Search

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.

The surface structures of the spores of Bacillus. cereus, B. thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. Diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.

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.

From the silty sediments of the Khadyn soda lake (Tuva), a binary sulfidogenic bacterial association capable of syntrophic acetate oxidation at pH 10.0 was isolated. An obligately syntrophic, gram-positive, spore-forming alkaliphilic rod-shaped bacterium performs acetate oxidation in a syntrophic association with a hydrogenotrophic, alkaliphilic sulfate-reducing bacterium; the latter organism was previously isolated and characterized as the new species Desulfonatronum cooperativum. Other sulfate-reducing bacteria of the genera Desulfonatronum and Desulfonatronovibrio can also act as the hydrogenotrophic partner. Apart from acetate, the syntrophic culture can oxidize ethanol, propanol, isopropanol, serine, fructose, and isobutyric acid. Selective amplification of 16S rRNA gene fragments of the acetate-utilizing syntrophic component of the binary culture was performed; it was found to cluster with clones of uncultured gram-positive bacteria within the family Syntrophomonadaceae. The acetate-oxidizing bacterium is thus the first representative of this cluster obtained in a laboratory culture. Based on its phylogenetic position, the new acetate-oxidizing syntrophic bacterium is proposed to be assigned, in a Candidate status, to a new genus and species: "Candidatus Contubernalis alkalaceticum."

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.

Comparison of the forms of parasporal inclusions in Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis under a scanning electron microscope showed that, in addition to bipyramidal and cuboid crystals, cells of the subspecies kurstaki are able to form crystals taking the shape of flat parallelopipeds typical of the subspecies tenebrionis. The inclusions in the subspecies tenebrionis vary in size and electron density. Examination of the structure of crystals in B. thuringiensis subsp. tenebrionis under a scanning tunneling microscope showed that delta-endotoxins in the crystals are assembled in globules 5.0 nm in diameter and 1.0 nm in height. The globules are connected by its bases and form chains. The subunits of tetragonal crystals in the subspecies tenebrionis are vertically oriented with respect to the chain axis, as was revealed by other methods for subunits of bipyramidal crystals of the subspecies kurstaki.

The influence of the carbon source on biological activity and morphological properties of the parasporal crystals of Bacillus thuringiensis subsp. kurstaki cultivated on three different media has been studied. Biological activity of delta-endotoxins contained in crystals was tested according to their impact on insects and microorganisms. Variations in carbon sources composition resulted in changes of biological activity and morphology of crystals, caused by alterations in synthesis rate of various delta-endotoxins contained in the crystals. Variations in the content of a certain carbon source (e.g. starch) in the medium produced no effect on the crystals properties, and the content variations of the delta-endotoxins content was proportional to the producer biomass. Control of the antibacterial effect of delta-endotoxins allows a valid evaluation of their biological activity under various growth conditions as well as comparison of the biological potential of entomopathogenic chemicals for plant protection containing delta-endotoxins with different specificity of insecticidal activity.

Spores and crystals of various Bacillus thuringiensis serotypes were studied by electron microscopy. The serotypes were shown to differ in the fine structure of the exosporium and crystals. The packing parameters of morphological subunits were determined by analysing electron photomicrographs by the technique of optical diffraction and filtration. Some of the strains are characterised by hypersynthesis of surface spore structures. The individual morphological properties of crystals can be used for their differentiation.

The formation of spores was studied with a morphologic type of the Actinomadura genus, Acd. verrucosospora (Nonomura a. Ohara, 1971. Spore formation begins with the division of a hypha by cross-partitions which, as a rule, appear as monolayered on cross-sections, thus differing from sporulation septae found in other actinomycetes. The structure and shape of septae change in the course of spore formation: first, they consist of one layer and have the same thickness as the cell wall; later, they consist of several layers and their thickness is uneven (the shape of dumbbels). In the course of spore maturation, thick regions of the septae are destroyed. Mature spores are characterized by the presence of vacuoles, the thichened cell wall and outer sheath; the latter bears rounded structures arranged in conglomerates which resemble grapes. The process of sporogenesis and the structure of spores in Actinomadura verrucosospora differ from those in other species of the Actinomadura genus (Williams et al., 1974; Soina et al., 1975); therefore, this genus of actinomycetes is heterogenous.

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.

Ultrastructural changes in the mycelium of Actinomyces (Streptomyces) chrysomallus 2703 producing the antibiotic chrysomallin in the process of submerged fermentation are described. When the antibiotic was produced at a high rate, most hyphae remained viable for a long period of time and had the ultrastructure typical of the young cells of actinomycetes. At the same time, a number of degenerative changes due to aging were observed; these resulted in autolysis. A decrease in the antibiotic titre was accompanied with intensive sporulation in a considerable number of the submerged hyphae. Structural changes in the sporeforming hyphae distinctly differed from those in the vegetative ones: the cell walls became thicker and numerous septa were formed. The exogenous formation of submerged spores in the culture under study has been studied in detail for the first time.

Vegetative cells and spores of the colonial morphological mutants of Bacillus subtilis A-50 were studied by electron microscopy. The ultrastructure of vegetative cells from both asporogenic colonial-morphological mutants and those which were capable of forming spores in the presence of high concentrations of nitrogen and carbon sources with a decreased activity and a modified spectrum of serine proteases differed from the parent strain by the presence of a microcapsule, the uneven thickness of a cell wall, and the absence of a distinct periplasmic space. Crystalline inclusions of a regular shape were detected in the sporeforming mutant in those cells which were devoid of spores. Spores of the mutant had additional layers.

The paper describes cultures that can be assigned to the genus Micropolyspora according to such properties as the presence of aerial mycelium, catenae of spores and galactose in the composition of sugars in the cell wall. However, the cultures contain xylose in the cell wall and therefore are related to the genus Micromonospora. These findings as well as the atypical proportion between DAPA and its isomers suggest that the cultures occupy an intermediate position between the genus Micromonospora and the genus Micropolyspora.

Both vegetative Bacillus stearothermophilus cells 3 and cells with mature spores undergo lysis during submerged cultivation in media with starch and maltose as was shown by light and electron microscopy. This can be caused by enzymes lysing cell wall and synthesized at the beginning of the culture growth. The fine structure of the strain 3 spore has certain peculiarities: it contains no exosporium and the inner envelope has a heterogeneous structure.

The submicroscopic organization and spore formation were studied in two strains of Desulfotomaculum nigrificans, 435 and 781. Cross-sections of cells fixed with osmic acid and contrasted with uranyl acetate and lead citrate revealed a cell wall devoid of the outer lipoprotein membrane, which was typical of gram-positive bacteria. The cell wall consisted of three electron-dense layers and two layers of lower electron density. A layer of a microcapsule having fibrillar structure and a thickness of 23-33 nm was detected on the surface of the cell wall. The cytoplasmic membrane and intracellular membrane structures were represented by the Robertson membrane about 9 nm thick. The cytoplasm contained electron-dense polyribosomal granules and inclusions having fine-granular structure. The nucleoid was located in the central part of the cell as an electron-transparent zone penetrated with electron-dense DNA threads 2.5 nm thick. The paper presents microphotographs of the main stages of spore formation which follows the mechanism described for bacteria of the Bacillus genus. The poles of spindle-like cells have electron-dense disk-shaped formations covered with a three-layer membrane and containing electron-dense granules, apparently, corresponding to polyribosomes.

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.

When amino acids are added to the culture of Bacillus polymyxa growing under the conditions of active spore formation, the rate of polymyxin biosynthesis and the quantity of spores being formed decrease. All cells in the population start to produce spores under these conditions, but only some of them pass through all of the differentiation stages. Most of the cells remain in the state of prospores. Some cells which have started to differentiate are characterized by ultrastructural anomalies, do not form prospores and undergo early lysis.

A new species of the Actinomadura genus, A. fastidiosa sp. nov., is described. The ultrastructure of the vegetative mycelium and spores of this organism was studied. The vegetative cells have a multilayered cell wall, often consisting of five layers with different thickness and electron density. The spores are similar to the vegetative cells by their inner structure but have a thicker wall.

The structure of spore surfaces was studied by electron microscopy in five species of actinomycetes, and two new types of elementary surface structures were discovered. The surface of spores in Streptomyces regensis consists of long flexible, closely interwoven, band-like structures 25 A wide. Ring-like structures 140--150 A in diameter can be observed on spores of Actinomadura verrucosospora; their clusters resemble grapes and the spore silhouette looks like a cone. Rod-like structures of spores in Streptomyces cavourensis and Str. streptomycini which form an ornament resembling the surface of a basket and tubular strucutres in Str. acrimycini of which hair-shaped spore protrusions are made are similar to the elementary structures of spore surfaces in other actinomycetes.

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.

Spore germination was studied in Bacillus thuringiensis subsp. galleriae. Optimal temperature regimes were selected for spore activation and initiation. The formation of spores and the rate of their subsequent germination were shown to depend on the composition of growth media in which the organism was cultivated. The paper describes changes in the fine structure of spores at different stages of their germination.

The spores of Bacillus cereus were treated with 5% NaOH at 50 degrees C and 0.25% NaOCl. As a result, 99% of the spores had lost their viability by the 11th and 19.5th minutes, respectively. Kinetics of the process has shown that the rates of elimination of calcium and dipicolinic acid from the spores are different, and intensive loss of RNA and DNA was registered. Progressive degradation of spore envelopes and inner structures has been revealed by electron microscopy. Lethal effect of the studied chemical substances is presumed to be related to changes in the spore envelopes, thus interfering with normal barriers of permeability.

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.

The structure of the omega-particle-bacteria, growing in the micronucleus of Paramecium caudatum (Ciliata, Protozoa), was studied by electrom microscopy in the course of their life cycle. The cytoplasm of the spindle-shaped vegetative cells contains a large number of dense particles and transparent regions comprising the fibrillar material. Such cells, via several intermediate stages, are transformed into elongated twisted cells that are regarded as spores. The spore consists of two parts: homogeneous, and that containing the membrane system and rounded light bodies. The membranes are often double and connected with the fibrils. The cell wall is constructed, during all stages, of the outer membrane layer and the inner electron-dense layer.

The ultrastructural organization of the red-orange cultures of Nocardia (Proactinomyces) asteroides was compared with that of a Nocardia-like variant of Actinomyces (Streptomyces) chrysomallus isolated upon the inoculation of the submerged parent culture. The comparison has demonstrated for the first time that the major difference consists in the structure of their cross partitions. In Nocardia, the septa is split at all stages of its formation; in the Nocardia-like variant, the partitions have the same structure as the cultures of the genus Actinomyces (Streptomyces). Therefore, the accepted terminology is conventional.

It has been established for the first time that autolysis observed when Actinomyces thermovulgaris T-54 is cultivated on liquid media of various composition is caused by the formation of submerged spores. These, in contrast to exospores of the majority of the actinomycetes, are endospores according to their structure and the following criteria: thermostability, the presence of dipicolinic acid, the structure of ultrathin cross-sections studied by electron microscopy, the resistance to novobiocin.

The ultrastructural organization of Sulfobacillus thermosulfidooxidans cells and spores was studied by electron microscopy using the techniques of negative contrasting and thin sections. The cell wall was found to be 20--25 nm thick and to consist of a rigid layer bound to the cytoplasmic membrane with crosspieces. A microcapsule about 20 nm thick having an ordered structure is closely associated with the cell wall. A layer of slime which can reach 0.2--0.3 mcm and more in thickness is located at the outside. The flagellar apparatus is absent. The membranous apparatus has a peculiar structure. On thin cross-sections of intact cells, the cytoplasmic membrane profile is stained sharply asymmetrically. Numerous membrane invaginates located in the cytoplasm are morphologically simple vesicular formations and structures of a rather complicated unusual configuration. Lamella-like structures are also encountered, which divide, as it were, the cytoplasm into compartments. The contours of the intracellular membranes look thicker as compared with the cytoplasmic membrane. Small vesicular formations are occasionally encountered in the periplasm. Vast areas with inclusions of unknown chemical nature having a fine granular structure are located in the cytoplasm. The process of sporogenesis comprises the same stages as in Bacillus, and spores have a strucutre typical of the endospores.