Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.     

Ultrastructure of the Exosporium and Underlying Inclusions in Spores of Bacillus megaterium Strains

Spores of selected strains of Bacillus megaterium were prepared by various methods and examined with the electron microscope. An exosporium like that of B. cereus, with a nap and basal layer, was found in spores of a B. megaterium strain that reportedly contains a capsule-like exosporium. The exosporium occasionally appeared to be doubled or have an apical opening. Pili-like filaments were discerned on the surface. Beneath the exosporium were found large deposits of planar inclusions, which in cross section appeared laminated and in surface views consisted of a patchwork of striated packets with a periodicity of approximately 5 nm. The inclusions were usually attached to the exosporium, but in ultrastructure they differed from both the exosporium and coat. In two other strains of B. megaterium, one or two coats occurred but a typical exosporium was not present.     

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum

The ultrastructural changes produced by ketoconazole on the yeast-phase ofH. capsulatum andP. brasiliensis were studied by means of scanning and transmission electron microscopy.The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure ofC. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole thanH. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 gmg/ml and in the latter at 1 g/ml.     

Drosophila virilis histone gene clusters lacking H1 coding segments

Summary Approximately 30–40% ofDrosophila virilis DNA complementary to clonedDrosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as theD. melanogaster core histone genes in the plasmid cDm500: . Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30–40% ofD. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution ofDrosophila. The ancestors of modernDrosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in thevirilis andmelanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes ofD. virilis andD. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently,D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes ofD. virilis andD. melanogaster are >25% divergent. Our estimate of sequence divergence in the H1 genes ofD. virilis andD. melanogaster seems high until one considers that the coding sequences of cloned H1 genes from the closely related speciesD. melanogaster andD. simulans are 5% divergent.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

An ultrastructural and cytochemical study of the wall-membrane apparatus of transfer cells using freeze-substitution

Summary A freeze-substitution technique is described which enables the ultrastructure of certain types of plant transfer cells to be preserved with minimal ice crystal damage. The ultrastructure of transfer cells fromFunaria, Lonicera, andSenecio after freeze-substitution has been compared with that of glutaraldehyde-osmium fixed material. The irregular clear zone between wall and plasma membrane, present in conventional preparations, is absent in freeze-substituted tissue. It is proposed that this interfacial zone is an artefact caused by expansion of wall ingrowth material during conventional fixation procedures. In transfer cells with a complex wall labyrinth the swelling of wall material severely disrupts the true structure of the wall-membrane apparatus and results in a large decrease in the surface to volume ratio of the protoplast. These findings are supported in the case ofFunaria by a freezefracture study. The reactivity of the plasma-membrane to the PTA/chromic acid stain is enhanced in freeze-substituted material. Use of theThiéry silver proteinate reagent in conjunction with freeze-substitution has revealed marked differences between the wall ingrowths ofFunaria sporophyte haustorium transfer cells and those ofLonicera nectary trichomes.     

Histone - induced changes in the cellular growth of synchronized chlamydomonas reinhardii are due to histone H1

The cellular growth ofChlamydomonas reinhardii is modified by the addition of a total exogenous histone fraction. These modifications may be related to chloroplast DNA replication; they are different according to the different classes of histones. The H₁ subfraction seems to be responsible for the effect of the total histone fraction.     

Thecal ultrastructure ofScrippsiella faeroense

Summary The thecal ultrastructure ofScrippsiella faeroense (Paulsen) Balech and Oliveira Soares as seen in the electron microscope is described. Additional structural detail of the thecal plate surface, plate connections, and the apical pore, is revealed.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Electron microscope autoradiography of14C photosynthate distribution at the haustorium-host interface in powdery mildew ofPisum sativum

Summary Haustorial complexes were isolated from leaves ofPisum sativum infected withErysiphe pisi and exposed to¹⁴CO₂ for 2 hours. The constituents of the isolated fraction were quantified and ultrastructurally described and the distribution of¹⁴C studied by electron microscope autoradiography and statistical treatment. Most (86%) of the isotope in the fraction was associated with haustorial complexes. Three classes of haustorial complexes were distinguished by degree of labelling and ultrastructure. Most of the haustorial complexes were termed healthy (i.e., they showed a normal ultrastructure) and were heavily labelled; necrotic complexes were unlabelled; and a class with intermediate labelling, modified extrahaustorial membrane and usually a normal haustorial cytoplasm was termed pre-necrotic. In healthy haustorial complexes the haustorial lobes and extrahaustorial membrane showed the highest grain densities and the body and extrahaustorial matrix were also significantly labelled. Comparison of the results suggest that ultrastructural modifications leading to necrosis were caused by dehydration which in turn determined reduction in photosynthate transfer. Other factors influencing transport into haustoria and the status of the extrahaustorial matrix are also discussed.U.V. fluorescence microscopy with acetamido-isothiocyanatostilbene salt was used to distinguish healthy and pre-necrotic haustorial complexes. It is recommended as a simple technique to monitor the functional quality of isolated haustorial complexes.     

Spores of microorganisms

Spores ofBacillus cereus (strain NCIB 8122) were germinated in a synthetic germination limited medium (GL-medium), which permitted germination but did not make the termination of post-germinative development possible. Incorporation of¹⁴C-diaminopimelic acid into the newly formed cell wall was followed in this culture. Morphological changes were studied by optical and electron microscopy. Germination was associated with the usual germination changes,i.e. depolymerization of the “bulky” cortex, differentiation of nuclear structure and mesosomes and ribosomes in the cytoplasm. At this stage the spore protoplast is surrounded by several layers: exosporium, laminated coat with four layers, residual spore wall and the protoplast membrane. During incubation in this limited medium the residual wall layer thickens and the nuclear structure, mesosomes and ribosomes were not more detectable. After enrichment of the GL medium (shift up) the thick-walled cells can form additional cell wall material, elongate and an atypical septum formation can occur. The cell wall material forms local thickenings. On long-term cultivation in the GL medium some of the cells in the GL medium lyze. If, in the course of 3–6 h the cells are transferred from the GL-medium to a solid complex medium (Difco Nutrient Agar) the thickwalled cells are transformed into dividing cells. When the cells are transferred later, their colony-forming ability rapidly decreases. The decrease of viability of the thick-walled cells derived directly from spores after their germination in the limited medium indicates that these cellular forms probably do not represent more stable cellular types that would be of considerable importance for survival of the populat ion of bacilli.     

Assembly of the outermost spore layer: pieces of the puzzle are coming together

Certain endospore‐forming soil dwelling bacteria are important human, animal or insect pathogens. These organisms produce spores containing an outer layer, the exosporium. The exosporium is the site of interactions between the spore and the soil environment and between the spore and the infected host during the initial stages of infection. The composition and assembly process of the exosporium are poorly understood. This is partly due to the extreme stability of the exosporium that has proven to be refractive to existing methods to deconstruct the intact structure into its component parts. Although more than 20 proteins have been identified as exosporium‐associated, their abundance, relationship to other proteins and the processes by which they are assembled to create the exosporium are largely unknown. In this issue of Molecular Microbiology, Terry, Jiang, and colleagues in Per Bullough's laboratory show that the ExsY protein is a major structural protein of the exosporium basal layer of B. cereus family spores and that it can self‐assemble into complex structures that possess many of the structural features characteristic of the exosporium basal layer. The authors refined a model for exosporium assembly. Their findings may have implications for exosporium formation in other spore forming bacteria, including Clostridium species.     

Differences in Protein Patterns in Suspension Cultures of Taxus cuspidata Induced by Cerium

The changes in soluble proteins induced by Ce⁴⁺ were analyzed in suspension cultures of Taxus cuspidata using two-dimensional polyacrylamide gel electrophoresis. The ultrastructure of cells obviously changed at day 4 after addition of Ce⁴⁺. Large amount of nuclear DNA fragments of about 200 bp were observed. Thirteen protein spots were different between the cultures grown with and without Ce⁴⁺ at day 4 as well as at day 6 after addition of Ce⁴⁺. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium ‘cap’ structure in Bacillus anthracis

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of an outer hair‐like nap layer and an internal basal layer. A major component of the hair‐like nap is the glycosylated collagen‐like protein BclA. A second collagen‐like protein, BclB, is also present in the exosporium. BclB possesses an N‐terminal sequence that targets it to the exosporium and is similar in sequence to a cognate targeting region in BclA. BclB lacks, however, sequence similarity to the region of BclA thought to mediate attachment to the basal layer via covalent interactions with the basal layer protein BxpB. Here we demonstrate that BxpB is critical for correct localization of BclB during spore formation and that the N‐terminal domains of the BclA and BclB proteins compete for BxpB‐controlled assembly sites. We found that BclB is located principally in a region of the exosporium that excludes a short arc on one side of the exosporium (the so‐called bottle‐cap region). We also found that in bclB mutant spores, the distribution of exosporium proteins CotY and BxpB is altered, suggesting that BclB has roles in exosporium assembly. In bclB mutant spores, the distance between the exosporium and the coat, the interspace, is reduced.     

Kinetics of cell deposition under the action of an external field

The kinetics of cellular depositon from a stagnant solution to a surface are studied, taking into account the combined effect of an interaction field between the cells and the surface and of an external field. Since the forces involved in the adhesion of cells to a surface are short ranged, the cells are conveyed to the vicinity of the surface only by the external field. The equations developed are general, in the sense that they are independent of any particular form of the potential energy function, provided that it presents an appreciable potential barrier between the cells and the deposition surface. The characteristic shape of the curve representing the decay of the fraction of cells in solution with time is shown to be affected by the value ofPt ^*, consisting of the probability per unit time,P, for the escape over the potential barrier, and the sedimentation time,t ^*. A simple inspection procedure of this curve can disclose the relative significance of the external field and of the potential barrier in the overall kinetics of deposition. In addition, such an inspection can reveal the existence of alterations in the cellular adhesiveness with increasing coverage of the deposition surface. By matching the equation obtained to experimental results, the cellular adhesiveness, in term ofP, and the sedimentation rate (in the case of very slow sedimentation) can be evaluated.     

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo‐inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo‐inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo‐inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium‐like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium‐like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter‐species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium‐producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo‐inactivation properties.     

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores

Background information. Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. Results and conclusions. In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo‐Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non‐pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde‐fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.