Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.     

Use of Yeast Spores for Microencapsulation of Enzymes

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose. This suggests that substrate selectivity could be altered by the encapsulation.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

Internal valves in populations of Meridion circulare from the A'er Mountain region of northeastern China: Implications for taxonomy and systematics

A population of Meridion circulare var.circulare (Greville) C.A.Agardh from Inner Mongolia was found to produce Innenschalen or internal spores.Examination of this population with light and scanning electron microscopy showed morphological differentiation between vegetative and spore morphologies.Vegetative valves typically bear costae and one rimoportula at the headpole.Spores lack costae and have two rimoportulae,one at the headpole and the other at the footpole.There is plasticity in the production of valve morphologies,and a variety of vegetative valve and spore combinations are evident.This population of M.circulare var.circulare has initial valves of over 90μm in length,and all of the initial cells encountered are acostate and bear two rimoportulae.These observations suggest that either spores are the product of sexual reproduction,or that initial valves may be produced parthenogenetically in Meridion.Spores as products of the sexual process have not been reported in diatoms previously,and parthenogenesis in Meridion was reported previously but discounted in other published reports.The plasticity of valve morphologies expressed in M.circulare var.ciculare,between vegetative valves and spores (and back) across a short temporal period suggests that diatoms can alter their cell wall structure dramatically and quickly in response to external variables.     

Adhesion of Clostridium cellulolyticum spores to filter paper

The adhesion of Clostridium cellulolyticum spores and cells to Whatman No. 1 filter paper was studied. A suspension of vegetative cells in exponential phase from a culture on cellobiose adhered at 60% whereas spores at the same initial concentration were bound to the Whatman filter paper at 90%. Adhesion of vegetative cellulolytic cells occurs on specific cellulosic sites and reveals a sigmoid type II curve. Non-cellulolytic vegetative cells from Clostridium butyricum do not adhere to the cellulose. Spore adhesion is a non-specific process since non-cellulolytic spores from Clostridium butyricum adhered almost in the same range to filter paper than cellulolytic spores.     

Differences in the surface membranes and water content between the vegetative cells and spores of Bacillus subtilis

Bacillus subtilis forms both vegetative cells and spores. The fluidity of the membranes in these forms was measured by using fluorescent anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH). The spores were more rigid than the vegetative cells, suggesting that the structure of the spores and vegetative cells was different. This difference was thought to be due to the structure of the cell membranes. The anisotrophy of DPH in the cell membranes of spores gave higher values at all temperatures. The anisotrophy of DPH in the cell membranes of vegetative cells was lower than that of the spores and the value depended upon the temperature. Time Domain Reflectometry (TDR) was used to measure the quantities of bound and free water in the vegetative cells and spores. The spores were dehydrated, and the amount of bound and free water in the spores was about two‐thirds of the levels in the vegetative cells. The spores have fewer sugars molecules on their cell surface membranes, but contained as much sugars within the cell. Almost 100 per cent of the vegetative cells wee absorbed toward chitin, but the spores were not absorbed toward it at all. It was felt that the surface membrane of the vegetative cell had a high mobility because it was sugar‐rich, while the surface membrane of the spore showed a lower mobility because there are fewer sugars on the outer membrane. The spores survive in high temperatures because the surface membrane of the spore is tight and has relatively few sugars. Dehydration causes the rigidity of the spores. On the other hand, the vegetative cells are sugar‐ and water‐rich, which makes them more fluid. The difference between the vegetative cells and spores is the glycosylation of their surface membranes. Copyright © 1999 John Wiley & Sons, Ltd.     

Procedure for mutagenizing spores of Saccharomyces cerevisiae

A procedure for inducing mutants of a homothallic strain of Saccharomyces cerevisiae is described. The essential parts of the procedure are long incubation in Glusulase, which preferentially kills vegetative cells instead of spores, and treatment in 9% ethyl methanesulfonate, which also preferentially kills vegetative cells instead of spores. Consequently, the viable population is virtually 100% spores.     

Distinctness of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli

A gas chromatographic analysis method was employed to determine the cellular fatty acid (CFA) profiles of spores and vegetative cells of some aerobic endospore-forming bacilli. The harvests of experimental strains were processed to obtain pure spores and acquire whole cell fatty acid methyl esters for the subsequent gas chromatographic analysis, and the corresponding vegetative cells were set as control. Evaluation of reproducibility of spore CFA components revealed that, provided under standardized experimental procedure, spore CFA composition was stable enough for research purposes. Fatty acids recovered in spores in greater quantities were saturated branched-chain acids containing 15 and 17 carbon atoms, similar to the vegetative cells. Commonly, the proportions of saturated branched-chain acids in spores were greater than in vegetative cells. The dendrograms obtained by cluster analysis provided some meaningful taxonomic information of the experimental strains. The fatty acids analysis of spores seems to be a promising supplementary tool for the chemotaxonomic research of aerobic endospore-forming bacilli.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor

An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-β-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.     

Presence of Bacillus coagulans spores and vegetative cells in rat intestine and feces and their physiological effects

ABSTRACT

This study was aimed to investigate the presence of Bacillus coagulans vegetative cells in the intestine and fecal samples in rats fed B. coagulans spores as well as to estimate the ratios of spores and vegetative cells in these samples. A two-step process has been developed to enumerate B. coagulans in different mixed bacterial samples, specifically (1) observation of yellow ring formation on modified GYEA medium upon incubation at 55°C, (2) microscopic examination of spore formation after 7 d of incubation. Our results have demonstrated the presence of vegetative cells in the intestinal and fecal samples in rats fed B. coagulans spores. The ratios of B. coagulans spores and vegetative cells in cecal fluid, colonic content, and feces were approximately 2:8, 2:8, and 4:6, respectively. The existence of B. coagulans vegetative cells improved the intestinal milieu through an elevated short-chain fatty acid concentrations, higher fecal moisture, and lower fecal pH.     

Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores. With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic-treated resting spores.     

ULTRASTRUCTURE OF TETRASPOROGENESIS IN THE MARINE RED ALGA PTILOTA HYPNOIDES1

Both tetrasporangia and dormant apical cells of short vegetative filaments of the marine red alga Ptilota hypnoides have been examined by electron microscopy. Various cytoplasmic inclusions readily distinguish the vegetative apical cells from the reproductive apical cells which become tetrasporangial mother cells. The transformation of tetrasporangial mother cells into mature tetrasporangia involves a series of cytoplasmic changes which can be correlated with specific changes in the investing wall layers. The extracellular changes provide the basic criteria for the division of tetrasporogenesis into 3 successive stages. The ultrastructure of each stage is described and discussed in relation to the current knowledge of red algal cytology. In addition, a possible mechanism for the liberation of spores and gametes of red algae is proposed.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Snow algae of the Windmill Islands, continental Antarctica 3. Chloromonas polyptera (Volvocales, Chlorophyta)

A new name, Chloromonas hohamii, is proposed to accommodate a common North American snow alga previously incorrectly referred to as Chloromonas polyptera. Chloromonas hohamii differs in having the motile vegetative cells with a cup-shaped chloroplast opening in the anterior end of the cell, shorter, narrower, ellipsoidal to elongate to somewhat fusiform, sexual spores with non-spiralled wall flanges, shorter and narrower daughter cells derived from the spores, and it grows in snow of significantly lower pH and conductivity. Received: 29 August 1997 / Accepted: 24 April 1998     

Comparisons of Cells, Refractile Bodies, and Spores of Bacillus popilliae

Spores of Bacillus popilliae from infected larvae and refractile bodies produced in a Trypticase-barbiturate medium were similar but distinct from vegetative cells of this organism in protein, nucleic acid, and enzyme composition. The spores and refractile bodies were found to have catalase activity, some of which was heat-resistant. This enzyme was not found in the vegetative cells. The spores contained dipicolinic acid, but the refractile bodies did not. The latter were similar to cells in having considerably higher levels of phosphate extractable with cold trichloroacetic acid and of poly-beta-hydroxybutyrate than had the spores. Electron microscopy demonstrated conclusively that the refractile bodies are distinctly different from either cells or spores of B. popilliae. The possibility that these bodies are formed as a result of an aborted sporulation process is discussed.     

Hydrophobicity of Bacillus and Clostridium spores

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.     

Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.