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A novel actinobacterium, strain K3-2T, was isolated in pure culture from a thawing ancient ice wedge at Mammoth Mountain (Eastern Siberia, Russia). Colonies of strain K3-2T were yellowish orange; cells had the fine structure typical of Gram-positive bacteria, were non-motile short rods and were non-spore-forming. Strain K3-2T was mesophilic (optimum growth at 28 °С), but capable of growing at 4 °С. The cell-wall peptidoglycan of strain K3-2T contained lysine (the diagnostic diamino acid), glutamic acid, alanine, ornithine, glycine and serine. The polar lipids were phosphatidylglycerol, lysophosphatidylserine, three unidentified phospholipids and glycolipids. The major fatty acids were anteiso-C15 : 0 and C16 : 0. The only menaquinone detected was MK-8(H4). 16S rRNA gene analysis indicated that strain K3-2T belongs to the genus Serinibacter . The closest taxonomically described relatives were Serinibacter salmoneus Kis4-28T and Serinibacter tropicus PS-14-7T, with 97.20 and 97.20 % 16 s rRNA gene sequence similarity, respectively. The average nucleotide identity value of the whole genome sequence between strain K3-2T and S. salmoneus Kis4-28T was 78.9 %. DNA–DNA relatedness values between strain K3-2T and S. salmoneus DSM 21801T (=Kis4-28T) and S. tropicus VKPM Ac 2044T (=PS-14-7T) were 41 and 47 %. Thus, strain K3-2T represents a novel species of the genus Serinibacter for which the name Serinibacter arcticus sp. nov. is proposed. The type strain is K3-2T (DSM 103859T=VKM Ас−2719T).

Analysis of the aerobic heterotrophic component of bacterial communities from samples of poorly studied ancient ice wedge from the Mamontova Gora section (Central Yakutia), was carried out. The abundance of colony forming cells (CFU) of aerobic heterotrophic bacteria detected by plating of the thawed samples onto an agar nutrient media varied within 103‒104 CFU/mL. At the same time, the total number of intact cells was only 1−2 orders of magnitude higher. The taxonomic position of bacteria was determined by analysis of the 16S rRNA gene sequences. These bacteria were represented by three phyla: Actinobacteria, Firmicutes, and Proteobacteria. The temperature growth range of 8–20°C, which is characteristic of psychrotolerant bacteria, proved to be a specific physiological feature of the isolated strains. Most bacteria were found to be sensitive to a wide range of antibiotics.

The estimation of nodulation competitiveness of industrial strains against the native nodule bacteria and also the analyses of distribution of strains genotypes which formed nodules on roots of 4 plant species was the purpose of this work. The objects of the research were rhizobium strains which formed nodules on roots of plant (the nodule-forming units NFU), obtained in field experiment with application of preseeding processing of seeds of pea (Pisum satіvum L.), fava beans (Vicia faba L.), lathyrus (Lathyrus sativus L.) and lentil (Lens culinaris L.). The mixture of the collection strains allocated from nodules of peas and beans, and having various combinations of chromosomal and symbiotic genotypes was used for inoculation of seeds. Identification of NFU was carried out with the use of the analysis of the emitted nodule total DNA on several chromosomal markers: the rpoB gene and the hin-region, and the plasmid marker nodD gene. It is established that only about 50% of nodules were formed by the strains used at inoculation of seeds. Besides, the combinations of chromosomal and symbiotic genotypes specific for a rhizobium symbionts of concrete host plants have been established: IA genotype with sym-2 for P. sativum; IB genotype with sym-4 for V. faba. The results of this study create prerequisites for selection of couples: macro- and microsymbiont for the purpose of increasing efficiency of plant-microbial systems, in which the nature of symbiotic interaction defines efficiency of partners.

A bacterial strain (FM3) that is closely related to Stenotrophomonas acidaminiphila and S. maltophilia, was isolated from the skin surface of the frog Xenopus laevis. Cytophysiological studies on vegetative cells and cyst-like cells (CLCs) that were obtained in model experiments addressed the dynamics of transition of vegetative cells to the dormant state and their reversion to vegetative growth. The ultrastructural organization of the vegetative and dormant cells of strain FM3 possesses unique properties. Cultures that developed after inoculating vegetative cells were characterized by: (1) resistance to physical factors and sterilization procedures; (2) high antimicrobial activity with respect to some gram-positive and gram-negative bacteria; (3) resistance to polypeptide antibiotics; (4) the presence of an easily detaching S-layer on the cell surface; (5) the ability to secret outer membrane vesicles into the intercellular space; and (6) formation of S-layerderived tubular structures associated with outer membrane vesicles that are regularly arranged within the tubes. Dormant cells were characterized by: (1) resistance to dehydration; (2) resistance to high temperatures; and (3) the preservation of the S-layer on the surface of cystlike cells (CLCs). Depending on experimental conditions, strain FM3 formed three CLC morphotypes, which differed in their abundance and ultrastructural organization. The experimental conditions used for CLC formation approximated those under which bacteria survive in hospitals. A model of intermicrobial parasitism is suggested that applies to motile FM3 cells during the development of their populations (cultures).

Our previous studies demonstrated the formation of structurally diverse DNA-containing microparticles (DNA MPs) in PCR with Mg-pyrophosphate (MgPPi) as the structure-forming component. These DNA MPs were referred to major structural types: microdisks (2D MPs) with nanometer thickness and 3D MPs with sophisticated morphology and constructed from intersecting disks and their segments. Little is known about factors that influence both the morphology and size of DNA MPs, and the present study was aimed at fulfilling this gap. We showed that the addition of Mn2+ cations to PCR mixtures caused the profound changes in MPs morphology, depending on DNA polymerase used (KlenTaq or Taq). Asymmetric PCR with 20-fold decrease in the concentration of one of two primers facilitated the predominant formation of microdisks with unusual structure. The addition of 1 mM Na-pyrophosphate to PCR mixtures with synthesized DNA and subsequent thermal cycling (10-15 cycles) were optimal to produce microdisks or nanometer 3D particles. Using electron microscopy, we studied also the structure of inorganic micro- and nanoparticles from MgPPi, formed during multiple heating and cooling cycles of a mixture of Mg2+ and Na-pyrophosphate in various regimes. Also, we found the conditions to yield planar (Mg·Mn)PPi nanocrystals (diameter ~100 nm and thickness ~10 nm) which efficiently adsorbed exogenous DNA. These inorganic nanoparticles are promising for DNA delivery in transfection studies. Mechanisms to be involved in structural modifications of MPs and perspectives of their practical application are discussed.

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species.

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.

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.

An issue on the cellular forms that ensure survival of pseudomonads is important due to wide occurrence of these bacteria in the environment and their role for clinical microbiology. The present work demonstrates the high survival potential of Pseudomonas aurantiaca and P. аeruginosa in the mass of exopolymers produced by cells. Exopolymer formation occurred only during incubation of the post-stationary phase cultures of P. aurantiaca (at 4°C) and P. aeruginosa (at 4 and 20°C). After storage for 1.5–12 months, the number of colony-forming units in the exopolymer was 30 to 68% of the viable cell titer in stationary-phase cultures. Antibiotic-tolerant persister cells that were revealed in the exopolymer cultures after treatment with ciprofloxacin (2.5–100 μg/mL) were more resistant to the antibiotic than persisters in suspension cultures, with the threshold doses of 25 and 2.5 μg/mL, respectively. The cells embedded in the exopolymer were found to be more resistant to 5-min heating at 60–70°C than the vegetative cells of suspension cultures, which did not survive such heat treatment conditions. Electron microscopic investigation revealed morphological heterogeneity of exopolymer-embedded pseudomonads, including the presence of the cells similar to cystlike dormant forms. The populations developing on solid media inoculated with the exopolymer mass with cells were found to contain 1.5 to 2 orders of magnitude more persisters tolerant to high ciprofloxacin doses (25 μg/mL for P. aurantiaca and 100 μg/mL for P. aeruginosa) than the populations developing after inoculation with second-transfer vegetative cells of the cells of planktonic cultures. The results obtained improve our understanding of pseudomonad survival in the environment.

To the present, different efficient but expensive, multistage, and time-consuming technologies have been developed to deliver ribonucleic acids (RNA) into eukaryotic cells. Here, we report a simple and feasible solution to design RNA nanocarriers based on nucleic acid condensation by bi- and trivalent metal ions during thermal cycling. Efficient RNA conversion to nanoparticles with small size (10-50 nm) suitable for transfection was achieved using cations Ni

Ice wedges differ from other types of surface and underground glacial bodies and are widely spread in perennially frozen sub-Arctic regions, but the bacterial and archaeal diversity in these permafrost features remains poorly studied. Here, we compared the prokaryotic community composition in the active layer and ancient, 13–19 kyr BP and ~ 40 kyr BP, ice wedge horizons from the same exposure profile of the Mammoth Mountain, using pyrosequencing 16S rRNA gene. The most abundant OTUs in the active layer were affiliated with Acidobacteria (31.81%) followed by Actinobacteria (18.29%), Proteobacteria (18.14%), Gemmatimonadetes (7.3%), Parcubacteria (7.13%) and Bacteroidetes (6.49%). The prokaryotic community in 13–19 kyr BP ice wedge differed at the phylum level by the predominance of Actinobacteria (29.15%) over Acidobacteria (19.52%), Proteobacteria (18.45%), Verrumicrobia (5.88%), Firmicutes (2.98%) and Gemmatimonadetes (2.87%). In contrast, the oldest (~ 40 kyr BP) ice wedge prokaryotic community was rather poor, and only three phyla Firmicutes (54.48%), Proteobacteria (31.42%) and Bacteroidetes (7.92%) constituted the major fraction of reads. Archaeal sequences contributed with no more than 0.6% to total reads in all studied samples. Apparently, the Mammoth Mountain exposure profile harbors insular microbial communities with specific structure that reflects the stratigraphy, properties and age.

A method is proposed for integral assessment of the propagation of microbial cells and phage particles during seasonal thawing of relic ice wedge layers. The results of field and laboratory investigation carried out in the upper part of permafrost exposure at Mamontova Gora (Yakutiya, Russia) are presented. Suspensions of yeast, bacteria, and two coliphages were introduced as biomarkers directly on the surface of thawing ice and in the meltwater flow. Microorganisms and phages were shown (a) to possess particular parameters of dissemination in the meltwater flow and (b) were able to move 132 m in 25–35 min with the stream water flow.

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.

This work is devoted to studying the mechanisms of formation of DNA-containing microparticles (MPs) during PCR. It was found that pyrophosphate, a byproduct of DNA synthesis, and magnesium cations are required for their formation, as evidenced by the results of biochemical and electron microscopy studies.

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.

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 non-spore-forming gram-positive bacterium Mycobacterium smegmatis mc2 155, related to M. tuberculosis, was revealed to be capable of forming different types of dormant forms (DFs) during the life cycle of its cultures. The relationship between the intraspecies diversity of DFs and the cultivation conditions of the mycobacterium was established. The DFs possessed the following common properties: (i) maintenance of viability for a long period of time (5 months), (ii) resistance to deleterious factors such as heat treatment, and (iii) morphological and ultrastructural peculiarities that distinguish DFs from vegetative cells. The diversity of M. smegmatis DFs manifested itself in differences in terms of structural organization, conditions required for growth renewal, and capacity to produce antibiotic-resistant variants upon germination on selective media. Well-differentiated cystlike dormant cells (CDCs) were formed in the cultures grown in synthetic SR1 medium with fivefold-decreased nitrogen content. The structural organization of CDCs differed from that of other DF types mainly in the presence of club-shaped cells, thickened lamellar cell walls, coarse cytoplasm texture, and large electron-transparent triacylglyceride inclusion bodies. It was possible to use mycobacterial CDCs as a source of PCR-competent DNA. CDC populations were heterogeneous in cell buoyant density, and the individual fractions, which we isolated, were found to differ in thermal stability and the ability to revert to growth under standard conditions. Coccoid DFs, which retained their colony-forming capacity for a long time but were less heat-resistant than the CDCs, were formed by mycobacteria grown in standard Sauton’s medium with initial pH value decreased to 6.2. Poorly differentiated DFs resulted from growing mycobacterial cultures in Sauton’s medium with a fivefold-decreased phosphorus content. Upon germination of various DF types, the variants resistant to kanamycin (200 μg/ml) and tetracycline (20 μg/ml) were obtained. CDC suspensions incubated for 5 months demonstrated the highest percentage (1.5%) of antibiotic-resistant clones. The data obtained on the DF diversity of M. smegmatis, a fast-growing relative of M. tuberculosis, contribute to our understanding of the flexibility of the survival strategy of this bacterium in nature and in the host organism.

The new model of obtaining of ovoid resting forms Mycobacterium smegmatis, which are morphologically different from vegetative (rod-like) cells, was developed. Ovoid forms were characterized by a drastically decreased level of metabolic activity, an increased stability to heat processing and antibiotics action, and also by prolonged (more than 2 months) storage time preserving colony-forming ability. Obtained resting forms of mycobacteria may be used in test-systems for checking efficiency of new medical agents against latent forms of tuberculosis and determination of role of these of those genes in entering rest state.

Reversible transition of the phytopathogenic gram-negative bacterium Erwinia carotovora, subsp. atroseptica, strain SCRI1043, to a dormant state was demonstrated; it was associated with a complete loss of cell ability to form colonies on the standard medium, i.e., with acquiring “non-culturability”. Entering of Erwinia cells to a nonculturable state occurred after long-term incubation (100–150 days) of the stationary-phase cell suspensions in either a fresh complete medium or in the carbon-free mineral medium or treatment with a chemical analogue of microbial anabiosis autoinducers (4 × 10−4 M of C12-alkylhydroxybenzene, AHB). However, confocal laser microscopy of the cells stained with the Live/Dead BacLight kit revealed that the majority of E. carotovora cells (90%) from long-incubated suspensions retained membrane integrity. In these suspensions, round cells of smaller size prevailed, with the envelope, containing an electron-dense outer layer and an underlying layer of lower density; the cytoplasm was coarse-granulated. Detection of “nonculturable” E. carotovora cells by quantitative real-time PCR analysis (Q-PCR) with specific primers by using standard procedures of sample preparation was shown to be inefficient. A special procedure including cell washing from the incubation medium in the absence of growth stimulation was developed, which promoted recovery of the colony-forming ability of the cells (up to 10% of the initial CFU number) and improved cell detection by Q-PCR from the number of genomic copies. The results provided further insight into the ways of long-term survival of phytopathogenic bacteria under environmental changes and carbon starvation.

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa+Gri+, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa+Gri− (swarming) phenotype and the Sm variant was nonmotile (Swa−Gri− phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa+Gri−), the mucoid M type showed the mixed type (Swa+Gri+) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.

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 Sauton's 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.

The effect of hexylresorcinol (HR), a chemical analogue of microbial anabiosis autoinducers of the alkylhydroxybenzene (AHB) group, on the stability of biological membranes and monolamellar liposomes formed of egg phosphatidylcholine (ePC) was studied. According to spectrophotometry and electron microscopy studies of HR-loaded liposomes in the presence of a surfactant Tween 20, the critical ratio between HR and ePC for liposome preservation was found to be close to equimolar. The trends in HR influence on membrane structural organization and stability of liposomes were also confirmed in experiments on intact bacterial cells explaining non-species-specific effect of AHBs. The demonstrated high efficiency of AHB biocides may be used in material and equipment protection against biocorrosion.

Differences in viscoelasticity (η) and molecular mass (M) values, as well as in the fatty acid profile of lipids in DNA supramolecular complexes (SC), isolated from Pseudomonas aurantiaca cultures at the exponential and stationary growth phases, were established for the first time. Typical characteristics of DNA SC from actively growing cells were the following: η = 315 ± 15 dl/g, MDNA = 39 × 106 Da, C16:0 > C18:0 > C18:1 present as basic fatty acids (FA) in a pool of loosely DNA-bound lipids; the tightly DNA-bound lipid fraction consisted of only two acids C18:0 > C16:0. Significantly higher values of viscoelasticity η = 779 ± 8 dl/g and MDNA = 198 × 106 Da were observed for DNA SC of the stationary phase cells; one more FA, C14:0, was detected in the loosely bound lipid fraction, while lipids tightly bound to DNA contained mainly C16:0 > C18:1 > C18:0 > C14:0 FA. The content of saturated FA in the DNA-bound lipids in the stationary phase cells was twice as high than in the exponential phase cells. The fraction of tightly bound lipids from the stationary phase cells contained nine times more unsaturated fatty acids than the fraction from proliferating cells. These differences in FA composition of DNA-bound lipids demonstrate the importance of lipids for the structural organization and functioning of genomic DNA during bacterial culture development.

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.

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.

The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is ≥ 80 nm, ∼40–50 nm, and ≤ 20 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40°C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg(2+) ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg(2+)-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.

The existence of Pseudomonas aurantiaca DNA-bound fatty acids and lipids is presented in this work. The isolation of DNA was carried out by two different procedures, namely, phenol and detergent-based phenol isolation in order to prove the presence of DNA-bound lipids. The lipid content of DNA is expressed in terms of fatty acid profile. A high level of 16:0, 18:0 and 18:1 is characteristic for tightly bound DNA lipids. On the other hand, the fatty acids such as 14:1, iso14:0 and iso16:0 are found in trace amounts only in DNA lipid fraction, but these fatty acids are not found in the whole-cell lipids. Absolutely no 3-hydroxy fatty acids were found in DNA lipids. However, both C16 and C18 species represent the main fatty acids of whole-cell and DNA-bound lipids. The presence of DNA-bound lipids even under tough treatment of DNA allows to conclude that these lipids represent a special pool among cellular lipids.

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.

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.

We conducted a comparative study of the effects of alpha-amino-gamma-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of gram-positive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10(-5)-10(-3) M HSL or 10(-5)-10(-4) M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10(-4)-10(-3) M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10(-4) to (1.5-2.5) 10(-4) M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10(-4) M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075-0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions on the microbial community level.

Successful missions to Mars, Europe and other bodies of the Solar system have created a prerequisite to search for extraterrestrial life. The first attempts of microbial life detection on the Martian surface by the Viking landed missions gave no biological results. Microbiological investigations of the Martian subsurface ground ice layers seem to be more promising. It is well substantiated to consider the Antarctic ice sheet and the Antarctic and Arctic permafrost as terrestrial analogues of Martian habitats. The results of our long-standing microbiological studies of the Antarctic ice would provide the basis for detection of viable microbial cells on Mars. Our microbiological investigations of the deepest and thus most ancient strata of the Antarctic ice sheet for the first time gave evidence for the natural phenomenon of long-term anabiosis (preservation of viability and vitality for millennia years). A combination of classical microbiological methods, epifluorescence microscopy, SEM, TEM, molecular diagnostics, radioisotope labeling and other techniques made it possible for us to obtain convincing proof of the presence of pro- and eukaryotes in the Antarctic ice sheet. In this communication, we will review and discuss some critical issues related to the detection of viable microorganisms in cold terrestrial environments with regard to future searches for microbial life and/or its biological signatures on extraterrestrial objects.

Peculiarities of the structural organization in non-spore-forming bacteria associated with long-term anabiosis were revealed both in laboratory cultures and in natural populations isolated from 1–3-Myr-old Eastern Siberian permafrost and tundra soil. Different advanced methods were used, including (a) high-resolution electron microscopy; (b) simulation of in situ conditions in the laboratory by varying the composition of growth medium and cultivation conditions; (c) low-temperature fractionation to isolate and concentrate microbial cells from natural soils; (d) comparative morphological analysis of microbial cells in model cultures and natural soils (in situ). Under laboratory conditions, the intense formation of resting cells by representatives of various taxa of eubacteria and halophilic archaea occurred in 2–9-month-old cultures grown in carbon-, nitrogen-, or phosphorus-limited media, in starved cell suspensions in the presence of sodium silicate, or on soil agar. Among resting cells, we revealed cystlike forms having a complicated structure and common features. These included a thick capsule; a thickened and multiprofile cell wall; the presence of large intramembrane particles on PF- and EF-fracture surfaces; fine-grained or lumpy cytoplasm; and a condensed nucleoid. The general morphological properties, ultrastructural organization, physiological features of cystlike cells, and their ability to germinate under the appropriate conditions suggest the existence of constitutive dormancy in non-spore-forming bacteria. It was found that the majority of microorganisms in permafrost and tundra soil are cystlike cells, very similar to those in laboratory cultures. Anabiotic (resting) cystlike cells are responsible for the survival of non-spore-formers in extreme Earth habitats and may be regarded as possible analogs of extraterrestrial forms of microbial life.

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.

Low-molecular-weight cell-to-cell communication factors are produced by various pro- and eukaryotes and involved in autoregulation of the growth and development of microbial cultures. As for some bacterial and yeast species, these factors were identified as isomers and homologues of alkylhydroxybenzenes (AHB). Depending on the concentration, they participate in controlling the transition to stationary phase, entering the resting state, and stress resistance of vegetative cells to gamma-irradiation, photooxidation (singlet oxygen), and heat shock. Chemical analogues of microbial AHB protected microbial cultures from stressful situations and exerted (1) the stabilizing activity toward macromolecules and (2) the ability to scavenge active oxygen species. The stabilizing effect of AHBs resulted from their complex formation with protected macromolecules due to intermolecular hydrogen bonds, hydrophobic and electrostatic interactions and was demonstrated on models of individual enzymes (trypsin). Particularly, AHBs protected the yeast from the action of (a) active oxygen species formed during gamma-irradiation (500 Gy, 1.96 Gy/s) or (b) singlet oxygen generated in cells photosensitized by chlorin e 6 (10 μg/L). It is important that microbial AHBs were not species-specific and defended cultured microbial and animal cells from the action of organic toxicants. The use of AHBs as protectants and adaptogens is discussed as well as perspectives of further investigations.

X-ray microanalysis showed that vegetative cells, viable resting forms, and nonviable forms (micromummies) of the bacteria Bacillus cereus and Micrococcus luteus and the yeast Saccharomyces cerevisiae differ in the content of elements S, P, Ca, and K and Ca/K and P/S ratios. Viable resting forms (cystlike refractive cells and bacillar endospores) had more calcium and less phosphorus and potassium than vegetative cells, the difference being higher for bacilli than for micrococci and yeasts. The distinctive feature of all viable resting microbial forms was their low P/S ratios and high Ca/K ratios. The differences revealed in the cellular content and ratios of elements probably reflect changes in ionic homeostasis accompanying the transition of vegetative microbial cells to the dormant state. Relevant potassium parameters indicate that the membranes of viable resting forms retain their barrier function. At the same time, the nonviable micromummies, even those morphologically intact, of B. cereus and S. cerevisiae exhibited an anomalously low content of potassium, while those of M. luteus had an anomalously high content of this element. This suggests that the cellular membranes of micromummies lose their barrier function, which results in a free diffusion of potassium ions across the membranes. The possibility of using the elemental composition parameters for the quick analysis of the physiological state of microorganisms in natural environments is discussed.

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d1 factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to an anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 μg/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C1-AHB) and hexylresorcinol (C6-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C6-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C1-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C6-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes: the phenotypic dissociation of microbial populations and the formation of resting microbial forms, is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.

This work has a focus on adaptive capabilities of the actinobacterium Rhodococcus ruber IEGM 326 to cope with drotaverine hydrochloride (DH), a known pharmaceutical pollutant. Cultivation of R. ruber in a nitrogen-limited medium with incubation at the ambient temperature resulted in the formation of cyst-like dormant cells (CLDCs). They maintained viability for 2–7 months, possessed the undetectable respiratory activity and elevated resistance to heating, and had a specific morphology. CLDCs are regarded to ensure long-term survival in various habitats and may be used as storage formulations. R. ruber IEGM 326 was tolerant to DH (MIC, 200 mg/l) and displayed different abilities to degrade this compound, depending on inoculum, temperature, and the presence of glucose as co-oxidized substrate. Thus, the loss of DH (20 mg/l) over 48 h at the optimal temperature (27 ± 2 °C) was 5–8 % in the absence of glucose after inoculating with vegetative cells. The addition of glucose (5 g/l) increased DH degradation up to 46 %. Noteworthy, CLDCs as inoculum were advantageous over vegetative cells to degrade DH at the non-optimal temperature (35 ± 2 °C) at reduced bulk respiratory activity. The obtained results are promising to improve the biodegrading capabilities of other Rhodococcus strains.

Under the influence of alkyl hydroxybenzene (C6-AHB) added to cell suspensions at concentrations of (1–5) × 10–3M, the cells of Saccharomyces cerevisiae, Micrococcus luteus, and Thioalkalivibrio versutusunderwent 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 Micrococcus luteusit 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.

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.

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg(2+)-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils - that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg(2+) - mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.