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The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.

Forespore formation in the anaerobic bacterium Anaerobacterpolyendosporus PS-1(T) was studied by phase contrast, fluorescence, and electron microscopy. It is concluded that in this bacterium the formation of all forespores in multispore sporangia occurs via the same mechanism as that operating in all known bacilli and clostridia during the single-spore variant of endogenous sporogenesis. Its cytological indicators are as follows: (1) formation of the forespore septum, (2) engulfment of the smaller prespore cell by the larger mother cell, (3) cortex synthesis, (4) assembly of the spore coats, (5) exosporium formation, and (6) lysis of the mother cell. Polysporogenesis in strain PS-1(T) is characterized by synchronous formation of all spores (siblings) in a given sporangium and by the absence of any indication of forespore division within the mother cell. These data suggest that multiple spores within a single PS-1(T) cell result not from division of the first forespores developing at one or two cell poles, as it was reported for another polysporogenic bacterium, "Metabacterium polyspora", but rather from simultaneous independent formation of several prespores in a single mother cell in the course of modified cell division.

We isolated 50 strains of free-living ultrasmall bacteria with a cell volume that varies from 0.02 to 1.3 microm3 from a range of extremal natural biotopes, namely permafrost soils, oil slime, soils, lake silt, thermal swamp moss, and the skin integuments of the clawed frog, Xenopus laevis. Of them, 15 isolates, characterized by a cell size of less than 0.1 microm3 and a genome size from 1.5 to 2.4 Mb, were subsumed to ultramicrobacteria belonging to different philogenetic groups (Alphaproteobacteria, Bacteroidetes, Actinobacteria) and genera (Kaistia, Chryseobacterium, Microbacterium, Leucobacter, Leifsonia, and Agrococcus) of the Bacteria domain. They are free-living mesophilic heterotrophic aerobic bacteria. The representatives of Kaistia and Chryseobacterium genera were capable of facultative parasitism on other species of chemo-organotrophic bacteria and cyanobacteria. The ultramicrobacteria differed in their morpholgy, cell ultrastructural organization, and physiological and biochemical features. According to the fine structure of their cell walls, the isolates were subdivided into two groups, namely Gram-positive and Gram-negative forms.

The number of spores formed in a single cell ofAnaerobacterpolyendosporus PS-1T is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one-two to five-seven. Investigation of spore formation by fluorescence and electron microscopy revealed that on media with 0.5-1.0% glucose or galactose most of the vegetative cells remained rod-shaped after cessation of cell division in the culture. Their nucleoids were localized at cell poles close to the polar site of the cytoplasmic membrane. Forespores were formed at one or both of these poles. A satellite nucleoid (operator) was detected close to each forespore. In the variant with bipolar organization of mother cells only one or two spores per cell were formed. In the second variant of cultivation, when the cells grew at low galactose concentrations (0.1-0.3%), most of the vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acids-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred during the early stationary phase. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in a number of the oval and spherical cells.

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

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1(T), as dependent on the cultivation conditions, are described in detail. High growth rates (0.22-0.30 h(-1)) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 degrees C on medium with inorganic (Fe(II), S(0), or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 degrees C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 degrees C and 12-14 degrees C) decreased two- and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of 14CO2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant sulfobacillus are compared versus moderately thermophilic sulfobacilli.

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 degrees 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.

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1-3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29-43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of < or =0.3 microm and volume of < or =0.014 microm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18-0.20 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400-800 nm and ultrasmall cells of 200-300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB: these include the following: (a) piles of rod-like subunits, ca. 30 A in diameter and 150-250 angstroms in length: (b) long bunches (up to 300-400 angstroms) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200-300 angstroms in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB eytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2-3) located directly on the mother cell, without formation of intennediate hyphae.

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 > or = 80 nm, 40-50 nm, and < or = 0 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40 degrees 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.

The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.

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.

The yeasts Saccharomyces cerevisiae and Pichia pastoris and the bacteria Micrococcus luteus, Bacillus subtilis, and Anaerobacter polyendosporus have been treated with the chaotropic agents guanidine hydrochloride and guanidine thiocyanate and certain detergents and studied using fluorescence microscopy. Studies with the use of fluorochromes that can selectively stain nucleic acids (diamidino-2-phenylindole (DAPI), propidium iodide, and acridine orange) show that treatment of the bacterial and yeast cells at 37 degrees C for 3-5 h induces a release of DNA from the cytoplasm and its accumulation in the cellular zone, known as ectoplasm, located between the cell wall and the remainder of the cytoplasm (called endoplasm) in the form of one or several large granules. After treating the cells with the chaotropic agents at 100 degrees C for 5-6 min, the DNA is diffusively distributed over the ectoplasm. The fluorochromes used do not allow the detection of RNA. These findings are in agreement with previous data obtained from electron microscopic study of thin cell sections. After 33 PCR cycles, a considerable portion of DNA leaves the cells; as a result, they show a low level of diffusive fluorescence when stained with DAPI. When endospores of B. subtilis are treated with the chaotropic agents, they become highly permeable to the fluorochromes. Fluorescence microscopic study of such endospores shows that they contain DNA in the central part of their cores.

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas auraniaca 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 elastoviscosity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their elastoviscosity 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 elastoviscosity 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 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 genotype and dissociate into distinct variants upon inoculation on solid media.

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground represented resting forms (spores, cysts, and cyst-like cells with specific organo-mineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy in studying microbial communities in situ.

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

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

The paper deals with the X-ray microanalysis of the elemental composition of bacteriomorphic particles in 170,000-year old Antarctic permafrost sediments and in indoor dust. A comparative analysis of the phosphorus, sulfur, calcium, and potassium contents and the Ca/K and P/S ratios in these particles and in reference microbial cells occurring in different physiological states showed that the absence of P and/or S peaks in the X-ray spectrum of an object may indicate that it is abiotic. Resting microbial forms can be revealed on the basis of the following characteristic features: an increased content of Ca, a high Ca/K ratio, and a low P/S ratio. Model experiments with nonviable bacterial and yeast micromummies with alterations in the structural and barrier functions of the cytoplasmic membrane showed that micromummies can be recognized by a super-high content of a marker element (e.g., P, K, or Si), accumulated due to facilitated diffusion along the deliberately created concentration gradient. Such an analysis of the permafrost sediment and dust made it possible to suggest the presence of mummified cells in these objects. The possibility of using X-ray microanalysis for the detection of microbial cells in natural habitats in order to enhance the efficiency of ecological monitoring of the environment is discussed.

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 contents of bioelements S, P, Ca, and K and the Ca/K and P/S ratios. Viable resting forms (cystlike refractory 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 bioelements 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 forms, even 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 quick analysis of the physiological state of microorganisms in natural environments is discussed.

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.

Under the influence of alkyl hydroxybenzene (C6-AHB) added to cell suspensions at a concentration of (1-5) x 10(-3) M, the cells of Saccharomyces cerevisiae, Micrococcus luteus, and Thioalkalivibrio versutus underwent 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 slit-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 slit-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 luteus it 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.

Some physiological and cytological properties of Pseudomonas putida strains resistant to arsenite and capable of degrading polycyclic aromatic hydrocarbons were studied. The resistance of P. putida BS202 (NPL-1) to arsenite proved to be determined by chromosomal genes, while the arsenite resistance of P. putida BS238 (pBS2; pBS3031) was by plasmid-borne genes. Arsenite affected the pattern and rate of growth of strain BS202 (NPL-1) in media with naphthalene or salicylate as carbon sources; particularly, it lengthened the lag phase. Electron-microscope analysis of the strains studied did not reveal any arsenite-induced destructive changes in the cell envelope. At the same time, arsenite in the growth medium induced some alterations in the structure of the outer membrane of strain BS202 (NPL-1) and the cytoplasmic membrane of strain BS238 (pBS2; pBS3031) and, in both strains, led to an increase in the density of intramembrane particles on the EF face of the freeze-fractured cytoplasmic membrane. Arsenite resistance probably evidently protects cells of both strains from greater damage. Physiological and cytological data suggest that the mechanisms of arsenite resistance in the strains studied are different.

Under experimentally selected conditions favoring spontaneous or induced autolysis of cell suspensions, the asporogenous bacteria Escherichia coli and Methylococcus capsulatus, the bacilli Bacillus cereus (under conditions of suppressed sporulation), and the yeast Saccharomyces cerevisiae were shown to be capable of forming cystlike resting cells. Their number was influenced by (1) cell density in the suspensions; (2) the presence of Ca2+ ions in nutrient-limited medium; (3) pH of medium; and (4) autolysis rate, dependent on the concentration of oleic acid (a chemical analogue of the autolysis-inducing d2 factor) introduced into the cell suspensions.

Under certain cultivation conditions, the bacteria Bacillus cereus and Micrococcus luteus form cystlike refractive cells (up to 60% of the total number) that retain viability over a long time, are metabolically inactive and thermotolerant and possess specific ultrastructure. These properties allow them to be attributed to a new type of resting forms of microorganisms.

The fine structure of Bacillus anthracoides spores is similar in general to that of other, taxonomically related species of spore forming bacteria. However, the former is characterized by a well-developed multilayered exosporium and the heterogeneous structure of an envelope. The lethal effect of a chloroactive disinfectant (2/3 of calcium hypochlorite basic salt) is caused by changes in the structural organization of spores, which interferes with the normal permeability barrier and metabolic processes.

Extracellular membrane-active compounds were shown to induce the transition of vegetative cells in the producing culture into resting refractile forms. The forms are characterized by the absence of energy, biosynthetic and destructive processes, by the capability to remain viable for a long time, and by a peculiar ultrafine organization. The physiological state and the ultrafine organization of B. cereus cells were found to change under the action of the autoregulatory factor introduced in the culture.

In the course of organoheterotrophous growth, Pseudomonas carboxydoflava Z-1107 and Bacillus cereus 504 were found to synthesize the autoregulatory factor d2, a membranotropic lipid metabolite, and to accumulate it in the growth medium. At a low concentration, the factor activated respiration of the cells: at a high concentration, it inhibited respiration and induced autolysis of the cells. The physiological action of d2 was shown to be due to the effect of free fatty acids, in particular, palmitic, stearic and oleic acids. Oleic acid displayed the highest physiological activity, P. carboxydoflava Z-1107 became more susceptible to high concentrations of d2 and oleic acid, and respiration of the cells was activated by low concentration of these compounds when the culture passed from the exponential growth phase to the linear one. These results as well as data reported in literature about the action of fatty acids on biological membrane suggest that low concentrations of the factor d2 uncouple respiration and oxidative phosphorylation whereas high concentrations of the factor disorganize the structure of the cytoplasmic membrane by increasing its fluidity.

The growth, development and changes in the chemical composition of Bacillus popilliae cells were studied in the course of their growth on a liquid chemically defined medium containing casein hydrolysate, complex vitamin mixtures and glucose. Growth of the culture on this medium was accompanied with diauxia which could be registered by changes in the rate of growth and synthesis of the main cellular polymers (DNA, RNA, protein, phospholipids). The rate of protein synthesis correlated with the specific growth rate which was maximal when the reserve substances of the cells or the compounds of partly digested biomass were used. The rate of protein synthesis in the experiment depended mainly on the protein synthesizing activity of RNA rather than on its content in the cells. The low rates of growth and protein synthesis, which suggest that the growth of the culture is being interested with in the course of utilization of the components of the nutrient medium, may account for the absence of spore formation by this strain on the medium used.

It has been found for the first time that an increase in the concentration of a specific autoregulatory factor in the medium is responsible for the hypometabolic state of vegetative cells; no principal cellular biopolymers are synthesized at this state (DNA, RNA, proteins and phospholipids), and no cellular structures are degraded. When the culture reverses to vegetative growth observed in the same medium, synthesis of the biopolymers is intensified and changes are detected in the ultrastructural organization of the cellular membranous apparatus, the cytoplasm, the nucleoide and the cell wall.

The localization of lipase was studied in the cells of Serratia marcescens 345. When the cells were treated with sodium cholate, the enzyme bound to the cellular structures was released in the solution. The supernatant obtained after the cells were treated with sodium cholate contained no glucose-6-phosphate dehydrogenase, a well known endocellular enzyme; therefore, the cytoplasmic membrane was not damaged by the treatment. Ultrathin sections of the intact cells and the cells treated with sodium cholate were examined using electron microscopy, and no disorders were detected in the cytoplasmic and outer membranes. It appears therefore that the lipase of S. marcescens 345 is located mainly on the exterior surface of the outer membrane.

Superoxide dismutase was found for the first time in the spores of the anaerobic bacterium Clostridium butyricum. The prosthetic group of the enzyme was shown to contain iron. The enzyme was demonstrated to be highly thermostable.

The presence, distribution and spatial arrangement of biopolymers in situ were studied in both a total intact spore and in a certain cellular layer using a spectroscopic technique of attenuated total refraction (ATR-IR) in the IR region. In contrast to vegetative cells, intact spores were characterized by isotropic distribution of protein components. This feature can be regarded as an index of the cryptobiotic state of spores. However, the distribution of protein components among individual layers of a spore was anisotropic. Bonds characterized by amide I and amide II bands were most often ordered in a layer which comprised cellular structures from the exosporium to the inner spore membrane.

A fraction of polar lipids was isolated from spores of the anaerobic bacterium Clostridium butyricum 35/11 exerting a noticeable radioprotective effect. The main biological activity of spore extracts was associated with this fraction. The fraction of polar lipids inhibited autolysis of the bacterial cell walls. The fraction was found to contain a phenolic glycolipid and a peptide component. The bacteriostatic and radiotherapeutic properties of the fraction are presumed to be due to its membranotropic activity.

The dormancy cyst-like forms of Bacillus cereus obtained under the action of an autoregulatory factor were shown to be more resistant to the action of such external factors of the medium as high temperatures, ultraviolet irradiation and low molecular weight alcohols comparing to the vegetative cells. Incubation at 80 degrees C for 15 min killed 98,5% vegetative cells and 86,5% of the dormancy refractile forms. U. V.--radiation treatment resulted in a loss of viability in 97% of the vegetative cells and in 75% of the refractile forms. Differences were also found in the resistance of the cyst-like forms and the vegetative cells to the action of ethanol, methanol and butanol. The resistance of the cyst-like forms of B. cereus to the action of the above damaging factors is consistent, in general, with the resistance of cysts, methylotrophic bacterial spores and other dormancy prokaryotic cells differing in the mode of their formation from endospores.

Contrary to other bacteria, those belonging to the genus Clostridium have their formation of vacuoles being strictly confined to the stage of spore production. In Clostridium, gas vacuoles are formed in the intraexosporial space, i. e. in the cytoplasm between the exosporial membrane and spore coats. Gas caps, being conical accumulations of gas vacuoles, impart increased floatation to spores and favour therefore their distribution in nature. Consequently, Clostridium species have ecological advantage if they can form gas vacuoles. The function of the exosporium and specific rod-shaped appendices of spore coats is discussed.

A chromatographically individual fraction with the biological activity of factor d1 was isolated from Pseudomonas carboxydoflava by the techniques of column and thin-layer chromatography. Alkyl hydroxybenzoles were shown to be the active principle of factor d1 inducing the transition of P. carboxydoflava vegetative cells into the hypometabolic or anabiotic state. As was found from the data of PMR, UV and IR spectroscopy and gas chromatography in combination with mass spectrometry, the active principle of factor d1 included alkylresorcins: 5-n-nonadecylresorcin and 5-n-heneicosylresorcin at a ratio of 1:3.

It has been found that, as soon as Bacillus cereus vegetative cells yield resting refractile forms under the action of specific autoregulating factors, the incorporation of labeled precursors of the main cellular biopolymers and lipids stops almost entirely and the level of cell endogenous respiration abruptly decreases. When the refractile forms revert to the vegetative state and growth, the processes of biosynthesis and respiration are restored. The level of metabolism typical of vegetative cells of the control cultures is reached within 90 min.

Peculiar trichome multicellular gram-negative bacteria were found in several types of soil, and were isolated as pure cultures. The bacteria are characterized by the following properties: (a) the presence of large regions of the periplasm, 200-300 nm thick, in the cells, the volume of the periplasm being greater than that of the cytoplasm; (b) a peculiar structure of multicellular trichomes in which the cells are surrounded by a common cell wall and a common periplasmic space; (c) a specific structure of trichome septa formed by membrane leaves and including no material of the cell wall; (d) the ability of protoplasts to divide within the trichome cell wall; (e) the ability to grow under anaerobic conditions. Therefore, these organisms belong to a new group of trichome multicellular bacteria.

The nucleotide composition of DNA from 12 studied species of anaerobic bacteria belongs to AT type, with G+C varying from 28.4 to 36.8 mole%. In the anaerobic group of Clostridium bifermentans, a correlation has been established between the nucleotide composition of DNA, the type of appendages on spores, and some physiologo-biochemical characteristics. The nucleotide composition of DNA in the spores of four anaerobic species is shifted toward GC type as compared to DNA in the vegetative cells. Data on the content of GC pairs in DNA of the spores may sometimes be of a higher taxonomic value than the corresponding evidence on DNA of the vegetative cells.