A. N. Kozlova

[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.

Regulation of the Functional Activity of Lysozyme by Alkylhydroxybenzenes

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10−7−10−3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.

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.

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.

Synthesis of Anabiosis Autoinducers by Non-Spore-Forming Bacteria as a Mechanism Regulating Their Activity in Soil and Subsoil Sedimentary Rocks

Non-spore-forming bacteria of the genera Arthrobacterand Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d1extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d1preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis245 produced an effect on the collection strain Arthrobacter globiformisB-1112 and also on Micrococcus luteusand Bacillus cereus.The d1preparation from the permafrost isolate of Arthrobacterdiffered from the chemical analogue of this factor, 4-n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d1preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d1anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural long-term cooling.

Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on the induction of phenotypic dissociation

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas aurantiaca and the phenotypic dissociation of this bacterium. The addition of 0.1–0.3 mM hexylresorcinol (C6-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their viscoelasticity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their viscoelasticity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C1-AHB). The DNA SC from actively proliferating cells were characterized by smaller viscoelasticity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C6-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C1-AHB produced a similar relaxing effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C6-AHB had a breaking point within the submillimolar (10−4 M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable phenotype and dissociate into distinct variants upon inoculation on solid media.

Properties of the phenotypic variants of Pseudomonas aurantiaca and P. fluorescens

Different capacity for phenotypic variation of Pseudomonas aurantiaca and P. fluorescens in populations of cyst-like resting cells (CRC) during their germination on solid media, was shown to be a characteristic trait of biodiversity for the dormant forms of these bacteria. This biodiversity manifests itself as qualitative and quantitative differences in the spectra and emergence frequency of phenotype variants, obtained by plating of CRC, and depends on the conditions of CRC formation and storage time. In P. aurantiaca, the variation was associated with transition of the wild-type S-colonial phenotype into the R-type or the more pigmented P-type. These transitions were most pronounced for the CRC obtained under nitrogen depletion (a twofold N limitation), as well as under the influence of a chemical analogue of microbial anabiosis autoinducers, C12-AHB. In the latter case, the frequency of S➝R and S➝P transitions (up to 70% and 80%, respectively) depended on the C12-AHB concentration (1.0 × 10−4 M and 2.5 × 10−4 M) and on the storage time of CRC suspensions (from 3 days to 1.3 months). In the CRC populations grown in nitrogen-deficient media, R-type appeared with a frequency of up to 45% after at least four months of storage. In the case of P. fluorescens, S➝R transitions depended not only on the storage time of CRC and C12-AHB concentrations, but also on the composition of the solid medium used for plating. Differences were shown between the R-, P-, and S-variants of P. aurantiaca in such morphological, physiological, and biochemical characteristics as the growth rate (μmax) in a poor medium, biomass yield (Ymax), resistance to streptomycin and tetracycline (LD50), and the productivity in extracellular proteases. The R-and S-variants of P. fluorescens differed in their growth characteristics, resistance to high salinity and oxidative stress, as well as in their sensitivity to exogenous introduction of chemical analogues of microbial autoregulators (C12-AHB and C7-AHB). Hence, both the formation of dormant forms of the various morphological types [1] and intrapopulation phenotypic variability observed during their germination are important for the survival strategy of pseudomonads under unfavorable environmental conditions.

Changes in Physicochemical Properties of Proteins, Caused by Modification with Alkylhydroxybenzenes

Kinetic characteristics of model enzymes and physicochemical properties of globular proteins modified by chemical analogues of low-molecular-weight microbial autoregulators (alkylhydroxybenzenes, AHBs) have been studied. C7 and C12 AHB homologues were used, differing in the length of the alkyl radical and the capacity for weak physicochemical interactions. Both homologues affected the degree of protein swelling, viscosity, and the degree of hydrophobicity. The effects depended on the structure of AHBs, their concentration, and pH of the solution, which likely reflects changes in the charge of the protein globule and its solvate cover. Variations of hydrophobicity indices of AHB-modified enzymes (trypsin and lysozyme) were coupled to changes in the catalytic activity. The values of KM, measured for the enzymes within both AHB complexes, did not change, whereas Vmax increased (in the case of C7 complexes) or decreased (C12 complexes). Possible molecular mechanisms of changes in the physicochemical and catalytic parameters of enzymatically active proteins, induced by modification with structurally distinct AHBs, are described, with emphasis on targeted regulation of functional activity.

The role of alkylhydroxybenzenes in the adaptation of Micrococcus luteus to heat shock

The response of the gram-positive bacterium micrococcus luteus to heat shock (4°C, 15 min) and the adaptogenic activity of alkylhydroxybenzenes (AHBs), which are extracellular growth-regulating substances of these bacteria, were studied. The perception of stress and the postshock behavior of M. luteus cells proved to depend on the growth phase and medium. The magnitude of the stress response was more pronounced in cultures grown on synthetic medium than in cultures grown on rich medium (nutrient broth). During exponential or linear growth, the cells were more sensitive to the temperature effect than during decelerated growth. In linearly growing m. luteus cultures, the amount of total intra- and extracellular alkylhydroxybenzenes, the anabiosis inducers, increased in response to heat shock. AHB redistribution between cells and culture liquid occurred in the course of stress and after stress. In micrococci exposed to heat shock, an increase in the AHB concentration both in cells and in culture liquid is likely a defense reaction of stress resistance. This conclusion was confirmed in experiments with the addition 30 min before the heat shock of a chemical analogue of the anabiosis inducer, C7-AHB (12 mM), which protected M. luteus cells so that their intense growth was observed after shock without any lag. The protective effect of AHBs is a result of their ability to form complexes with enzyme macromolecules and stabilize them. The data obtained extend the knowledge of the stress-protective functions of low-molecular-weight autoregulators and of the role of intercellular communications in the stress response of bacterial cultures.