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The resistance of microorganisms’ biofilms to antibacterials is a problem both for medicine and for many industries. Increasing the effectiveness of antimicrobial agents is an urgent task. The goal of the present work was to develop a new approach to development of anti-biofilm compositions based on conventional disinfectants in combination with enhancers (adjuvants). Methods of microbiology (viable cells count, model biofilms) and electron microscopy were employed. This research formulates the principles for selection of adjuvants. The adjuvants should: (1) increase the efficiency of decomposition of the biofilm matrix or/and (2) suppress the microbial protective mechanisms. For testing anti-biofilm compositions, two models of biofilms have been developed, on a solid surface at the interface with air or liquid. It was demonstrated that hydrogen peroxide, ethanol, isopropanol, and 4-hexylresorcinol enhanced the biocidal effect of disinfectants based on oxidants (peroxides and chlorine-containing) and quaternary ammonium salts by three to six orders of magnitude. Mechanisms of adjuvant action were mechanical decomposition of the matrix (by oxygen bubbles formed inside a biofilm in the case of hydrogen peroxide), coagulation of matrix polymers (in the case of alcohols), and a decrease in metabolism (in the case of 4-hexylresorcinol). The use of approved chemicals as adjuvants will accelerate the design of effective anti-biofilm antiseptics for medicine, social hygiene, and food manufactures and other industries.

Bacterial survival upon sharp fluctuations of environmental parameters and exposure to lethal doses of stressors (antibiotics, disinfectants, heat shock, and others) is ensured by the use of different strategies of resistance, an important place among which is occupied by the forms with reduced or stopped metabolism, antibiotic tolerant (AT) persister (P) cells, anabiotic dormant forms (DFs), and viable but non-culturable (VBNC) cells. Elucidating the role of these forms of bacterial resistance to an impact of chemical and biological toxicants and physical stressors is of great fundamental and practical interest. The aim of this research was to study the dynamics of the resistance forms in bacteria developing in biofilms and, for comparison in liquid media, upon exposure to lethal doses of antibiotics and heat shock (80 °C, 15 min). In the trials, the experimental model of the development of monospecies and binary forms of bacterial biofilms including contaminants of meat products (eight strains of genera Pseudomonas, Escherichia, Salmonella, Staphylococcus, and Kocuria) on the fiberglass filters was used. It was established that survival of populations in the presence of lethal doses of antibiotics and upon heating was ensured by persister cells forming in bacterial populations and, at the late stages of the development of biofilm or planktonic cultures (28 days of incubation), by anabiotic DFs. With that, the number of thermoresistant (TR) DFs (103–104 CFU/mL) in dying biofilms (28 days) developing in the standard conditions (composition and volume of a medium, pH, growth temperature) weakly depended on the bacterial taxonomic status. This study demonstrates the heterogeneity of DF populations of biofilm bacterial cultures in terms of the depth of dormancy, as a result of which the number of thermoresistant DFs after heating can exceed their total number before heating (due to the effect of DF revival, resuscitation). When studying the dynamics of TR cells and P cells in bacterial biofilm and planktonic cultures, it was found that their number (CFU/mL) in populations decreased up to the absence of TR cells and P cells on the 21st day of growth and was restored on the 28th day of growth. The revealed phenomenon can be explained only by cardinal changes in the ultrastructural organization of cells, namely, cytoplasm vitrification due to a sharp decrease in an amount of free water in a cell, which, according to the results obtained, occurs in the period between the 21st and 28th days of incubation. A high degree of correlation between the number of AT cells and TR cells (0.5–0.92) confirms the hypothesis that regards P cells as precursors of DFs.

While liquid biopreparations for water and soil bioremediation are convenient and economically attractive, microbial survival under standard environmental conditions is poor. Microbial immobilization is a common and efficient method to preserve high viable cell titers. We tested the effect of three new materials on the survival of various hydrocarbon oxidizers during long-term storage. The suspended (planktonic) cultures stored under the same conditions served as a control when assessing the viability of immobilized cultures after long-term storage. The cells of Acinetobacter seifertii, Pseudomonas extremaustralis, P. aeruginosa, Rhodococcus erythropolis, and Dietzia maris grown with microcapsules (MCs) of chitosan-modified polyurea (represented by crumpled spheres, 40–200 µm in diameter) attached abundantly to the MC surface. After several months of storage with MCs, CFU titers were two to five times higher than in the control. Cultivation of P. aeruginosa and R. erythropolis with polylactide (PLA) MCs (represented by slightly crumpled thin-walled spheres 25–100 µm in size) resulted in the formation of thick biofilms on MC surface. After 30-day storage with these MCs, CFU titers of R. erytropolis were up to one thousand times higher than in the control. In the presence of PLA MCs, the respiration rates of both cultures were five to eight times higher than in the control without MCs. The reasons of the better survival of immobilized cells are being discussed. After storage for up to 12 months, CFU titers of bacteria (R. erythropolis, A. seifertii, P. aeruginosa, P. extremaustralis) and yeasts (Yarrovia lipolytica) immobilized in the gel based on silanol derivatives of humic substances were ten to one hundred times higher than in the control. After 4-month storage, hydrocarbon degradation by the stabilized cultures began earlier and was faster and more complete than in the control. The tested materials may be used to extend the storage time for the preparations of biotechnologically important bacteria and for application for petroleum bioremediation.

Background: The biological activities of dark Chinese teas are largely due to their microbial post-fermentation. Herbal teas are traditional Russian beverages that hold special value, owing to their taste and useful medicinal properties. However, no data are available in the literature on using microbial post-fermentation for enhancing their biological activity. The goal of this work was to demonstrate that the fungus Eurotium isolated from Chinese black teas can be used for the post-fermentation of herbal teas produced from bay willow and apple leaves.Methods: Eurotium cristatum was isolated from brick Chinese tea Fujan and identified using conventional methods of microbiology and molecular biology. Low molecular weight metabolites (phenols, amines, sugars, and amino acids) were determined by HPLC. E. cristatum was grown in association with the bacterium Bacillus amyloliquefaciens.Results: It was revealed to exhibit valuable biosynthetic features, such as a lack of mycotoxins, zero antimicrobial activity, and the capacity to synthesize neuroactive amines. B. amyloliquefaciens displayed a wide spectrum of antibiotic (antimicrobial and antifungal) activities that anifested themselves even with antibiotic-resistant bacteria). While growing on green unfermented tea (Camellia sp.) E. cristatum produced and modified neuroactive amines, such as dopamine, serotonin, and epinephrine. The fungus efficiently grew during the post-fermentation of herbal teas from both bay willow and apple leaves. Even though Camellia leaves substantially differed from bay willow and apple leaves in terms of phenol content, the growth of E. cristatum on Camellia was also sufficiently good. This suggests that the growth of Eurotium fungi is not influenced by the phenolic compounds. The data obtained on the composition of phenolic compounds, carbohydrates, and amino acids in the fermented plants and raw material provide evidence that the growth of the fungus proceeds depends on the hydrolysis of high molecular weight phenols and cell biopolymers in the fermented material.Conclusion: Thus, the ability of E. cristatum to grow on plant leaves of significantly different biochemical composition provides foundations for new technologies aiming to obtain post-fermented herbal teas with high biological activity that are enriched in low molecular weight compounds including biogenic amines.

Persisters (P) are the cells surviving, but not reproducing in the presence of lethal doses of antibiotics (AB). These are the cells of a scarce (0.001–1.0%) subpopulation, emerging due to a phenotypic transition, which may be stochastic or induced by stress impacts. Growing in fresh medium, P reproduce the structure of the parent population, which is considered the cause of: (a) recurrent infections; (b) inefficiency of antibiotic treatment; and (c) development of antibiotic resistance, as was discussed in numerous articles and reviews. The goal of the present work was to investigate P formation by bacteria contaminating human skin and their properties, which have not been studied previously. Two approaches were used to measure the frequency of occurrence was determined for P in the populations of stationary-phase cultures of Staphylococcus capitis Р205-1 (UNIQEM, Russia), which is sensitive to polymyxin, Na sulfacyl, and co-trimoxazole, and Staphylococcus epidermidis 14990 АТСС, which is resistant to these antibiotics: (1) traditional, by application of lethal AB doses and (2) by using a lysis solution (LS), which killed the ordinary vegetative cells and selected the persister cells resistant to AB and LS (PAB and PLS, respectively). This is the first report on applicability of the method involving the lysis solution for determination of P abundance in strains with multidrug AB resistance (for the S. epidermidis strain). Unlike most bacterial strains retrieved from collections , the share of P in stationary-phase cultures of staphylococci inhabiting human skin was quite high: ~10% for both S. epidermidis PLS and S. capitis. Heterogeneity in resistance to the lysis solution was revealed in the PAB obtained by the standard procedure: PLS share in S. capitis was ~1% of PAB abundance. These PLS were resistant to AB cross-action. The differences in PLS abundance in the studied strains (10% for S. epidermidis and below 1.0% for S. capitis) correlated with their AB resistance. Long-term incubation (4 months) under standard conditions resulted in the cycle of staphylococci development ending by emergence of dormant forms (DF); their share for both strains was ~0.1% of the number of viable cells in the stationary-phase cultures. The DF of staphylococci had all characteristics of dormant cells: emerged in the cycle of culture development; preserved viability under nongrowth conditions; had no experimentally detectable metabolism (absence of respiration); had specific ultrastructural organization; and were heat-resistant (200‒600 times higher than the vegetative cells at 90°C for 15 min). The correlation between PLS abundance in the cultures of staphylococci and the share of heat-resistant DF confirms our earlier suggestion on persisters as DF percursors.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pmr in mice. Complex therapy with polymyxin B (1 mg/kg) and 4-hexylresorcinol (30 mg/kg) led to cure in 80%; in 20% of these mice, no bacterial cells were found. After treatment with polymyxin B alone, only 50% animals survived and all of them contained bacterial cells. Comparative analysis of the results of monotherapy and combined treatment indicates that 4-hexylresorcinol not only increases the efficiency of antibiotic, but also minimizes persistence of the infection agent and therefore, the risk of development of antibiotic resistance.

A new approach is described to increasing the numbers of viable cells in long-term stored populations (cultures) of the hydrocarbon-oxidizing bacterium Acinetobacter junii, which involves application of humic substances (HSs). HSs are produced due to condensation and oxidation of phenolic compounds, including alkylresorcinols, factors of intercellular microbial communication with stress-potentiating and antioxidant activity. HS addition to the stationary-phase bacterial culture was shown to result in 10- to 15-fold increase in cell viability for bacterial preparations stored for 1 to 4 months under provocative conditions (free air supply, growth medium, and temperatures of 20–25°C). Analysis of death curves for experimental and control A. junii cultures revealed two phases, with the initial stage of rapid death (0.167 log CFU/mL day), usual for autolysis of the regular stationary cells, and the stage of slower death (log 0.015 CFU/mL day), typical of persister cells. This was previously shown to be the feature determining this type of phenotypic heterogeneity in the populations of opportunistic bacteria. The concentration dependence of HSs effect on persisters formation was determined. While treatment of A. junii stationary cultures with hydrogen peroxide (0.03–0.3%) as a stress factor also resulted in enhanced persisters formation (fourfold), unlike the HS-treated variants they survived for not more than 1.5 to 2 months. Plating of the control and experimental variants (with HSs or H2O2) after long-term storage (2–4 months) revealed their phase variation spectrum to change due to replacement of the dominant colony morphology phenotype by the minor ones (40% and more), which is one of the features of the dormant cystlike bacterial forms. The ratios of the variants in the control and experiments (with HSs) were different. Comparison of the chemical composition of HS preparations indicated that the most oxidized hydrophilic HSs affected formation of persister cells, while less oxidized humates with higher antioxidant activity were responsible for the viability prolongation. Thus, the effect of HSs on bacterial populations includes induction of persister cell formation (their increased number), prolongation of the colony-forming capacity in the cells surviving under provocative storage conditions, and alteration of the phase variant spectrum of the stored populations. Formation of stress-induced persister cells developing into mature dormant forms supporting survival of the population (species) was established for saprotrophic bacteria (in the case of A. junii). In practical terms, HS-induced formation of persister cells may be recommended for development of diverse bacterial preparations.

Addition of humic compounds (9 preparations) to the cultures of hydrocarbon-oxidizing bacteria was shown to result in up to 15-fold increase in the number of viable cells in the cultures stored in the growth media with access to air at 20‒25°C. The most oxidized humates exhibited the highest activity in induction of persister formation, which subsequently matured to dormant forms, while unoxidized humates with antioxidant activity supported viability persistence.

—Approaches to stabilizing the cells of lactic acid organisms (LAO) investigated in the present work resulted in maintaining the viable cell titer at 107–108 cells/mL, the value recommended for the LAO titer in foodstuffs, for 12 months. Natural LAO associations selected for long-term preservation of LAO cell viability exhibited high efficiency. Lactic acid bacteria Lactobacillus plantarum and yeasts Candida parapsilosis were isolated from the association isolated from marketed curdled milk and selected for 6 months. The death rate of these LAO in binary culture (50% of each component) was several times lower than in monocultures. Adding other approaches for cell stabilization in a binary LAO culture (anoxic conditions and stabilizing antioxidant and biopolymer supplements) resulted in a 5- to 6-fold increase in the number of viable cells after 12-month storage. The algorithm for combined application of several approaches to prolongation of cell viability in the cultures of lactic acid microorganisms during long-term storage (up to 12 months) was not reported previously. It may be recommended for LAO cell stabilization both in fermented milk products and in leaven cultures. This is the first report of C. parapsilosis as the yeast component of a stable association with lactic acid bacteria.

Anabiotic dormant forms (DF) of gram-negative bacterial epibionts of the smooth clawed frog Xenopus laevis—Stenotrophomonas sp. strain FM3 and Morganella morganii subsp. sibonii strain FF1, were obtained and characterized. The ultrastructural features of the dormant cells of strains FM3 and FF1 (large periplasmic space, dense heterogeneous cytoplasm, and ordered reorganization of the compacted nucleoid) were typical of the cystlike cells (CLC) of the previously studied other taxa of gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, P. fluorescens, Azospirillum brasilense , etc.). Preservation of potentially active enzymes of the antioxidant defense systems was not previously reported for anabiotic dormant forms. They are probably responsible for DF survival at early stages of their germination, providing for resistance to new medium stress and other stressors in the absence of de novo enzyme synthesis. Our results are of interest for both basic microbiology and biomedicine, since strains FM3 and FF1 are phylogenetically related to bacterial agents of nosocomial clinical infections and emergence of their dormant forms may explain resistance of nosocomial infections to sanitary treatment.

One of the adaptive strategies for the constantly changing conditions of the environment utilized in bacterial cells involves the condensation of DNA in complex with the DNA-binding protein, Dps. With the use of electron microscopy and electron tomography, we observed several morphologically different types of DNA condensation in dormantEscherichia colicells, namely:nanocrystalline, liquid crystalline, and thefolded nucleosome-like. We confirmed the presence of both Dps and DNA in all of the ordered structures using EDX analysis. The comparison of EDX spectra obtained for the three different ordered structures revealed that innanocrystallineformation the majority of Dps protein is tightly bound to nucleoid DNA. We demonstrated that the population of the dormant cell is structurally heterogeneous, which allows cells to respond flexibly to environmental changes. It increases the ability of the whole bacterial population to survive under extreme stress conditions.

Nontraditional sources were shown to be promising for obtaining bacterial isolates characterized by efficient production of antibiotics, including those affecting the strains with multidrug resistance. From Antarctic permafrost, 32 bacterial strains were isolated, and their ability to synthesize antimicrobal compounds efficient against 12 test strains, including the ones with multidrug resistance, was studied in submerged cultures. Out of 13 strains producing antibiotic compounds with different spectra of action, seven were chosen for further study. Their species identification was carried out by microbiological techniques and by analysis of their 16S rRNA gene sequences. Six strains of spore-forming bacteria were identified as Bacillus species (B. licheniformis, B. mojavensis, B. safensis, and B. subtilis), while one strain was identified as Gordonia terrae. Antimicrobial activity of two B. mojavensis strains (INA 01149 and INA 01151), two B. safensis strains (INA 01153 and INA 01154), and B. licheniformis strain INA 01155 against the antibiotic-resistant tester strain Leuconostoc mesenteroides VKPM B-4177 (VR) was revealed. Antibiotic activity of two B. safensis strains (INA 01153 and INA 01154) against tester bacteria, including Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus INA 00761 with multidrug resistance has not been reported previously. Antibiotic producers isolated from Antarctic permafrost samples are of interest to medicine due to the global issue of increasing antibiotic resistance of pathogenic bacteria.

A series of X-ray diffraction experiments were performed for the first time to study stress-induced biocrystallization (structural response to stress) in the bacteria E. coli, the spore-forming bacteria Bacillus cereus, and in cells and spores of the mycelial fungus Umbelopsis ramanniana. High-intensity areas with spacings of 90 and 44 A are indicative of a periodically ordered arrangement (most likely nanocrystalline) of the bacterial nucleoid. For the starved bacteria Bacillus cereus, a peak at a spacing of 45 A is also assigned to nanocrystalline complexes of DNA with the Dps protein. The spores of the fungus Umbelopsis ramanniana VKM F-582, as well as the spores of Bacillus cereus, form ordered arrays of DNA molecules with DNA-condensing acid-soluble proteins SASPs. Starved dehydrated mycelial cells of the fungus Umbelopsis ramanniana form ordered structures with spacings from 27 to 55 A. A series of peaks reflect the formation of a number of ordered protein arrays, apparently with DNA, with continuously varying characteristic interplanar spacings.

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2‒3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.

This work was aimed at studying the response of soil non-spore-forming actinobacterial strain Arthrobacter agilis Lush 13 to changing natural conditions, such as nutrient availability and the presence of degradable and recalcitrant aliphatic and aromatic substrates. The A. agilis strain Lush13 was able to degrade octane, nonane, hexadecane, benzoate, phenol, and 2,3-, 2,4-, 2,5-, 2,6-dichlorophenols, but not grew on 3,4-dichlorophenol, 2,3,4-, 2,4,5-, 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP), 2-chlorobenzoate, 3-chlorobenzoate, 3,5-dichlorobenzoate, 2,4-dichlorobenzoate. Under growth-arresting conditions due to nitrogen- or multiple starvation or recalcitrant (non-utilizable) carbon source, the studied strain preserved viability for prolonged periods (4-24 months) due to transition to dormancy in the form of conglomerated small and ultrasmall cyst-like dormant cells (CLC). Dormant cells were shown to germinate rapidly (30 min or later) after removal of starvation stress, and this process was followed by breakdown of conglomerates with the eliberation and further division of small multiple actively growing daughter cells. Results of this study shed some light to adaptive capabilities of soil arthrobacters in pure and polluted environments.

Structural, biochemical, and genetic changes caused by stress factors are known to be largely similar for cells of all modern organisms, which inherited the basic strategies of adaptation to different types of stress from their ancient ancestors. In the present work, the adaptation process is considered for the simplest example of the bacterial E. coli nucleoid. Experimental studies performed recently on prokaryotic bacterial cells, the simplest living organisms, have demonstrated that, under unfavorable environmental conditions (for example, starvation), bacterial cells can use biocrystallization, a special mechanism of protection of the genetic apparatus (nucleoid), generally untypical of living organisms. This mechanism helps to protect the nucleoid from damage and resume the activity of the bacterial cells later, upon improvement of the external conditions. The results of studying the structure of the nucleoid of E. coli bacteria (BL21-Gold strain (DE3)) subjected to starvation stress by using synchrotron radiation at the ESRF beamline ID23-1 are reported.

Cellular organization of the cystlike cells (CLC) of non-spore-forming heterotrophic actinobacteria isolated from soils contaminated with chemical plant waste (Bereznyaki, Russia) and identified as Microbacterium foliorum BN52 was studied. CLC were obtained in laboratory conditions in limited or starving cultures. Two morphotypes of CLC capable of reversion to the vegetative growth were revealed and characterized in detail. The morphological, ultrastructural, and physiological peculiarities of germination and transition to the vegetative growth were studied at first for the dormant forms of M. foliorum BN52. Germination of morphotype II CLC was associated with gradual recovery of the cell shape and subcellular structures. In contrast to the germination of morphotype II CLC, during the first hours of germination of morphotype I CLC cell volume increased significantly with the subsequent formation of very large cell with sizes several times larger than the typical vegetative cells of the strain. The obtained cells were characterized by polynucleoidity, being polyploids undergoing fission at the next stage of germination, resulting in formation of numerous small and ultrasmall viable cell forms. Formation of cyst-like dormant cells, germinating in the form of polyploid cells is assumed to be the basis of survival and adaptation strategies of heterotrophic bacteria, which are incapable of toxicant degradation in natural habitats, under the conditions of the toxic pressure.

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl2 concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C7AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50–60%). In both cases, the optimal C7AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (–20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C7AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C7HOB-modification in the preservation of activity in immobilized amylases after four freeze–thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.

Ever decreasing efficiency of antibiotic treatment due to growing antibiotic resistance of pathogenic bacteria is a critical issue in clinical practice. The two generally accepted major approaches to this problem are the search for new antibiotics and the development of antibiotic adjuvants to enhance the antimicrobial activity of known compounds. It was therefore the aim of the present study to test whether alkylresorcinols, a class of phenolic lipids, can be used as adjuvants to potentiate the effect of various classes of antibiotics. Alkylresorcinols were combined with 12 clinically used antibiotics. Growth-inhibiting activity against a broad range of pro- and eukaryotic microorganisms was determined. Test organisms did comprise 10 bacterial and 2 fungal collection strains, including E. coli and S. aureus, and clinical isolates of K. pneumoniae. The highest adjuvant activity was observed in the case of 4-hexylresorcinol (4-HR), a natural compound found in plants with antimicrobial activity. 50% of the minimal inhibitory concentration (MIC) of 4-HR caused an up to 50-fold decrease in the MIC of antibiotics of various classes. Application of 4-HR as an adjuvant revealed its efficiency against germination of bacterial dormant forms (spores) and prevented formation of antibiotic-tolerant persister cells. Using an in vivo mouse model of K. pneumoniae-induced sepsis, we could demonstrate that the combination of 4-HR and polymyxin was highly effective. 75% of animals were free of infection after treatment as compared to none of the animals receiving the antibiotic alone. We conclude that alkylresorcinols such as 4-HR can be used as an adjuvant to increase the efficiency of several known antibiotics. We suggest that by this approach the risk for development of genetically determined antibiotic resistance can be minimized due to the multimodal mode of action of 4-HR.

One of the adaptive strategies for the constantly changing conditions of the environment utilized in bacterial cells involves the condensation of DNA in complex with the DNA-binding protein, Dps. With the use of electron microscopy and electron tomography, we observed several morphologically different types of DNA condensation in dormant Escherichia coli cells, namely: nanocrystalline, liquid crystalline, and the folded nucleosome-like. We confirmed the presence of both Dps and DNA in all of the ordered structures using EDX analysis. The comparison of EDX spectra obtained for the three different ordered structures revealed that in nanocrystalline formation the majority of the Dps protein is tightly bound to nucleoid DNA. The dps-null cells contained only one type of condensed DNA structure, liquid crystalline, thus, differing from those with Dps. The results obtained here shed some light on the phenomenon of DNA condensation in dormant prokaryotic cells and on the general problem of developing a response to stress. We demonstrated that the population of dormant cells is structurally heterogeneous, allowing them to respond flexibly to environmental changes. It increases the ability of the whole bacterial population to survive under extreme stress conditions.

Current concepts concerning social behavior of the microorganisms inhabiting human gastrointestinal tract, as well as their role in the formation of integrated supracellular structures and in intercellular communication in the host-microbiota system are reviewed. Analysis of the literature data and the results obtained by the authors indicate an important role of neuromediators (biogenic amines, amino acids, peptides, and nitric oxide) in the intra- and interspecies microbial communication, as well as in the microbiota-host dialogue. The role of this dialogue for human health, its effect on human psyche and social behavior, and the possibility of construction of probiotic preparations with a goal-directed neurochemical effect are discussed.

Effect of human inherent immunity factors of, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL1) on formation of antibiotic-tolerant persister cells surviving in the presence of ciprofloxacin (Cpf, 100 μg/mL) and ampicillin (Amp, 100 μg/mL) in submerged bacterial cultures (Staphylococcus aureus FGA 209P, Escherichia coli K12, and Pseudomonas aeruginosa PAO1) was studied. While Ind in physiological concentrations (0.3 and 3.0 μg/mL) introduced to the lag- or exponential-phase cultures of test organisms exhibited no reliable effect on population growth, the number of persisters increased at 3.0 μg/mL. Bactericidal Ind concentrations (9 μg/mL) suppressed S. aureus growth (-0.1% of surviving cells) with subsequent recovery due to development of the more antibiotic-tolerant white variant. Treatment with Cpf after Ind addition resulted in mutual potentiation of their antimicrobial activity, with the number of S. aureus persisters 2 to 3 orders of magnitude lower than in the case of the antibiotic alone. IL1, another immunity factor, when introduced (0.1-1 ng/mL) to the exponentially growing S. aureus culture (but not to the lag phase culture) had a temporary growth-static effect, with the number of persisters surviving Cpf treatment (100 μg/mL) increasing by 1 to 2 orders of magnitude. Electron microscopy revealed significant alterations in the outer cell envelope layer of surviving S. aureus cells, which should be associated with their changed antigenic properties. Thus, the factors of human inherent immunity have a dose-dependent effect on the growth of bacterial populations. In combination with antibiotics, they exhibit synergism of antimicrobial action (indolicidin) and minimize (indolicidin) or increase (interleukin 1) the frequency of formation of persister cells responsible for survival of a population subjected to an antibiotic attack.

Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 μg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent infections and, together with antibiotic-resistant cells, are important as components of test systems to assay the of efficiency of potential pharmaceuticals against resistant infections.

Currently, limited information is available on the enzymatic activities in ancient paleosols and permafrost, which are recognized as unique depositories of extant microorganisms. In this work, samples from buried and modern top Kastanozems (Haplic Calcisol), modern tundra (Gleysol) soils, and permafrost were tested for amylase activity based on hydrolysis of starch to low-molecular sugars that are important energy sources for microorganisms. Our study demonstrated the preservation of amylase in buried paleosols (300 and 2000 years old) and permafrost (15,000–60,000 years old). The enzyme activity in buried soils was 5–7 times lower than in top soils and did not significantly (P > 0.05) differ from permafrost and tundra soils. Exogenously added alkylresorcinols, valine, and glycine influenced amylolytic activity in soil and permafrost samples in the dose-dependent manner. The amylase response to stimulating concentrations of alkylresorcinols and amino acids was greater in buried paleosols (4.3–11.5-fold) than in top soils (1.9–3.8-fold) over the enzymatic activities in the controls. Positive response of soil amylase even to little changes in concentrations of the low-molecular-weight compounds may be important during recovery of biological activity of microorganisms. Results of this study contribute to a more complete evaluation of hidden biological potential of ancient soils and permafrost and are relevant to paleomicrobiology and soil biology.

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45 degrees C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli 12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10(-4) to 10(-2)% of the intial CFU number) surviving lethal doses of an antibiotic (a mpicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete.

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions, the similarity between the MALDI profiles was shown to increase in the row: genus–species–strain–variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and Sm) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

Growth of members of most of the studied genera of gram-positive (Dietzia, Kocuria, and Rhodococcus) and gram-negative bacteria (Pseudomonas and Chromobacterium) in biofilms exhibited higher resistance to a translation inhibitor, azithromycin compared to the growth of planktonic cultures of the same strains. Low concentrations of azithromycin were found to stimulate biofilm formation by the studied saprotrophic strains. The rate of synthesis of the polysaccharide matrix component exceeded the rate of cell growth, indicating implementation of the biofilm phenotype under these conditions. It was found that an alkylhydroxybenzene (AHB) compound 4-hexylresorcinol was capable of almost uniform suppression of growth of both planktonic cultures and biofilms of the saprotrophic strains under study. In some cases, combined action of azithromycin and AHB resulted in an additive inhibitory effect and prevented the stimulation of biofilm growth by subinhibitory azithromycin concentrations. Thus, AHB may be considered a promising antibiofilm agent.

Molecular and cellular luminescent biotests were used to reveal the effects of five alkylresorcinol homologues (C7-, C9-, C11-, C12-, and C18-AR) on the thermally-induced denaturation and refolding of bacterial luciferases, as well as on the synthesis of heat shock proteins. The ARs activities were found to depend on their fine structure and concentration. The direct heat-protective effect of short-chain C7- and C9-AR on the chromatographically pure Photobactrium leiognathii luciferase/oxidoreductase was shown within a broad range of concentrations (10−3–10−3 M). The long-chain ARs homologues exhibited a similar heat-protective effect at micromolar concentrations only, while their millimolar concentrations increased the sensitivity of the model proteins to thermal treatment. The recombinant strain Escherichia coli K12 MG1655, bearing constitutively expressed Vibrio fischeri luxAB genes was used to investigate the ARs effect on the intracellular chaperone-independent refolding of bacterial luciferase. The functional activity of heat-inactivated enzyme was restored by micromolar concentrations of short-chain ARs, while long-chain homologues inhibited refolding over the wide concentration range. The recombinant luminescent E. coli strain bearing the inducible ibpA’::luxCDABE genetic construction was used to determine the effect of ARs on the synthesis of heat shock proteins (HSP). The preincubation mode of bacterial cells with long-chain alkylresorcinols led to the dose-dependent stimulation of HSP synthesis (2.7 to 4 times), which confirmed that ARs function as “alarmones.” Subsequent thermal treatment resulted in a 5- to 15-fold decrease of the following HSP induction compared to the control, while the number of viable cells opposite increased by 1.5- to 4-fold. Thus, pretreatment of the bacterial cells with long-chain ARs resulted in their preadaptation to subsequent thermally induced stress. Short-chain ARs caused less pronounced HSP suppression, although this was still was accompanied by increased heat resistance of the AR-pretreated bacterial cells.

The structure and specific characteristics of the hydrolytic microbial complexes from chestnut paleosols buried under the barrows of different ages (∼4500 and ∼3500 years) was compared with their modern analogue in microcosm experiments. Potential activity of the hydrolytic complex of the microbial community of the barrow paleosols was found to be higher than in the modern soil complex. The share of metabolically active cells revealed by FISH after the introduction of a growth-stimulating polysaccharide into the paleosol microcosm was 50% of the whole prokaryotic cell number. The paleosol community exhibited a more pronounced response to addition of the substrate than the modern soil community. The differences in the phylogenetic taxonomic structure of the prokaryotic metabolically active hydrolytic complex in the buried and modern soils were revealed. The hydrolytic complex of modern soil was more diverse, while the dominant hydrolytic organisms revealed in paleosols were unicellular and mycelial Actinobacteria, as well as Proteobacteria.

The effects of two alkylresorcinols, namely, methylresorcinol (MR) and hexylresorcinol (HR), which are chemical analogues of the natural anabiosis factors of some bacteria, on the catalytic activity of β-amylase has been investigated. When water-soluble potato starch is used as a substrate, MR in a concentration ranging from 0.8 to 12.9 mmol/L raises the activity of β-amylase to 170%. HR at a concentration ranging from 0.05 to 0.35 mmol/L stimulates a weak increase in the activity of β-amylase to 115%. A further increase in the concentration (0.75 mmol/L) leads to inhibition by 50%. The interaction of MR with β-amylase allows one to increase the general yield of the hydrolysis products of potato and malt starch by 1.5 to 1.6 times, to enhance the rate of hydrolysis by 4 to 5 times, and to extend the temperature and pH ranges of catalysis. Non-competitive activation in the presence of MR and non-competitive inhibition in the presence of HR are observed upon determining the kinetic parameters of the starch hydrolysis by β-amylase modified with alkylresorcinols.

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

Cross protection of members of the domains Bacteria, Archaea, and lower Eukaryota from stress factors due to the action of extracellular low-molecular metabolites with adaptogenic functions was shown. The adaptogen produced by Luteococcus japonicus subsp. casei and described previously as a reactivating factor (RF) was shown to protect the yeasts Saccharomyces cerevisiae, archaea Haloarcula marismorti, and the cells of higher eukaryotes (HeLa) against weak stressor impacts. Production of an archaeal extracellular metabolite with a weak adaptogenic effect of the producer cells and capable of a threefold increase in survival of heat-inactivated yeast cells was discovered. Our results confirm the similarity of the compensatory adaptive reactions in prokaryotes (bacteria and archaea) and eukaryotes.

The changes in physiological and biochemical properties of Pseudomonas fluorescens 26K, a degrader of chlorinated aromatic compounds, were revealed after existing in a dormant state as cyst-like cells (CLC). The CLC maintained the ability to form colonies after long-term storage, possessed enhanced resistance to damaging factors (heating and drying), and had specific ultrastructural organization. In populations grown from CLC on solid media, we observed the appearance of phenotypic variants, which differed from the dominant type in the shape, consistency, and pigmentation of colonies. The emerging phenotypes had higher growth rates on some aromatic substrates, which required the enzymes with broadened substrate specificity for their utilization.

In liquid media, rhizobacteria Azospirillum brasilense are motile by means of a polar flagellum and in semisolid media, also by means of additionally synthesized lateral flagella. In the presence of 4-n-hexylre-sorcinol (0.04–0.10 mM), the swimming rate of A. brasilense Sp245 decreased significantly. At the concentrations over 0.10 mM, motility was completely suppressed. The presence of 4-n-hexylresorcinol in liquid medium also had a negative effect on motility of the cells of Sp245 and other A. brasilense strains (Sp7, Cd, and SR75). The concentrations of 5-methylresorcinol which negatively affected the rate of bacterial spreading in semisolid media were an order of magnitude higher than those of 4-n-hexylresorcinol. Alkylresorcinols, which belong to alkylhydroxybenzene autoregulators, probably affect the cellular mechanisms (systems) responsible for flagella rotation in azospirilla.

The unified method of template preparation for PCR in the form of DNA covered by permeabilized cell envelopes was used for the cells of different physiological status (vegetative, dormant forms of different types, and nonviable micromummies). The procedure for the preparation of template DNA included one-stage (boiling in a buffer with chaotropic salts) or two-stage (boiling in a buffer with chaotropic salts followed by treatment with proteinase K) sample preparation. The proposed method proved effective for detection of not only vegetative cells but also of the bacillary spores and the cystlike dormant cells (CLC) of non-spore-forming bacteria. For example, the two-stage sample preparation of Bacillus cereus spores resulted in the PCR sensitivity increase up to the detection level of 3–30 spores per sample; the one-stage sample preparation was three orders of magnitude less efficient (104 spores per sample). An increase in the sensitivity of PCR detection (4–10-fold) owing to the use of the two-stage sample preparation was shown for bacillary, staphylococcal, and mycobacterial CLC. The possibility of PCR detection of staphylococcal micromummies with irreversibly lost viability, which were therefore undetectable by plating techniques, was also demonstrated. The application of the unified sample preparation method ensuring efficacious PCR detection of bacterial cells, irrespective of their physiological state, may be a promising approach to more complete detection of microbial diversity and the overall insemination of natural substrates.

The effect of the extracellular peptide reactivating factor (RF) synthesized by Luteococcus casei on stress response of Escherichia coli cells subjected to UV irradiation was studied. For these studies, we constructed a test strain carrying the umuD-lacZ operon. The expression rate of this operon reflects the rate of SOS response. Protective effect of RF, defined as the number of cells retaining the colony-forming activity (CFU) after UV irradiation (49–1166 J/m2), was dose-dependent, species-nonspecific, and increasing with increase of the stress load. RF was demonstrated to possess the properties of a direct adaptogen: 15 min of preincubation with RF caused a 1.5–6-fold decrease in expression of the umuD SOS response gene in UV-treated cells, concurrently with a 1.2–7.5 times increase in the number of viable cells (those having retained their colony-forming activity). The probable mechanisms of the protective effect of RF are being discussed.

We investigated the impact of bacterial regulators homoserine lactones (HSLs) and alkylhydroxybenzenes (AHBs) (which are present in human fluids at pico- and nanomolar concentrations) on neutrophile oxidative metabolism. The HSL and AHB effects were determined using a test based on induced luminol-dependent chemoluminescence of neutrophiles in human peripheral blood. In this test, neutrophiles were preincubated with chemical analogs of bacterial autoregulators with different lengths of the hydrocarbon radical, such as HSL · HCl, C6- and C12-HSL, and C1-, C6-, and C12-AHB. We revealed that they suppressed the chemoluminescence and, accordingly, the oxidative metabolism of neutrophiles. This effect was more significant with HSLs than with AHBs. Within each of the two groups, the effect increased with an increase in the length of the hydrocarbon chain of the homologues.

Впервые для бактериальных клеток различного физиологического статуса (вегетативных, покоящихся форм разных типов и нежизнеспособных микромумий) был апробирован единый метод получения матриц для ПЦР в виде ДНК-содержащих клеточных чехлов с пермеабилизованными клеточными оболочками. Процедура получения ДНК-содержащих чехлов включала одностадийную (кипячение в буфере с солями-хаотропами) или двустадийную (кипячение в буфере с хаотропами с последующей обработкой протеиназой К) пробоподготовку клеток. Предложенный метод оказался эффективным для детекции не только вегетативных клеток, но и спор бацилл и цистоподобных покоящихся клеток (ЦПК) неспорообразующих бактерий. Например, при двустадийной пробоподготовке спор Bacillus cereus чувствительность ПЦР повышалась до уровня обнаружения 3–30 спор в пробе, а при одностадийной пробоподготовке – оказалась на три порядка меньше и составила 104 спор в пробе. Повышение чувствительности ПЦР-детекции (в 4–10 раз) за счет применения двустадийной проподготовки было показано для ЦПК бацилл, стафилококков и микобактерий. Аналогичным образом была продемонстрирована возможность ПЦР-детекции микромумий стафилококков, необратимо утративших жизнеспособность и потому не выявляемых методами посевов. Применение единого метода пробоподготовки, обеспечивающего эффективную детекцию в ПЦР бактериальных клеток, вне зависимости от их физиологического состояния, представляется перспективным для более полного выявления микробного разнообразия и общей обсемененности природных субстратов.

A comparative study was conducted on the adaptive mechanisms of the strains Arthrobacter oxydans K14 and Acinetobacter lwoffii EK30A isolated from permafrost subsoil sediments and of those of the analogous collection strains (Ac-1114 Type and BSW-27, respectively). In each pair of the strains compared, the strains differed in terms of (i) growth-related, physiological, and biochemical properties; (ii) resistance to stress factors; (iii) capacity for generation of dormant forms (DFs) under growth arrest conditions, and (iv) intrapopulation production of phase variants. The strains isolated from permafrost displayed a lower growth rate but were more resistant to repeated freezing-thawing treatment than the collection strains. Under the same growth conditions, the permafrost strains formed larger numbers of cystlike anabiotic DFs, extraordinarily small cells, and forms that became nonculturable during long-term storage. Resuscitation of the nonculturable forms resulted in a 2- to-7-fold increase in the percentage of FISH-detectable metabolically active cells. The permafrost strains were also distinguished by increased genome lability. This facilitated their dissociation into intrapopulation variants with phenotypically distinct colonial and morphological properties and different antibiotic resistance. The phenotypic variability was more prominent in Arthrobacter (for which it was not reported previously) than in Acinetobacter. In the populations produced by plating the dormant bacterial forms, the qualitative and quantitative characteristics of the phase variant spectra varied depending on the formation conditions and the composition of the solid media used for the plating. Thus, the permafrost isolates of A. oxydans and Ac. lwoffii were distinguished from their collection analogs by a more manifest adaptive potential including stress resistance, the intensity of DF generation under growth arrest conditions, and increased intrapopulation variability.

This work is concerned with the role of alkylhydroxybenzenes (AHBs), chemical analogs of the autoregulatory microbial d 1 factors, on the development of the stress response of bacterial cells to UV irradiation, including SOS system induction, preservation of cell viability, and S → R phase transitions of the Escherichia coli test strain with the bioluminescence genes cloned under the control of the recA gene promoter. UV irradiation, a natural stress factor, and an increase in AHB concentrations were found to elicit uniform responses in bacteria, indicating that AHBs function as alarmones, i.e., alarm signals. It was revealed that preincubating bacteria with alkylhydroxybenzenes considerably enhanced their viability upon irradiation with lethal UV doses; this was accompanied by a relative decrease in the SOS response activity and a concomitant increase in the frequency of phase transitions. The efficiency of the protective action of AHBs increased with an increase in their hydrophobicity degree. The probable mechanism of the protective effect of AHBs is discussed, based on their capacity for the interaction with biopolymers, which results in changing their structural organization and conferring resistance to a broad spectrum of stress factors. Such a “passive” protective mechanism reduces the susceptibility of DNA to UV irradiation, causing a decrease in the parameters related to the SOS system induction that is responsible for the “active” protective mechanism in bacterial cells. As a result, viability retention under the lethal influence of UV irradiation is possible at minimal values of repair activity and is accompanied by an increase in the phenotypic variability of the surviving part of a bacterial population.

The influence of hexylresorcinol on the structure, equilibrium fluctuations, and functional activity of water-soluble enzyme lysozyme was studied over a wide range of hexylresorcinol concentrations. Hexylresorcinol was found to be not only a stabilizer of lysozyme. At low hexylresorcinol concentrations (2 to 10 molecules per lysozyme globule), the activity of lysozyme sharply increased; activity began to decrease as the concentration grew. The influence of hexylresorcinol on the structural, dynamic, and functional lysozyme characteristics is well described by models of preferential hydration and preferential protein interaction with hexylresorcinol. The hexylresorcinol molecule consists of hydrophobic (alkyl radical) and hydrophilic (aromatic nucleus) moieties, which has additional regulatory action on the functional activity of lysozyme. As the concentration of hexylresorcinol increases, the effect of regions with preferential hydration begins to noticeably predominate over the effect of preferential interaction with hexylresorcinol. At hexylresorcinol concentrations higher than 100 molecules per lysozyme globule, the activity of lysozyme is fully inhibited. This is caused by the preferential hydration of the protein with the displacement of hexylresorcinol from direct contacts with it. The displacement of hexylresorcinol causes the formation of high-density hexylresorcinol micelles. Dense micelles interfere with the approach of substrates to the protein and fully inhibit its functional activity. The complete inhibition of lysozyme activity occurs at hexylresorcinol concentrations lower by an order of magnitude than glycerol inhibiting concentrations.

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.

The adaptogenic effect of the chemical analogues of alkylhydroxybenzenes (AHBs), bacterial extracellular autoregulators (the individual compound C7-AHB and its technical preparation Sidovit), was demonstrated for two pseudomonad species, Pseudomonas aeruginosa and P. fluorescens. The protective effect of AHBs resulted in increased growth rate and biomass accumulation in bacteria grown under suboptimal conditions within the species tolerance range. The adaptogenic effect of AHBs (10–50 μmg/l) was more pronounced under more unfavorable growth conditions. In the case of P. fluorescens, the individual compound C7-AHB increased the biomass yield by 30% under alkaline conditions (pH 9.5), when the growth rate decreased by 80–90% compared to the optimum (pH 5.5–7.5). The Sidovit preparation, containing a mixture of natural AHBs with C7-AHB as the main component, increased the growth rate of P. aeruginosa by 40–60% at nonoptimal temperatures (45 and 10°C) or under enhanced salinity (1% NaCl). The action of AHBs as regulators of the rpoS and SOS response stress regulons was demonstrated to be among the mechanisms of their adaptogenic effect, as was demonstrated with the relevant reporter genes in the model strains E. coli C600 thi, thr, leuΔ(pro-lac) with the osmE-lacZ and umuD-lacZ hybrid operons, respectively. AHBs are technologically and economically acceptable as adaptogenic supplements for bacterial preparations used in soil bioremediation and oil spillage removal under conditions unfavorable for microbial growth, including increased salinity, extreme pH, and fluctuating sub- or supraoptimal temperatures.