By Alexander BORONIN, RAS Corresponding Member,

Vitaly DUDA, Dr. Sc. (Biol.),

Natalya SUZINA, Cand. Sc. (Biol.), G. Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS

The ideas of the biological diversity and molecular genetic bases on the biological cell structure, functioning, and evolution have changed appreciably due to the progress in studies of the world of microorganisms, attained during the recent decade.An important result of studies of many new and rare microbiotic species is an ample information on the range of bacterial cell sizes; ultrasmall forms attract special attention of specialists.

NOT RANDOM INTEREST

Today the largest bacteria discovered are Thiomargarita namibiensis, a giant spherical aero-biobacterium, - 750 μm in diameter, and Epulopiscium sp., an anaerobic bacillus, - 80 μm diameter, which are larger than even some multicellular eukaryotes*. It is noteworthy that previously it was assumed that an extremely small diameter of a prokaryotic cell

* Eukaryotes - uni- or multicellular plants, animals and some microorganisms, whose cell body is differentiated by cytoplasm and nucleus, limited by a membrane. Prokaryotes are microorganisms that have no typical cell nucleus and chromosome system. - Auth.

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can serve as the main phenotypical distinction of bacteria. By the way, it has become known recently (from publications by Claude Courties et al. in 1994 and 1998, as well as by Motomichi Matsuzaki in 2004, from the Tokyo State University, Japan) that the smallest one-cell independent eukaryotic organisms (Ostreococcus tauri and Cyanidoschizon merolae algae) are not larger than 0.6 - 2.0 μ. The smallest prokaryotes are Mycoplasma genitalium and Pelagibacter ubique bacteria, which are < 0.3 μm (300 nm). Let us note that the largest virus is 0.4 μm, while the majority of bacterial cells are 1 -2 um in size.

The interest of scientists in ultrasmall bacterial forms is increasing, as the interpretation of the phenomenon of bacterial cell minimization and existence a world of such organisms in nature became a complex fundamental problem, which has applied and gnoseological significance. The solution of this problem is closely connected with the progress in at least six priority trends of research. The aim of the first of them is to find out the percentage of ultrasmall organisms in the microcenoses of various natural media and their role in the substances turnover in nature. The second trend is aimed at investigation of the bacterial species diversity, distinguishing heretofore not cultured microorganisms, and revealing metabolic processes, which remain unknown. The third one is aimed at approaching the discovery of the mystery of life emergence on the Earth and obtaining new data on its evolution. The fourth extremely important trend of research is determination of the minimum set of genes and a complex of the main metabolic processes providing the reproduction of prokaryotic cells, which will be significant for the creation of an artificial living cell. Fifth, we should remember about scientific search for extraterrestrial life. And finally, the sixth important task is creation of model ultramicrobacterial species fit for use in biotechnology (genetic engineering, microbiological industry, nanotechnologies).

"DWARFS", "LILLIPUTIANS", OR NANOBACTERIA?

The term "ultramicrobacteria" was for the first time used by scientists Francisco Torella and Richard Morita in 1981 to denote extremely small bacteria with cells of < 0.3 μm in diameter, isolated from sea water. In 1982 Frits Shut et al. introduced the threshold value of cell volume (< 0.3 μm³), irrespective of the geometrical shape of the bacterium, as the most important and accurate sign of these organisms. Using this criterion, new species of independent saprotrophic (neither parasitic nor symbiotic) ultramicrobacteria were isolated and described: Sphingopyxis alaskensis, Pelagibacter ubique, and an actinobacteria under the laboratory number of MWH-Tal (the species name is still not specified). Thus, the new notion is distinguished on the basis of the metric and taxonomic criteria.

The term "nanobacteria" is sometimes used as a synonym of the discussed term. However, the majority of scientists refer to nanobacteria the smallest representatives of ultramicrobacteria with cell volume of - 0.004 μm³. A typical example is Mycoplasma genitalium, a pathogenic mycoplasm without a cell wall.

In geological literature supersmall bacterium-like particles (structures) in rocks and meteorites are called nanobacteria, in medicine this term is used for description of hypothetical obligate parasites detected in human tissues, serum, and organs and presumably causing atherosclerosis and formation of renal stones. However, many scientists do not share this opinion as there are no sufficiently exact proofs in favor of existence of these pathogenic organisms. Claims on discovery of nanobacteria with cell size of - 50 μm, containing no DNA and ribosomes, are doubtful, and application of the strict term "bacterium" to these particles is not correct.

Hence, there are no universally acknowledged terms and notions in this sphere of research. Some authors call ultramicrobacteria "dwarfs", "Lilliputians", minicells, etc., but the latter term is doubtful, as microbiologists and geneticists use it to denote nucleus-free cells forming as a result of abnormal cell division. In addition, minicells of not all bacterial species are so small. By contrast, the prefix "nano-" is quite justified for cells of ≤ 0.3 μm in diameter, occurring in natural material or bacterial cultures.

What are the advantages of ultramicrobacteria? First, huge surface area (area of the envelopes absorbing nutritives and releasing metabolites) of these miniature cells promotes their better development in natural media with low concentration of nutrient ingredients. Second, due to ultrasmall size these creatures can penetrate through supersmall pores and locuses of natural substrates and into larger cells of other living organisms, thus promoting development of niches, inaccessible for other creatures. And, the last but not the least, ultra-small cells are more effectively reproduced and populate the natural substrates, thus, no doubt, promoting their survival.

METHODS FOR INVESTIGATION OF AN UNUSUAL OBJECT

Now it is just good time to recollect the prophetic words of the great Russian scientist Mikhail Lomonosov: "Due to the microscope, many mysteries have been discovered - invisible particles and fine veins in the body". Using direct microscopic methods (primarily electron microscopy, showing objects invisible in an optic microscope; using this method, the scientists study the fine structure of these objects), Russian and foreign scientists A. Kriss (1948), D. Nikitin et al. (1964, 1966), from the RAS Institute of Microbiology, L. Casida (1969), H. C. Bae et al. (1972, 1973), from the Pennsylvania State University, R. Morita (1985) from

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Photographs of NF1 strain ultramicrobacterial cells (under-electron microscopy, length of the scale 300 nm): CL: capsular layer, N - nanocells, OM - outer membrane, P - periplasm, Pp - polyphosphate granules, B - bud, BPS - bodies with periodical structure, CM - cytoplasmic membrane, CC - central cell, SA - spheroid aggregations of nanocells; a - ultrathin section of spheroid aggregations ofnanocells developing in oil slime, b - fragment of peripheral part of two spheroid aggregations with fine structure of compartments and separating spherical nanocells, c - budding cell, d - formation of cell aggregations, similar to spheroid ones, shown in Figs, a and b.

NF1 strain cells, chains and grapes of budding nanocells. Phase-contrast photography (scale length - μ10 pm).

Ultrathin section of NF3 strain cell. SP - spherical protrusions on cells. Black granules - bodies with periodical structure (scale = 0.5 μm).

Oregon State University, Frits Shut et al. (1977) from Microscreen BV (Groningen, Netherlands) demonstrated the presence of numerous ultrasmall cells in natural media (soil, silt, sea and lake water). The greatest breakthrough was made by the US scientists H. C. Bae, L. Casida, and D. Balkwill (Pennsylvania State University, USA): using an original method of microorganism fractionation, they characterized in detail ultramicrobacteria in situ in soils of different types and thus proved the existence of cells smaller than 0.3 μm in these soils. The

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Scheme of cell cycle variants (1 - 4) for NF1 and NF3 strains. Blue circles - initially existing individuals, red circles - cells formed DE NOVO by budding of mother cells. 1 - formation of a large "final" cell through budding of an intermediate small cell; 2 - formation of a cell equal by volume to the initial (maternal) cell; 3 - formation of two buds simultaneously on mother cell; 4 - formation of three buds simultaneously on mother cell; 5 - development cycle of an indigenousultramicrobacterium in soil.

number of these cells reached about 60 percent of total number of cells in the samples.

The nature of these microorganisms (taxonomic appurtenance, physiology, phylogeny, etc.) remained unclear for a long time. Only in 1997 P. Janssen et al. from the Max Planck Institute (FRG), and Takashi Iizuka et al. from Ajinomoto Co. (Kawasaki, Japan) in 1998 isolated and characterized soil ultramicrobacteria in pure cultures, including the representatives of new species, which have not yet been classified.

Indigenous (natural, in situ) forms of microorganisms in soils, oil slime, and permafrost soil were studied at the Laboratory of Structural and Functional Adaptation of Microorganisms of our Institute. In order to clear up the ultrastructural organization of their cells, we used new methods and approaches, including spot electron microscopy*, study of soil micromonoliths, and low-temperature method for microorganism fractionation (cell separation) in combination with fluorescent** and high-resolution transmission electron microscopy, which allowed to identify cell ultrastructures. Due to these original approaches we managed to establish that the percentage of ultramicrobacteria in oil slime and permafrost soil amounted to 30 - 40 percent of total content of bacteria. However, one should strictly differentiate between cells and such cell-like structures as cytoplasm fragments, aggregations of organic macromolecules, vesicles of external membranes of gram-negative bacteria, minicells, etc.

Electron-microscopic study of ultrathin sections of fractionated indigenous microorganisms and oil slime micromonoliths showed 7 morphological types of ultrasmall bacteria, including one bacterium of unique ultra-structure and multiplication type, with coccus-like cells of - 200 - 300 nm diameter (sometimes - 180 nm - less than 0.2 μm - virtually indiscernible in a common optic microscope). These cells formed spheroid aggregations, separated from other bacteria by large capsules. Analysis of these formations showed that the majority of specimens in them were at the stage of division, while a part of small peripheral cells separated after the formation of their membranes was over and were released into the environment. After maturing these aggregations disintegrated into individual cells or elements consisting of 2 - 4 cells.

Thus, free-living ultramicrobacteria are really present and developing in slimes and soils.

IT IS IMPOSSIBLE TO UNDERSTAND THE NATURE OF THE ORGANISM WITHOUT KNOWING ITS STRUCTURE

It is obvious that the role of an ultramicrobacterium in the biosphere, its structural and metabolic varieties, and probability of realization of heretofore unknown biochemical processes by this microorganism can be evaluated only after isolation of this ultramicrobacterium in pure culture and characterization of its geno- and phenotypical properties. Using original methods, we isolated 15 strains of ultramicrobacteria, including representatives of various morphological, systematic and physiological groups of bacteria: Gram positive and Gram negative species, vibrios, cocci and bacilli, aerobic and anaerobic bacteria.

Strains NF1 and NF3 were studied best of all. The former was isolated as follows: spheroid aggregations of ultrasmall cells were transferred (under a microscope) from slides on the surface of sterile agarized oil slime, serving as nutrient medium. First, enrichment culture was grown in this medium, and then pure culture was

* Spot electron microscopy implies the use of a complex of preparative methods due to which it is possible to examine a certain microscopic locus in natural substratum. - Auth.

** Fluorescent electron microscopy is a study of cell fluorescence stimulated by exposure to rays of a certain spectrum. - Auth.

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Lyzed cyanobacterial (Chlorogloeopsis fritschii) cells (LC) under fluorescent microscope. BC-NF1 strain bacterial cells. DAPI fluorochrome treatment (scale on figures = 5 pm).

obtained after several passages onto agar-containing medium.

The strain NF1 characteristic diagnostic signs are as follows. It has budding cells, the surface of which is covered by spherical or conical protrusions. In addition, it forms spheroid aggregations and on agar media forms characteristic wax-like round convex mucous colonies of 2 - 5 mm in diameter. Due to this information we isolated one more pure ultramicrobacterial culture (strain NF3) from aqueous culture of the Pedilantus tithymaloides roots.

Both strains form two cell types in synthetic carbohydrate media: large oval cells 400*800 nm (0.4 - 0.8 μm) in size and ultrasmall cocci (rarely bacilli) of - 300 nm (0.3 μm) in diameter, penetrating through membrane filters with 0.2 - μm pores and multiplying by budding. One to three budds emerge directly on parental cells, no hyphaes being formed between them.

The structure of cell membrane in these strains is characteristic of gram negative bacteria and consists of an outer membrane, mureine layer*, and periplasmic space**; the cell is enveloped in a fine microcapsule, particularly pronounced for cells co-cultured with cyanobacteria.

The periplasmic zone is locally dilated, due to which the cell surface has spherical and conical protrusions - periplasmic prosthecaes***. These are sites for the formation of buds or extracellular vesicles, enveloped in the outer membrane, and for contact with the cyanobacterium cell surface (the development of ultramicrobacteria is closely connected with them; we shall speak about it later).

The nucleoid zone and reserve inclusions are easily discernible in the cytoplasm: poly-β-hydroxybutyrate and polyphosphates. In ultrathin sections the former look as spheroid electron-optically transparent bodies surrounded by a thin unilamellar wall - 30 A thick. The latter more often look like spheroid electron-dense bodies located in the cytoplasm and nucleoplasm, with

* Mureine layer consists of peptidoglycane (mureine). - Ed.

** Periplasm is a cell compartment in gram negative bacteria, located between the cytoplasmic and outer membranes. - Ed.

*** Prosthecaes are cell protrusions due to which the bacteria increase the surface/volume ratio; this helps cells survive in media with low content of nutritives. - Ed.

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Ultrathin sections (a, b, c). Ultramicrobacteria adhere to and incorporate in CHLOROGLOEOPSIS FRITSCHII cyanobacterial cells (mark on the figure = 0.3 &mu;m. BC - NF1 strain bacterial cells, EL - external layer of the envelope, IL - internal layer of cyanobacterial envelope, C - capsule (scale on figures = 0.3 μm).

granular structure, though sometimes their central part has an electron-transparent core.

In addition, unique crystal-like formations - bodies with periodical structure, representing stacks of compactly packed stab-like subunits - 30 A thick and up to 150 - 300 A long (according to cytochemical data, these are protein structures)werw detected in the cytoplasm. Mycoplasma pneumoniae terminal organelle, also characterized by structural periodicity, is the only analog of these bodies, described in literature. The functions of these bodies and of another uncommon structure, detected in NF3 strain cells (long, up to 50 - 60 nm, bundles of threads - 35 - 40 A thick) remain unknown. All these inclusions are present in the cells of spheroid bacterial aggregations as well.

One more unique ultrastructural feature of the isolated bacterium is its capacity to form large (up to 20 - 30 nm in diameter) electron-dense spherical bodies in the periplasm, particularly often seen in co-culturing with cyanobacteria. Let us note that we describe this intricate structural differentiation of the periplasm for the first time.

THEIR "VITAL STRATEGY"

NF1 and NF3 strains are heterotrophs, aerobes, catalase- and urease-positive, metabolizing a limited number of organic compounds: they utilize simple carbohydrates as a source of carbon, for nitrogen they utilize ammonium and nitrate or amino acids, polymers (cellulose, pectin, agar). They do not digest proteins (gelatin, casein) and need growth factors added into the media: yeast extract, vitamins (pyridoxine, thiamin, riboflavin, nicotinic, p-aminobenzoic, and folic acids, biotin, and nicotinamide). These strains produce cytochromes* a, b, and c, as well as a spectrum of fatty acids intrinsic to alpha-proteobacteria.

Incubated together with cultures of some cyanobacteria, ultramicrobacterial strains lyse them irrespective of illumination. Cyanobacteria Chlorogloeopsis fritschii, str ATCC** 27193, and Chlorogloeopsis sp,. str. S. whose cells have sheaths, are the most sensitive to this treatment. Strains Anabaena variabilis str. ATCC 29413, Nostoc museorum str. 3, and Spirulina sp. str. 287 are less sensitive.

The green color of cyanobacterial biomass is reduced after addition of ultramicrobacteria, and after 15 - 20 days the biomass is completely bleached. In parallel with this we observed in an epifluorescent microscope reduction and then complete disappearance of red fluorescence of chlorophylls induced by ultraviolet irradiation of cells.

Fluorescent and electron microscopy showed that the first stage of lysis consisted in adhesion of ultramicrobacterial cells to the cyanobacterial cell surface. Then ultramicrobacteria penetrate into the sheath, first into its surface capsular loose layer, then into the internal electron-dense layer with lamellar structure. At the third (last) stage these bacteria penetrate inside, causing deep lysis of the host cell cytoplasm. It is obvious that carbohydrate-carbohydrate relationships are essential for cell-to-cell interaction, as at the beginning we observed a close contact between cyanobacterial capsular fibrils and ultramicrobacterial microcapsular fibrils.

It is essential that NF1 and NF3 grow well under conditions of co-culturing with cyanobacteria even in agar media for phototrophs (devoid of nitrogen and organic carbon sources) and retain viability during more than 12 months, but do not grow at all without cyanobacteria. Hence, they use nutrient elements (including vitamins) produced by cyanobacteria. A similar picture is observed during the growth of ultramicrobacteria on other prokaryotic cells, for example, on Bacillus subtilis. Thus, in this case we have facultative ecto- and endocellular parasites.

Let us note that microorganisms causing extracellular lysis of cyanobacteria (Bdellovibrio and some Cytophaga strains) were described previously, but not parasites penetrating inside their envelopes and into the cell cytoplasm, which have never been isolated and described up to the present time.

Analysis of nucleotide sequences of 16S rRNA gene showed high similarity (99.4 percent) of NF1 and NF3 strains, which seem to belong to the same species. By this character and by molar percentage of DNA guanine and cytosine NF1 and NF3 are very much similar to the type strain of a recently described Kaistia adipata species. It is most likely that these microorganisms should be

* Cytochromes are conjugated proteins, performing in living cells gradual transmission of electrons and (or) hydrogen from oxidizable organic substances to molecular oxygen. - Auth.

** ATCC: American Type Culture Collection. - Auth.

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referred to this species, but the final conclusion can be made only after we have data on the level of their DNA homology. It is noteworthy that in synthetic nutrient media the studied strains form, in addition to nanocells, large cells, with 3 - 4 - fold larger nucleoids. They can be regarded as undivided multicellular individuals. The representatives of K. adipata seem to be highly prevalent in nature: they were detected in oil slime from Tatarstan (NF1), near-root zone of Pedilanthus tithymaloides (NF3), in light chestnut soil of the Caspian lowland, and in the Lake Baikal silt (NF2).

We should also like to mention one more ultramicrobacterial strain NF4, isolated from the silt of the Lake Baikal. Along with rod forms, it produces ultra-small spherical cells. Phylogenetic analysis based on the data on the nucleotide sequence of 16S rRNA gene showed that this bacterium belongs to a new species of the Chryseobacterium genus (Phylum 'Bacteroidetes'), for which we suggest the name of Chryseobacterium 'nanocystis'

NEW PARADOXES AND "OPEN" QUESTIONS

At present the number of isolated and well-studied species of ultramicrobacteria differs much from the number of their free-living species detected by genetic methods in natural substrates. Closing this "gap", we shall better understand the variety of bacterial world and its role in nature.

Let us discuss the main properties of some cultured ultramicrobacteria. For example, Sphyngopyxis alaskensis is a free living heterotroph; the size of its genome is 3.2 Mb, cells are 0.2 - 0.5*1.0 - 1.5 μm large, their mean volume is 0.12 μm³, cell wall is of Gram negative type. A specific feature of cells of this species is the presence of the only operon* copy coding the formation of ribosomal RNA and not many (from 200 to 2,000) ribosomes in a cell.

One more species worthy of note is Pelagibacter ubique, a free living facultative heterotroph with a genome of 1.3 Mb in size, cells of 0.12 - 0.2*0.37 - 0.89 um in size, 0.01 μm³ volume, and Gram negative cell wall. Its characteristic feature is the absence of pseudo-genes**, introns***, transposon****, and extrachromosomal elements*****.

The third ultramicrobacterium we should like to mention is an MWH-Ta1 strain actinobacterium with heretofore unidentified species status. It is a free-living heterotroph. The size of its genome is not yet known. The cells reach 0.15 - 0.24*0.5 - 1.5 μm in size, their mean volume is 0.03 μm³. Cell wall is Gram positive, has an S layer (surface layer consisting of orderly positioned protein subunits).

Let us also mention Mycoplasma genitalium, a parasite with genome of 0.58 Mb in size and cells (without cell wall) 0.2 μm in size and 0.004 μm³ volume. The genome

* Operon is a site of genetic material consisting of 1, 2, or more linked structural genes, coding proteins (enzymes) realizing successive stages of some metabolite biosynthesis. - Ed.

** Pseudogenes are inert but stable genome elements emerging as a result of mutations in a previously working gene. - Ed.

*** Intron is a gene site carrying no information on the primary protein structure and located between coding sites (exons). Detected mainly in eukaryotic genes. - Ed.

**** Transposons (mobile genetic elements of eukaryotes) are DNA fragments capable of translocation in cell genome or between genomes and containing the genes of enzymes essential for transposition. Incorporating in various sites of chromosomes, they modify the genes' activity and cause mutations of various types, promoting instability and variability of the genome. - Ed.

***** Extrachromosomal elements are bacterial plasmids. - Ed.

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Ultrathin section of NF4 strain ultramicrocells.

of this ultramicrobacterium has just 468 genes coding for proteins.

The last species we should like to mention here is Nanoarchaeum equitans, a symbiont of hyperthermophilic archaea Ignicoccus. The size of its genome is 0.5 Mb, cells (with a wall consisting of just an S layer) are 0.4 μm in diameter, and their mean volume is 0.0033 μm³. This ultramicrobacterium has no lipid synthesis genes.

Hence, it is obvious that the well-known species of ultramicrobacteria differ greatly. Our data indicate that some of these microorganisms are characterized by new, heretofore unknown physiological properties and uncommon cell ultrastructures. The findings of American specialists, who studied sea ultramicrobacterium Pelagibacter ubique recently isolated in pure culture, confirmed our data (S. Giovannoni et al., 2005). During uncommon photosynthesis this ultramicrobacterium utilizes bacteriorhodopsin; its biomass in the seas is greater than that of all fish. P. ubique has the smallest genome of heretofore known free-living ultramicrobacteria (it ranks behind of only a few symbiotic and pathogenic prokaryotes, developing in nature only inside cells or tissues of other living organisms). As was mentioned above, the genome of this species is extremely compact, which is in agreement with the ultrasmall size of the bacterial cell.

Analysis of the characteristics of well-studied cultured ultramicrobacteria revealed a paradox: there is no direct correlation between the cell and its genome sizes. For example, recently described ultramicroarchea Nanoarchaeum equitans and P. ubique: the former has a very small genome and rather "large" cells, while the latter, vice versa, small cells and "large" genome. The causes of this disagreement are unclear. Presumably, some of these microorganisms have rather large nucleoids (prokaryotic analog of the cell nucleus) due to the many chromosomal repeats in them. Hence, the problem of the nucleus/cytoplasm ratio for various species of these bacteria remains unsolved.

One more problem which is still not clear is the probable existence of chemolithoautotrophic ultramicrobacteria and free-living anaerobic ultramicroprokaryotes devoid of cell wall and other envelopes. Discovery of these forms can make an important contribution to our knowledge of the structural, functional, and genomic diversity of the microbiotas and to the expansion of potentialities of biotechnology.

The need of profound studies of not only structural and functional organization of genomes, but also of ultrastructural and molecular cytology of ultramicrobacterial cells is obvious. If research in this direction progresses, we shall approach the solution of the megaproblem of modern biology: creation of an artificial living cell.

These studies were carried out with the financial support of the Russian Fund of Basic Research, grant No. 06 - 04 - 49463

Illustrations supplied by the authors