NUCLEAR MIGRATION AND ASYMMETRIC CELL DIVISION IN ONOCLEA SENSIBILIS SPORES: AN ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY

A method of preparation for electron microscopy of fern spores in early stages of germination is presented. The cytochemistry and fine structure of Onoclea spores during the early stages of germination are described. The cytoplasm of the hydrated spore is filled with lipid droplets, protein granules and chloroplasts. During the early stages of development ribosomes and mitochondria increase in the area surrounding the central nucleus, and a new peripheral wall forms around the protoplast. Microtubules and large, branching mitochondria are associated with the nucleus during migration from its original central position in the spore to the proximal face and then to one end of the spore. There is no morphological polarization of cytoplasmic organelles of the spore before migration of the nucleus.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm³) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.     

SPORE ULTRASTRUCTURE OF THE MYXOMYCETE PHYSARUM POLYCEPHALUM

Spores of the true slime mold Physarum polycephalum were examined at several stages of their development by means of scanning and transmission electron microscopy. The spores were globose, spine-covered structures produced within a sporangium enclosed in a tough, noncellular peridium. Cytologically, the spore represented a typical eukaryotic cell, having discrete organelles similar to spores of other myxomycetes. The presence of dictyosomes, helical filaments, and microbodies in these cells, as well as the further elucidation of the cell wall and the “polysaccharide-containing” areas, represent new contributions to the ultrastructure of the myxomycete spore. Of special interest were observations of metaphase nuclei just prior to spore cleavage, interphase nuclei in young spores, and nuclei in mature spores containing synaptonemal complexes. These observations indicate that in Physarum polycephalum mitosis occurs just prior to spore cleavage, and meiosis takes place after spore cleavage.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

ssp Genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus

It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae.     

Spore Germination Patterns in the Ferns, Cyathea and Dicksonia

Cell division patterns during germination of spores of Cyatheaaustralis, C. cooperi and Dicksonia antarctica were examinedby light microscopy of sectioned materials and by the scanningelectron microscope. In C. australis and C. cooperi the rhizoidwas traced to a small cell formed by an asymmetric divisionof the spore by a wall parallel to its equatorial plane. Incontrast, the rhizoid was formed by a division of the sporeparallel to its polar axis in D. antarctica. In spores of bothgenera, a second division wall oriented in a plane perpendicularto the first gave rise to the protonemal cell. Certain aspectsof germination described here in spores of Cyathea and Dicksoniaare in conflict with the published accounts of spore germinationin these genera. Cyathea, Dicksonia, spore germination, cell division pattern     

Dry mass of Fusarium roseum spores before and after germination

The total dry mass of Fusarium roseum spores and contained lipid bodies were determined before and after spores germinated using quantitative interference microscopy. The mean for spore dry mass before germination was about 57 pg. Lipid bodies accounted for about 61% of that. Areas of lipid bodies in spores before and after germination were about 23 % but the contents of the lipid bodies accounted for only 10% of the spore dry mass after germination. The total dry mass of the spore and germ tube(s) greatly exceeded that of the spore before germination. We infer that nutrients for germ tube growth are derived from within the germinating spore and from the medium which must contain nutrients leached from non-germinating spores.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Peculiarities of Exogenous Dormancy of Aspergillus nigerConidia

Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.     

Uninucleate spores of Phycomyces

Summary Under most culture conditions only 0.3% of the vegetative spores of Phycomyces blakesleeanus are uninucleate. On an acidified minimal medium, the uninucleate fraction can be raised up to 4.5% of the spores. The spore population can be fractionated in a gradient under gravity (1xg) yielding fractions that contain over 80% uninucleate spores. These uninucleate spores are fully viable. When the spores to be fractionated are obtained from a heterokaryotic mycelium, the uninucleate fraction produces homokaryotic mycelia.     

Effects of Macroalgal Chemical Extracts on Spore Behavior of the Antarctic Epiphyte Elachista antarctica Phaeophyceae

Most macroalgal species along the Western Antarctic Peninsula (WAP) are defended against predation, many using chemical defenses. These subtidal communities are also mostly devoid of free living filamentous algae. However, one endo/epiphyte, Elachista antarctica, is found growing exclusively out of the palatable rhodophyte Palmaria decipiens. To understand this unusual and exclusive epiphytization, we tested whether macroalgal secondary metabolites such as those responsible for deterring sympatric grazers, affect the behaviors of the epiphyte's spores. Settlement, germination, and swimming behaviors of the epiphyte's motile spores were quantified in the presence of fractionated lipophilic and hydrophilic extracts of host P. decipiens and other rhodophytes from the shallow subtidal. Host P. decipiens was the only alga tested that did not inhibit spore settlement or germination. We also examined whether extracts from these chemically rich algae affect spore swimming behaviors and found spores to be chemotactically attracted to seawater soluble extract fractions of host P. decipiens. These results indicate that chemosensory behaviors of the epiphyte's spores to metabolites associated with these chemically defended macrophytes can explain this exclusive epiphyte–host interaction.     

Cellular and molecular approaches to understanding primary adhesion in Enteromorpha: an overview

The attachment of motile spores of the green alga Enteromorpha to the substratum is an active process involving an irreversible commitment to adhesion and the secretion of an adhesive. This paper provides an overview of the spore adhesion processes and outlines the results of an experimental approach towards the molecular characterisation of the adhesive, based on the use of monoclonal antibody (mAb) technology. Hybridomas were produced to settled spores displaying secreted adhesive. Candidates producing mAbs to putative adhesive were selected using a range of criteria based on cellular localisation, time of secretion and functional inhibition of adhesion. MAb Ent 6 immunolabelled fibrillar material which was secreted during the early stages of adhesion and low (nM) concentrations of this mAb, or its F(ab)₂ fragments, strongly inhibited the attachment of zoospores. A related antibody (Ent 1) also labelled the spore adhesive apparatus, but the antigen appeared to be secreted later during the adhesion process and was predominantly associated with the developing cell wall. Ent 1 also inhibited settlement in spore adhesion assays but the effect was most pronounced at later time points which suggests that this antigen does not have a role in the earliest stages of adhesion. Immunolocalisation showed that both antigens were absent from the cytoplasm or organelles of vegetative tissue but labelled the vegetative cell wall, suggesting a relationship between cell wall components and materials involved in primary adhesion. Both mAbs labelled the Golgi region of settled spores, suggesting continued synthesis of both antigens after adhesion. Both mAbs recognised a 110 kDa N‐linked polydisperse and heterogeneous glycoprotein in extracts of swimming spores under denaturing conditions. In native form the antigens behaved as high molecular weight aggregates (M_r>1.3 × 10⁶). The antigens became progressively insoluble after zoospore attachment. Taken together, the data suggest that the two antibodies recognise closely related, polydisperse, self‐aggregating cell wall glycoproteins in which there is some structural variation to suit alternative roles in primary adhesion and cell wall formation. The two mAbs Ent 1 and Ent 6 partially discriminate between these structural and functional variants. A model for zoospore adhesion is discussed in which adhesion is viewed as an extension of cell wall synthesis, with cross‐links between glycoproteins and other cell wall matrix components providing a strong physical continuum between the cell and the adhesive at the substratum interface.     

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.     

Endospores of Sporosarcina halophila: characteristics and ultrastructure

Sporosarcina halophila forms endospores. Electron micrographs revealed ultrastructural similarity to spores of S. ureae. Spore germination indicated by loss of refractility, darkening, swelling and formation of new vegetative cells was followed by phase contrast light microscopy. To induce spore germination, the endospores needed to be heat avtivated. After activation, they were inoculated into nutrient broth medium supplemented with sea-water. Double concentrated sea-water was found to be optimal for germination. Similar to other bacterial endospores, the spores were found to be resistant to heat and ethanol. An ultraviolet absorbing substance was isolated from suspensions of free spores; it was identified to be pyridine-2,6-dicarboxylic acid (DPA) usually present in bacterial spores. DPA was detected in amounts ranging from 5–7% of the spore dry weight; it was not detected in extracts of vegetative cells.Abbreviation DPA 2,6-pyridine-dicarboxylic acid     

Sporogenesis in Streptomyces melanochromogenes

The mode of spore differentiation in a strain of Streptomyces melanochromogenes was followed by analysis of ultrathin sections of sporulating aerial hyphae at various stages of sporogenesis. A special accent was laid on the formation of the sporulation septum and its alterations in the course of spore delimitation and separation. Distinct differences in formation and substructure have been observed between the cross walls of vegetative hyphae and the sporulation septa.Cross walls of vegetative hyphae are formed in a way typical for Gram-positive bacteria by a centripetal annular ingrowth of cytoplasmic membrane, on which wall material immediately is deposited. The development of the sporulation septa is characterized by the accumulation of amorphous material in addition to the newly synthesized wall layer inside the invaginating cytoplasmic membrane. This amorphous septal material will later be decomposed presumably by two lytic systems which cause the separation of the spores. The central region of the finished sporulation septum is perforated by microplasmodesmata. Spores are released by a break down of the surface sheath. The complete spores are enveloped by a twolayered cell wall and the spiny surface sheath.     

Resting spore formation in the antarctic diatoms Coscinodiscus furcatus Karsten and Thalassiosira australis Peragallo

Summary The resting spore morphology of the antarctic diatoms Coscinodiscus furcatus Karsten and Thalassiosira australis Peragallo are described. Both species form endogenous resting spores. The spore valve of C. furcatus differ from those of the vegetative cells primarily by (i) a greater convexity and (ii) a coarser and more distinctly fasciculated areolation. This resting spore is identical to the diatom traditionally identified as C. stellaris var. symbolophorus (Grunow) Jørgensen in the Antarctic. The resting spore of T. australis differs from the vegetative cells by (i) a lack of clusters of strutted processes in a modified ring on valve face, (ii) a coarser areolation and tangential rows of areolae and (iii) a narrower and more simply structured girdle. The resting spore valve of T. australis has been described as belonging to a separate species, Actinocyclus excentricus Peragallo.     

Resting spore formation ofLeptocylindrus danicus (Bacillariophyceae) during short time-scale upwelling and its significance as predicted by a simple model

Resting spore formation during short time-scale upwelling and its significance were investigated in the field and by a simple theoretical model. Field observations of spore formation ofLeptocylindrus danicus were made off Izu Peninsula, Japan. A rapid increase in ratio of resting spore to vegetative cell numbers indicated thatL. danicus formed resting spores quickly as a response to nutrient depletion in the upwelled water, although only a very low number of resting spores was found in the upwelling. A simple model was constructed to investigate the possible advantages of spore formation during short time-scale upwelling. This showed that there is a critical time-scale for resting spore formation to be advantageous. The nutrient depletion period of the upwelling off Izu was shorter than the critical time-scale determined by the model. Rapid-sinking of resting spores may increase further the critical time-scale, unless spores return with upwelling water. For short time-scale upwelling, the vegetative cell may be better suited than the resting spore for enduring a short period of nutrient depletion. Contribution from Shimoda Marine Research Center, University of Tsukuba, No. 475.     

The ultrastructure of Prototheca wickerhamii

Two clinical isolates of Prototheca wickerhamii were freeze-dried, fixed and studied by electron microscopy and were also examined growing in culture medium by phase contrast light microscopy. Resting spores placed on fresh medium developed cytoplasmic extensions which sequestrated before proliferation occurred. In the presence of adequate nutrients vegetative spores grew and subdivided to form up to 12 endospores within large sporangia which ruptured to release free spores every 5–6 hours. These proliferating or vegetative spores contained much more endoplasmic reticulum and mitochondria than the resting spores which contained more lipid, and often starch granules as well, but relatively few membranous organelles.