V. I. Duda

Adaptogenic functions of extracellular autoregulators of microorganisms

Information about the functions of extracellular autoregulators, which adapt microorganisms to the stresses “scheduled” in the development cycle of microbial cultures (stresses of new medium, starvation, or space exhaustion (high cell density)) is summarized in the review. In a number of bacteria and yeasts, derivatives of alkylhydroxybenzenes (AHB), particularly of the class of alkyl resorcinols, act as autoregulators with adaptogenic functions. The chemical structure of AHB determines their amphiphility; capacity for physical and chemical interaction with membrane lipids, proteins, and DNA; properties as natural modifiers of biological membranes and enzymes; and the expression of antioxidant activity. Increase of AHB concentration up to the critical level (10−5-10−4 M) results in cessation of cell division and in transition of the microbial culture to the stationary phase; further increase to 10−4-10−3 M induces a transition of some of the cells of a post-stationary culture to the anabiotic state with the formation of cystlike resting cells (CRC), even in non-spore-forming bacteria. AHB participate in the regulation of the phenotypic variability of bacteria. The dynamics of extra-and intracellular concentrations of AHB in growing microbial cultures and the polymodality of their effect determine the adaptogenic functions of AHB as autoinhibitors of culture growth, autoinducers of anabiosis, and autoinhibitors of germination of resting forms. Manifestation of any given function depends on the concentration of AHB, the physiological state of the recipient cells, and on environmental factors. The species nonspecificity of AHB effects points to their significant role in the regulation of the development and functioning of microbial communities.

Dormant forms of Mycobacterium smegmatis with distinct morphology.

Cultivation of Mycobacterium smegmatis cells in a nitrogen-limited minimal medium (SR-1) followed by prolonged storage at room temperature without shaking resulted in the gradual accumulation of morphologically distinct ovoid forms characterized by (i) low metabolic activity; (ii) elevated resistance to antibiotics and to heat treatment; and (iii) inability to produce colonies on standard agar plates (non-platable cells). Detailed microscopic examination confirmed that ovoid cells possessed an intact cell envelope, specific fine structure and large electron-transparent bodies in the cytoplasm. Cell staining with Nile red and analysis of the lipid content by TLC revealed the presence of significant amounts of apolar lipids in these bodies. The ovoid forms could be stored for significant periods (up to 5 months) and resuscitated afterwards in a modified Sautons medium. Importantly, resuscitation of ovoid cells was accompanied by their transformation into the typical rod-shaped cells. We suggest that the observed ovoid cells represent dormant forms, resembling morphologically distinct cells of Mycobacterium tuberculosis previously isolated from tuberculosis patients and infected animals.

[Ultrastructure of resting cells of some non-spore-forming bacteria].

Using electron microscopy (ultrathin sections and freeze-fractures), we investigated the ultrastructure of the resting cells formed in cultures of Micrococcus luteus, Arthrobacter globiformis, and Pseudomonas aurantiaca under conditions of prolonged incubation (up to 9 months). These resting cells included cystlike forms that were characterized by a complex cell structure and the following ultrastructural properties: (i) a thickened or multiprofiled cell wall (CW), typically made up of a layer of the preexisting CW and one to three de novo synthesized murein layers; (ii) a thick, structurally differentiated capsule; (iii) the presence of large intramembrane particles (d = 180–270 Å), occurring both on the PF and EF faces of the membrane fractures of M. luteus and A. globiformis; (iv) a peculiar structure of the cytoplasm, which was either fine-grained or lumpy (coarse-grained) in different parts of the cell population; and (v) a condensed nucleoid. Intense formation of cystlike cells occurred in aged (2- to 9-month-old) bacterial cultures grown on diluted complex media or on nitrogen-, carbon-, and phosphorus-limited synthetic media, as well as in cell suspensions incubated in media with sodium silicate. The general morphological properties, ultrastructural organization, and physiological features of cystlike cells formed during the developmental cycle suggest that constitutive dormancy is characteristic of non-spore-forming bacteria.

Ultramicrobacteria: Formation of the concept and contribution of ultramicrobacteria to biology

Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of proliferating cells (less than 0.1 μm3 in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various natural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects. Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea (members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm3. The term nanobacteria is used in the literature also to denote ultrasmall bacterium-like particles occurring in rocks, sands, soils, deep sub-surface layers, meteorites, and clinical samples. The systematic position and the capacity for self-reproduction of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphology, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according to the type of cell wall structure and the reaction to Gram staining: (1) gram-negative, (2) gram-positive, and (3) cell wall-lacking. Their cells divide by constriction, septation, or budding. The unique processes performed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that synthesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest relatives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the currently known UMB species and the reductive mode of their evolution.

Structural and physiological diversity among cystlike resting cells of bacteria of the genus Pseudomonas

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C12-AHB, 10−4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.

Dormant forms of Micrococcus luteus and Arthrobacter globiformis not platable on standard media

The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in the course of 3–9 month incubation to the level of 106–107 CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although preincubation of CRC suspensions in a buffer solution of 0.1 M K2HPO4 (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation of macrocolonies by a small portion of nonplateable CRC of M. luteus (50−80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 105 mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number (MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’ micrococcal CRCs to microcolony formation was observed (up to 2.3 × 107 mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary methods is of great importance for advanced monitoring studies.

Diverse morphological types of dormant cells and conditions for their formation in Azospirillum brasilense

Differences in generation of dormant forms (DF) were revealed between two strains of non-sporeforming gram-negative bacteria Azospirillum brasilense, Sp7 (non-endophytic) and Sp245 (endophytic strain). In post-stationary ageing bacterial cultures grown in a synthetic medium with a fivefold decreased initial nitrogen content, strain Sp7 formed two types of cyst-like resting cells (CRC). Strain Sp245 did not form such types of DF under the same conditions. CRC of the first type were formed in strain Sp245 only under phosphorus deficiency (C > P). The endophytic strain was also shown to form structurally differentiated cells under complete starvation, i.e. at a transfer of early stationary cultures, grown in the media with C > N unbalance, to saline solution (pH 7.2). These DF had a complex structure similar to that of azotobacter cysts. The CRC, which are generated by both azospirilla strains and belong to distinct morphological types, possessed the following major features: absence of division; specific ultrastructural organization; long-term maintenance of viability (for 4 months and more); higher heat resistance (50–60°C, 10 min) as compared with vegetative cells, i.e. the important criteria for dormant prokaryotic forms. However, CRC of non-endophytic strain Sp7 had higher heat resistance (50, 55, 60°C). The viability maintenance and the portion of heat-resistant cells depended on the conditions of maturation and storage of CRC populations. Long-term storage (for 4 months and more) of azospirilla DF populations at −20°C was optimal for maintenance of their colony-forming ability (57% of the CFU number in stationary cultures), whereas the largest percentage of heat-resistant cells was in CRC suspensions incubated in a spent culture medium (but not in saline solution) at room temperature. The data on the intraspecies diversity of azospirilla DF demonstrate the relation between certain type DF formation to the type of interaction (non-endophytic or endophytic) with the plant partner and provide more insight into the adaptation mechanisms that ensure the survival of gram-negative non-spore-forming bacteria in nature.

Fine Structure of Mummified Cells of Microorganisms Formed under the Influence of a Chemical Analogue of the Anabiosis Autoinducer

Under the influence of alkyl hydroxybenzene (C6-AHB) added to cell suspensions at concentrations of (1–5) × 10–3M, the cells of Saccharomycescerevisiae, Micrococcusluteus, and Thioalkalivibrioversutusunderwent dramatic changes in the ultrastructural organization of cell membranes, cytoplasm, and inclusions. In yeast suspension, the first changes were observed after 15 min in the structure of pocket-like invaginations in the cytoplasmic membrane (CM): they were shortened and thickened. In the subsequent 30 to 60 min, CM ruptures were formed in the regions devoid of intramembrane protein particles and in the pocket-like invaginations. After 24 h, complete disintegration of the intracellular membrane structures and conglomeration of the ribosomal part of the cytoplasm occurred. Similar changes were observed on the exposure of gram-positive and gram-negative bacteria to AHB. However, the cell wall in all the microorganisms studied was not destroyed, and in Micrococcusluteusit was even thickened. These mummified forms were preserved as morphologically intact but nonviable cells for more than three years of observations. By their ultrastructural characteristics, these mummified forms of microorganisms were similar to the fossilized microorganisms discovered by us in fibrous kerite. The concept of micromummies was formulated. AHB are supposed to play an important role in the process of fossilization of microorganisms in nature.

Ultrastructural Characteristics of Natural Forms of Microorganisms Isolated from Permafrost Grounds of Eastern Siberia by the Method of Low-Temperature Fractionation

The method of isolation of microbial cells from natural solid substrates was suggested and successfully tested in studies of common soil microorganisms [6] and microorganisms of permafrost grounds [7]. However, to study Arctic deposits, this method had to be modified, because all procedures of separation and concentration of microbial cells in this case should be performed at the low temperature corresponding to the natural temperature of these deposits (–12 ° C). To meet this requirement, soil samples were diluted and resuspended (after centrifugation) in 35, 40, and 50% glycerol solutions precooled to –12 ° C. Generally, the process of fractionation consisted of series of sequential procedures of dilution, dispersion, sonication, and centrifugation (a total of 14 procedures took about 2 h altogether). Soil samples were dispersed and microbial N2 3 cells were desorbed by sonication of the soil suspension for 1 min at an ultrasound frequency of 15 kHz and an ultrasonic generator output current of 0.44 A [8]. This method of fractionation was preliminarily tested using samples of meadow soil inoculated with the indicator yeast strain Cryptococcus albidus as a substrate. This strain has been earlier isolated from the permafrost samples studied in this work [3]. Microscopic analysis (phase-contrast microscopy) demonstrated that as much as 80% of soil yeast cells could be isolated as an individual fraction using this method of fractionation. Samples for electron microscopic cryofractography were prepared using a JEE-4X vacuum freezer with the sample cooling rate of 10 6 K/s [9]. Ultrathin sections were prepared as described earlier [3]. Electron-microscopic examination revealed that the fractions isolated from the soil samples studied contained both apparently intact cells (i.e., cells without visible disturbance of ultrastructural morphology and integrity) and partially degraded cells (ruptures of cell walls and membranes, lysis and bleaching of cytoplasm, etc.). Empty cells (“ghost” cells) containing only cell walls were also found. The majority of cells with undamaged integral structure were found to be encapsulated in thick ( d = 0.15–0.4 μ m) laminated covers (cocoon-like envelope) (Figs. 1a–1c). Individual layers of the laminated envelope differ from each other in structural parameters. The structure of the inner fibrillar–granular layer adjacent to the cell wall is very much similar to the structure of the capsule of many cultivated bacteria. Two sublayers were observed in the ultrathin sections of the inner layer. The electron-transparent sublayer is aligned immediately against the cell wall, whereas the electron-dense fibrillar–granular sublayer is located above the electron-transparent sublayer. The outer layer of the cover consists of a capsular or slime-like material encrusted with mineral crystalloid structures. These structures (needle crystals, plates, and large granules) are of high electron density. Therefore, these mineral crystalloid structures are thought to contain metals (primarily, iron). A high mechanical strength of such cocoon-like structures is evidenced by the fact that they are not disintegrated during sample MICROBIOLOGY