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, Pushchino State University

The majority of microorganism species inhabiting the Earth have not yet been isolated and studied. This indicates that our knowledge of the life on our planet is incomplete and drastically limits our potentialities to control biospheric processes and inhibits development of biotechnologies. Therefore, new information about predaceous ultramicrobacteria, realizing a unique heretofore unknown in biology method of cohesion (drawing together and strong coupling of parasite cells and victim cells) is particularly valuable.

Tens of earlier unknown in science representatives of the microflora were discovered in 2010, the year proclaimed the International Year of Biological Diversity by the UNO. Our ideas on the role of bacteria in the biosphere and involvement of microorganisms in the synthesis of compounds useful for man have been extended. Studying some ultrasmall (cell volume 〈0.3 µm³) bacteria* at the Laboratory of Microorganism Cytology and Plasmid Biology at our Institute, we have found out that this population includes true predators, parasitizing on other heterotrophic and phototrophic bacteria. They behave as epibionts–exist and develop tightly attached to the victim cells, captured beforehand in their "molecular network", like moths in a cobweb.

THE WAY PROKARYOTES PARASITIZE

Bacteria and archea, attacking and destroying living prokaryotic (without a true nucleus) cells, are considered parasites or predators, in contradistinction to triv-

* See: A. Boronin, V. Duda, N. Suzina, "Ultramicrobacteria", Science in Russia, No. 5, 2007.–Ed.

KAISTIA SP. NF1. Ultrathin section of cells. Pr-protrusions, EM-external membrane, P-periplasm, CM-cytoplasmatic membrane, N-nucleoid, M-murein layer in the cell wall.

ial pathogenic bacteria attacking eukaryotic (having a true nucleus) multicellular organisms.

The prokaryotic predators have been known in microbiology for a long time. In 1963 Heinz Stolp and Mortimer Star, American scientists, described Bdello-vibrio, an obligate parasitic bacterium developing inside cells and causing their destruction in some gram-negative* bacteria. In 1982, Veronika Lambina, Cand. Sc. (Biol.), from our Institute with a team of colleagues published an article about another bacterium with a similar vital strategy; it was referred to a new genus Micavibrio. This work has became the classics. The same year scientists from the laboratory headed by Prof. Lester Casida (University of Pennsylvania, USA) isolated from the soil and described two optional bacterial parasites from the previously unknown Ensifer and Cupriavidus genera. The discovery in 2002 by Harald Haber with his colleagues (University of Regensburg, Germany) of the first and by the present time the only representative of obligate  parasitic archaeus**, called Nanoarchaeum equitans, became an important event.

Isolation and description in 2008-2010 of new optional parasitic bacteria from Kaistia and Chryseobacterium genera by scientists from our Institute supplemented our knowledge of this predatory microorganisms.

Thus, the group of parasites attacking living cells and having no true nucleus by the present time includes organisms of different morphology: bacillary, coccoid forms, and vibrios. Philogenetically they are heterogeneous and do not form a single root, but are representatives of prokaryote branches far from each other-from orders to domains. They developed later than their philogenetic relatives (except, presumably, Nanoarchaeum equitans).

Our analysis has shown that the majority of these predators are ultrasmall organisms, except the representatives of Ensifer and Cupriavidus genera. The volume of Micavibrio aeruginosavorus ARL13 cells (the index here and further on indicates certain strains of this or that

* Gram-negative bacteria, colored with aniline dye-stuff with iodine fixation, due to the biochemical characteristics of their cell wall are decolorized if washed in ethanol–in contrast to gram-positive bacteria.–Ed.

** Archea are unicellular prokaryotes of the Archaea domain (upper level of grouping of organism in the system of scientific classification), differing significantly at the molecular level from bacteria and eukaryotes.–Auth.

CHRYSEOBACTERIUM SP. NF4 ultramicrobacterium (UMB) attacks BACILLUS SUBTILIS (B.s.). 1-20 mm, 2-12 h, 3-two days (intact bacillus cells and immature (dark) spores (S).

bacterium) is, according to our estimates, just 0.05 µm³. The Bdellovibrio bacteriovorus cells are not much larger (~0.13 µm³) and are 5-6-fold smaller than those of its victims, for example, Escherichia coli. The Micavibrio, Kaistia NF1 and NF3, Chryseobacterium NF4 and NF5 are also very small. Nanoarchaeum equitans obligate parasite can also be referred to ultramicroarchaea.

All these organisms have no problems with disposition in case of contact type parasitic interactions: the host cell can shelter and provide multiplication of not just one, but of several unwanted guests. In addition, it is easier to penetrate into small ecological niches for small predators.

An important distinctive feature of Kaistia genus NF1 and NF3 and Chryseobacterium NF3 and NF5 strains, isolated from various natural habitats, is immobility of their cells and absence of flagella and fimbria*. These structures play an important role in intercellular interactions and in our case might help the predators in search of victims and contacts with them. But, as we see, it is possible to do without them.

ULTRASTRUCTURE OF PREDACEOUS CELLS

Examination of ultrathin cell sections of these microorganisms shows that the structure of their cell wall corresponds to the gram-negative type: an external membrane, periplasmatic space**, and murein layer***.

The nucleoid inside is well discernible: a clear zone in the center filled by DNA fibrils. Each cell is surrounded by a capsule-like structure in the form of a rete of filaments and cords 5.5-6.0 nm and 18-35 nm thick, with ~25-nm granules, threaded on them at a distance of 50-300 nm. All these formations are well stained by inorganic dye-stuff Ruthenium Red (complex ruthenium ammonium chloride), which indicates their polysaccharide nature. The NF1 and NF3 strain cells have also round or cone-shaped protrusions. Electron microscopic studies have shown that these are special suckers attaching the predators to the victims. In other words, parasitic ultramicrobacteria form a "molecular cobweb" for capturing bacteria. True "nanospiders", aren't they?

Phase contrast**** and fluorescent microscopy clearly shows that in liquid media they "suck" to the victim very rapidly–during the first minutes after mixing of suspensions. Up to 10, and sometimes even more nanospiders of this kind can be located on one host cell, often almost completely covering it. It has been established that the "aggressors" "stick" to the victim very strongly, so that they cannot be washed away by flowing solutions.

Some of them make local vibrating movements on the surface they occupy (presumably just during the initial stage of adhesion to it) by means of extracellular polysaccharide filaments. Later on, with stronger adhesion, they "die away". Electron microphotos show the victims enveloped by retes consisting of separate filaments or

* Fimbria (from Latin fimbria-fringe) are long thin microvilli in the cell wall, facilitating adhesion of bacteria to substrates or other cells,–Auth.

** Periplasm is a cell compartment between cytoplasmatic and external membranes in gram-negative bacteria.–Auth.

*** Murein layer is a rigid component of the ceil wall, consisting of pep-tidoglycane (murein) with protecting and antigenic functions.–Auth.

**** Phase contrast microscopy is a method based on obtaining (by means of special devices) contrast images of structures of colorless transparent microobjects, differing by density, for example, living microorganisms and tissue cultures,–Ed.

KAISTIA SP. NF1 ultramicrobacterial molecular cords capturing ACIDOVORAX DELAFIELDII cells. Electron microscopy. F-polysaccharide filaments-cords, G-sticky granules on filaments; EM-extemal membrane of the victim cell, VC-victim cell.

bundles originating from the nanospiders. In addition, globular nodules of the "cobweb" stick to the attacked cell membranes, whose number is increasing as the predator gets closer to its "prey".

Periplasmatic cone-shaped protruding suckers of parasitic microorganisms, filled by granular matter of high electron density, play a leading role at stage 2 of cohesion. Their external membrane touches the surface layer of the victim's membrane, causing its destruction at close contact points. The fact that ultramicrobacteria very rapidly "capture" the "prey", though they are incapable of active movements, indicates that they have a special mechanism of drawing together, "mooring", and tight adhesion of cells.

MOLECULAR COBWEB

Due to experimental findings, we can schematically present this mechanism. At the earliest stage of interaction due to liquid flows in nutrient medium and to Brownian movement, the cells come closer to each other, so that the distal terminals of polysaccharide filaments, originating from the parasite, touch the victim's surface. Then the nodules at these terminals (sticky globular particles) are attached to the outer layer of its membrane. Thus the victim is captured by the nanospider. At this stage the parasitic cells perform local rocking vibrations near the surface they have occupied; these vibrations are well discernible in a fluorescent and phase contrast microscopes.

The convergence is in progress. New granules stick to the victim's membrane, located at the base of the "threads of web", quite close to the nanospiders' surface; vibrations at this stage are poorly discernible or completely undetectable. At the final stage of adhesion, the aggressor and attacked cell couples are "tightened" (strong cohesion) and, as electron microscopy has shown, there forms a close contact between the outer layers of their membranes (later on leading to the victim's death). This cohesion is presumably realized largely due to the carbohydrate-carbohydrate interactions, playing an important role in intercellular "recognition" processes. Our previous data on ultramicrobacteria parasitizing on cyanobacteria also confirm this fact. Similar interactions between the nanospiders and heterotrophic bacteria Bacillus subtilis, Acidovorax delafieldii are also possible, as their membranes have the so-called C-layer, consisting of orderly protein subunits.

Thus, we have found out that bacterial parasites of the Kaistia and Chryseobacterium genera have special adhesive structures and use an original mechanism of victim capturing. Their subsequent symbiosis is realized as an

Schematic picture of the mechanism of capturing, pulling, and adhesion (cohesion) of parasite cell (P) and victim cell (VC). CW-cell wall, G-sticky granules, PF-polysaccharide filaments, L-glycoprotein layer on the cell wall, UMB-parasitic ultramicrobacterium cell.

epibiosis: predaceous ultramicrobacteria tightly adhere to the surface of cells they attack for subsequent joint coexistence. Having no flagella and incapable of active movements, they use the Brownian movement energy for drawing closer to their "prey". At a required moment, the sticky granules of their threads of web stick one-by-one to the chosen surface, providing a close contact and subsequent adhesion of the membranes of interacting organisms. These nanospiders with a cell diameter of 180-400 nm and volume of 0.004-0.1 urn³, "knit" nets, differing from actual cobwebs by polysaccharide molecules, constituting their filaments, which function as cords pulling the cells to each other.

The efficiency of the discovered and first described here cohesion mechanism consists in utilization of the inexhaustible energy of Brownian movement by the ultramicrobacteria. They need no great complex of genes ensuring the synthesis, assembly of locomotor and adhesive structures and regulation of their activity. This fact and presumably the low metabolic potential explain the small size of the genome (~1.7 Mb) of Chryseobacterium sp. NF4.

The existence in an attached state (epibiosis) is very beneficial, as the adsorbed cells are in the "epicenter" of the host cell lythic activity (dissolution of the substrate by this cell) and concentration of metabolites, later on released into the environment by host cells. However, it is not yet clear what kind of substances are "sucked out" by the predators from the victim. These are most likely amino acids and peptides, as only such nutrition is used by the parasites to grow autonomously. Thus, the object of our future studies is clarification of this problem. It is obvious that nanospiders use their "prey" economically, their strategy consisting in maximally long exploitation of the "moth", which got into their cobweb.

Covered by parasites, the attacked microorganisms live for a rather long time (up to several days) and remain green when stained by the fluorescent dye-stuff (this is how dead cells are revealed). It is noteworthy that if, say, a bacillus becomes a victim, its sporulation is strongly inhibited or even blocked–as otherwise, an appreciable portion of nutrients and energy would have been spent for spore formation and the maternal cells would cease metabolism and die off. Certainly, this "is not planned" by the nanospiders, which need a permanent source of organic compounds.

It remains unknown what substance the sticky granules, located on the polysaccharide filaments of the "cobweb", consist of. Identification of the nature of this "biological glue" has theoretical and, presumably, practical importance.

Studies of the possibility of utilization of predaceous ultramicrobacteria for reducing populations of parasitic microorganisms in various ecotopes are also in the focus of our attention.

The studies were supported by the Analytical Departmental Program "Development of Scientific Potential of Higher School", grant No. 2.1.1/1187.