Plasma membrane alterations as a result of heat activation in Dictyostelium spores

At the end of heat activation the distribution of spore plasma membrane particles between the two fracture faces (PF and EF) is drastically changed. While in dormant spores the particle number ratio of PF/EF was about 1:1, it increased up to 9:1 in heat activated spores, indicating a subtle change in plasma membrane properties. The permeability of spores increased within 30 min following heat activation as determined by efflux measurements of radioactively labelled spores. At the onset of swelling this efflux was accelerated. During germination the osmotically active material within the spores increased, part of which could be recovered from the supernatant. The combined experiments point to the plasma membrane as possible target site of heat activation in this system.     

Ultrastructural and Cytochemical Observations on Sporogenesis of Myxobolus sp. (Myxosporida: Myxobolidae) from the Common Shiner Notropis cornutus*

SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.     

On the Cytology of Toxoglugea chironomi (Debaisieux, 1931) Jírovec 1936 (Microspora, Thelohaniidae)

ABSTRACT. The light microscopic and ultrastructural characteristics of a microsporidium provisionally identified as Toxoglugea chironomi (Debaiseux, 1931) Jírovec, 1936, is described. It was isolated from oenocytes and adipose tissue of a midge larva of the genus Dicrotendipes . Merozoites are diplokaryotic. The sporogony produces, by fragmentation, eight monokaryotic spores in a sporophorous vesicle. Mature spores are horse-shoe shaped. The total length is about 5.8 μm, the width 0.8-0.9 μm, the external height of the curve 2.3-3.5 μm, and the external width of the curve 3.5-5.2 μm. The polaroplast has lamellar compartments of two types: narrow and closely packed anteriorly, and wider and more loosely arranged posteriorly. The isofilar polar filament is arranged in 8–10 coils in the posterior fourth of the spore. The external nuclear membrane is sometimes continuous with the endoplasmic reticulum. Lamellar and tubular material of exospore construction are present in the episporontal space from the beginning of sporogony. Teratological and normal spores sometimes occur together in the sporophorous vesicle. The identification of the species is discussed and the ultrastructure is compared to Toxoglugea variabilis , the only further species of the genus with known ultrastructural cytology.     

Honey-coloured,sessile Endogone spores

Summary Wall structure is described in the parent and resting spores of an Endogone sp. with honey-coloured, sessile spores. Wall thickness increases in the parent spore and subtending hypha by passage of material through the plasmalemma, or by formation of an apparently separate inner wall and degeneration of the trapped cytoplasm. Structure and development of the multi-layered wall of the mature resting spore are described. Unusual features are: 1. the incorporation of many pigment granules into the coloured outer wall, 2. the presence between the outer coloured and inner transparent walls of a tripartite membrane and adjacent layer with a regular periodicity and 3. a sectored layer with a crystalline component. The structure of the wall is discussed with reference to that of other mucoraceous fungi, to spore germination and to the mechanism of wall formation.     

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.     

Fungal spores: dormancy, germination, chemical composition, and role in biotechnology (review)

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.     

Fungal spores: Dormancy, germination, chemical composition, and role in biotechnology (review)

This review is focused on one of the stages of ontogenesis distinctive by its particular tolerance to the action of unfavorable factors and ability to retain the genomic material for a long period of time, i.e., fungal spores. The major part is devoted to the characterization of the specific stage typical for spores, which is called dormancy. Data are presented characterizing the carbohydrate and lipid composition of spores, with special attention being paid to the role of carbohydrate protectors, in particular, trehalose and mannite, as well as to the role of rafts in the process of sporogenesis. The role of special compounds called autoinhibitors and autostimulators in the process of exit from dormancy is discussed. The final section deals with the role of spore seeding material in biotechnological processes. Data on the correlation between the chemical composition of spores, their ability to remain dormant, and the germination process are considered. Special biotechnological approaches are presented for the first; they allow for the preservation of the germinating ability of spores, intensification of sporogenesis, changes in the ratio of final fermentation products, and an increase in their yield.     

Development, ultrastructure, and mode of transmission of Amblyospora sp. (Microspora) in the mosquito

Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merogonic cycle, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simultaneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.     

Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers

A variant strain that produced spores lacking exosporium was isolated from a culture of Bacillus megaterium QM-B1551. Two additional spore morphotypes were obtained from the parent and variant strains by chemical removal of the complex of coat and outer membrane. Among the four morphotype spores, heat resistance did not correlate with total water content, wet density, refractive index, or dipicolinate or cation content, but did correlate with the volume ratio of protoplast to protoplast plus cortex. The divestment of integument layers exterior to the cortex had little influence on heat resistance. Moreover, the divestment did not change the response of either the parent or the variant spores to various germination-initiating agents, except for making the spores susceptible to germination by lysozyme. The primary permeability barrier to glucose for the intact parent and variant spores was found to be the outer membrane, whereas the barrier for the divested spores was the inner membrane.     

Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio.

Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.     

Life cycle of tetrodotoxin-producing <Emphasis Type="Italic">Bacillus</Emphasis> sp. on solid and liquid medium: Light and electron microscopy studies

The lifecycle of the Bacillus sp. 1839 cultivated during a long period on solid and liquid Youschimizu-Kimura medium was investigated, and then bacteria and spores were studied by light and transmission electron microscopy. Sporulation in this strain is distinguished by engulfment of forespore by mother cell. In the liquid medium, bacteria have the decondensed nucleoid and the loose granular component of cytoplasm; bacteria and spores are generally smaller; the outer coat of spores includes 2 concentric rings. On the solid substratum, the nucleoid is condensed, and the cytoplasmic region is extensive and dense; a longer cultivation stimulates transition of vegetative cells into the spore form; spores have a thicker outer coat with 3–5 rings. On the solid substratum, sporulation in Bacillus sp. 1839 is spontaneous, without additional stimulation; spores have a larger diameter and thicker layers than those in the liquid medium. This research contributes to the current understanding of biotechnological tetrodotoxin production from a bacterial raw material.     

Development,Ultrastructure, and Mode of Transmission of Amblyospora sp. (Microspora) in the Mosquito*

SYNOPSIS. Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merognic cylce, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simulatneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

Glucose-C14 metabolism of dormant and activated ascospores of Neurospora

Budd, Kenneth (The University of Michigan, Ann Arbor), Alfred S. Sussman, and Frederick I. Eilers. Glucose-C(14) metabolism of dormant and activated ascospores of Neurospora. J. Bacteriol. 91:551-561. 1966.-Dormant and activated ascospores of Neurospora tetrasperma, incubated in C(14)-labeled glucose, absorb and metabolize this sugar. At the same time, up to 55% of the CO(2) production from endogenous substrates is quenched, whereas total CO(2) production is unchanged. Glucose-carbon appears in CO(2), lipids, and ethyl alcohol-soluble and -insoluble material in both dormant and activated ascospores, although the proportions entering these fractions differ in the two groups of spores. With few exceptions, the identifiable intermediates of glucose metabolism are the same in dormant and activated ascospores, indicating that the principal pathways may be identical. During glucose metabolism, dormant ascospores accumulate a nondialyzable, ethyl alcohol-soluble polymer, or polymers, which is either absent from activated spores or present in much smaller amounts. This material contains glucose, ribose, and at least nine amino acids, and may represent precursors of more complex cell material which accumulate because of an enzymatic deficiency in the dormant spore. Radioactivity is incorporated into all fractions of the dormant spores and into CO(2) without a noticeable lag, indicating that most, if not all, of the enzymes for glucose utilization are present. A lag in incorporation is observed in the activated spores, which most probably is due to rapid endogenous production of glucose from trehalose, resulting in dilution of lable. After absorption of labeled glucose, two pools of trehalose are found in dormant spores, one of which is extractable without breaking the spores, and the other, only after the spores are disintegrated. The widely differing specific radioactivity of the two pools indicates that these are separated in the intact spore.     

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.     

Effect of trehalose on the viability of sporangiospores of the mucorous fungus <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

Sporangiospores of Blakeslea trispora are in a state of exogenous dormancy, and water is the key factor controlling their germination. A wide range of carbohydrates, ammonium salts, and yeast extract had a weak stimulating effect (less than 50%) on spore germination, whereas amino acids could significantly inhibit this process. Cultivation of B. trispora on sporogenous sucrose- and trehalose-containing media (S and T spores, respectively) resulted in significant changes in spore formation, as well as in the chemical composition of spores and their viability. In the presence of trehalose, the amount of spores increased twofold; spore viability during storage increased as well. All changes in the carbohydrate composition of the cytosol and in the composition of the spore membrane lipids indicated that the dormancy of T spores was deeper than that of S spores, which has a favorable effect on their viability.     

Ultrastructure of the ascospores of some species of the Torulaspora group

Development and germination of the ascospores in species of the Torulaspora group of yeasts have been described. Most species had warty spores which, in sections, showed a dark outer layer consisting of the outer unit membrane of the prospore wall and a layer underneath formed at an early stage of development of the spores. In mature spores the light inner layer of the wall was delimited at the outside by a thin dark layer. The warts often contained dark material. The ascospores of two Pichia and three Debaryomyces species were studied for comparison; they differed in sections from the Torulaspora spores. The taxonomic implications of the ultrastructural observations have been discussed.     

Transport and localization of protein S, a spore coat protein, during fruiting body formation by Myxococcus xanthus.

Protein S, the most abundant soluble protein synthesized by Myxococcus xanthus FB during early fruiting body formation, accumulates in the soluble fraction of developing cells, reaching a peak at about 24 h; at late stages of fruiting body formation, protein S is found on the surface of spores (M. Inouye et al. Proc. Natl. Acad. Sci. U.S.A. 76:209-213, 1979). In this study, the transport and localization of protein S were investigated. Cells were fractionated to give osmotic shock, membrane, cytoplasmic, and spore fractions. The various fractions were then analyzed for protein S. Protein S was first detected in the cytoplasmic fraction at about 3 to 6 h of development. However, transport of protein S through the cytoplasmic membrane was not observed until 15 to 18 h of development. Thus, protein S is unusual among translocated proteins in that it accumulates as a soluble cytoplasmic protein before translocation. Biosynthesis of protein S ceased after 48 h; by 72 h, protein S was only found on the surface of spores. Pulse-chase experiments were performed to determine the transport kinetics of protein S. The results showed that in 24-h developing cells, the transport of protein S across the cytoplasmic membrane was rapid, occurring in less than 2 min. However, transport across the outer membrane was slow, requiring 10 to 15 min. Pulses of 15 s with [35S]methionine failed to reveal any short-lived precursor form in immunoprecipitated material separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also failed to detect any precursor form of protein S. Thus, protein S appears to be translocated in the absence of a cleaved signal peptide.