanoparticles of bacterial origin (magnetic globules). A working collection has been produced of required cultures with highly productive particles of this kind, with methods elaborated for the latter to be removed from the cells.


We should like to point out in conclusion that nanotechnologies and nanomaterials were included in the official list of Russia's critical technologies, approved by the President of the Russian Federation in 2006. They have also been included in the list of priority trends of development of science and technology in our country. Biotechnologies with the use of nanobacteria should also occupy their place among them.

Опубликовано на Порталусе 20 октября 2018 года

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