A. L. Mulyukin

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 ovoid cells of Mycobacterium tuberculosis are formed in response to gradual external acidification

It is believed that latent tuberculosis is associated with the persistence of Mycobacterium tuberculosis (MTB) in a dormant-like state. Dormant cells of MTB with coccoid morphology were produced in some in vivo studies, but similar forms were not produced in the known in vitro models in sufficient amounts to permit their characterization. This work demonstrates the efficient formation of phase-dark ovoid cells in MTB cultures within 150 days after the onset of stationary phase. During this time the medium underwent gradual acidification (pH 8.5 → 4.7) as a result of cellular metabolism. A rapid change in the external pH resulted in cell degradation and death. In common with the dormant forms found in other organisms, the ovoid cells had thickened cell walls, a low metabolic activity and elevated resistance to antibiotics and heating. The ovoid cells had lost the ability to form colonies on solid medium and were thus regarded as operationally «non-culturable». At an early stage in the acidification process (about 40 days post inoculation), the ovoid cells self-resuscitated when placed in fresh liquid medium. However, ovoid cells, stored for a prolonged time, required supernatant from active MTB cells, or externally added recombinant form of resuscitation promoting factor (Rpf) for successful resuscitation. It is suggested that the adaptation of cellular metabolism leading to gradual acidification of the external medium results in the formation of morphologically distinct dormant MTB cells in vitro. The model of MTB dormancy developed here could be a useful tool for the development of new drugs against latent TB.

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.

Autoregulation of stress response in microorganisms

Examples are considered of the involvement of low-molecular-weight autoregulators in the development of resistance of proliferating microbial cultures to unfavorable environmental impacts of various intensity, including impacts programmed to occur in the developmental cycle (“new medium stress,” starvation stress) and nonprogrammed impacts. It was shown that extracellular adaptation factors control the reversible adhesion of cells in submerged cultures and the processes of cell reactivation in the poststress period and are involved in the stabilization of cellular biopolymers (proteins and DNA) and subcellular structures (membranes); the adaptogens of the phenolic type also act as efficient scavengers of reactive oxygen species. The protective effect of the adaptogenic autoregulators is manifested in the increase of resistance of microbial cells to stressors of various nature and in the preservation of the cell proliferative ability.

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.

Protection of Saccharomyces cerevisiae against Oxidative and Radiation-Caused Damage by Alkylhydroxybenzenes

The effects of C7-alkylhydroxybenzene (С7-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under γ irradiation of cell suspensions using doses of 10–150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensitized with chlorin e6 (10 μg/l). C7-AHB was found to exert a protective effect. The addition of 0.05–0.16 vol % of C7-AHB to cell suspensions 30 min before irradiation protected yeast cells from γ radiation (50 krad). The protective effect of C7-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C7-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C7-AHB demonstrated that the protective mechanism of С7-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial С7-AHB are not related to its chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.

The Role of Microbial Dormancy Autoinducers in Metabolism Blockade

Alkyl-substituted hydroxybenzenes (AHBs), which are autoinducers of microbial dormancy (d1 factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C6-AHB at concentrations from 10−4 to 10−2 M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C6-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C6-AHB and their mixtures and in the surface tension isotherms of C6-AHB and trypsin solutions. Data on the involvement of dormancy autoinducers in the posttranslational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.

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.