Sporulation of Bacillus subtilis in Binary Cultures with Ultramicrobacteria.

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.

[transversion of cell polarity from Bi- to multipolarity is the mechanism determining multiple spore formation in Anaerobacter polyendosporus PS-1T].

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.

[Fluorescence microscopic study of the microorganisms treated with chaotropic agents].

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.

[Ultrastructure of resting cells of some non-spore-forming bacteria]. / Tonkoe stroenie pokoiashchikhsia kletok nekotorykh nesporoobrazuiushchikh bakterii.

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.

[Changes in the fine structure of microbial cells induced by chaotropic salts]. / Izmenenie tonkoi struktury kletok mikroorganizmov pod vozgeistviem solei-khaotropov.

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.

[Fine structure of mummified cells of microorganisms formed under exposure to a chemical analog of anabiosis autoinducer]. / Tonkaia struktura mumifitsirovannykh kletok mikroorganizmov, obrazuiushchikhsia pod vliianiem khinicheskogo analoga autoinduktora anabioza.

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.

[Artificial immobilization of the two-membered bacterial system Bdellovibrio bacteriovorus 109D--Escherichia coli B in polyacrylamide gel]. / Iskusstvennaia immobilizatsiia dvukhkomponentnoi bakterial'noi sistemy Bdellovibrio bacteriovorus 109D--Escherichia coli v poliakrilamidnom gele.

The interaction of a parasite with a host was studied in the two-membered bacterial system, Bdellovibrio bacteriovorus 109D and Escherichia coli B, immobilised in polyacrylamide gel (PAAG). The parasite localised inside the host cells was found to be more resistant to the toxic action of PAAG components than free B. bacteriovorus. The latter lost its mobility and was inactivated in the matrix of the carrier whereas the intracellular parasite had a normal cycle of development in the periplasm of the infected cells. The dynamics of B. bacteriovorus and E. coli incidence in the liquid phase and in PAAG granules was studied while the immobilised system was incubated. The interaction in the immobilised system could be intensified by growing more bacterial host cells in PAAG particles. The immobilisation was shown to favour the survival of the parasite and the host in the two-membered system.