Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study

We utilize the fluorescent molecular rotor Bodipy-C₁₂ to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4 ns, upon viscosity increase from 1 to 1500 cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000 cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600 cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.     

Anamorphs in Mycocalicium albonigrum and M. subtile (Caliciales)

Anamorphs of Mycocalicium albonigrum and M. subtile have been obtained in culture from ascospores, conidia collected in the field, and conidia produced in culture. The ontogenies of the conidiomata and conidia are described. Differences between the species with respect to cultural characteristics, septation of ascospores during germination, occurrence of chlamydospores and pyriform cells as well as ultrastructural details of conidiogenous cells and conidia were studied. Conidia are suggested to possibly be important in the dispersal of the species.     

Study on the overwintering of cleistothecia and conidia of Erysiphe betae causal agent of sugar beet powdery mildew in Iran

Sugar beet leaves covered by sexual (cleistothecia) and asexual forms (mycelia and conidia) of Erysiphe betae were gathered at harvest time and maintained under natural outdoor conditions. In order to determine the function of cleistothecia and also conidia in the overwintering of E. betae some experiments were performed. The results showed that ascospores were unable to be released in petri dishes but their release under natural conditions occurred after 4 months. Under In vitro conditions ascospores did not germinate but on the leaves germination was rarely possible, however these ascospores were degraded after 7 days and didn't have pathogenicity. Conidia could induce pathogenicity after 3 but not 4 months. The period after inoculation until appearance of disease symptoms increased with aging of conidia. The results for conidial germination showed that fresh conidia had 80 percent germination on glass slides but it decreased sharply after 2 weeks and reached to 0 percent after 4 weeks. Although their germination on the leaves was not more than 46 percent of fresh conidia but they had good germination after 2 and 4 weeks. The results for the experiment to observe the first appearance of the disease in the field suggested that the first conidia were trapped by spore-trap in early June and the first symptoms appeared 20 days later. The conclusive results showed that ascospores had no function in the survival of the fungus and air-borne conidia are the main source of primary infections.     

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2 M external Ni²⁺ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50–60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3–8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5–2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.     

Comparison between internal microviscosity of low-density erythrocytes and the microviscosity of hemoglobin solutions: an electron paramagnetic resonance study.

The hypothesis that the internal viscosity of erythrocytes is governed by the intracellular hemoglobin (Hb) concentration is examined. Here viscosity is determined by labeling of the cytoplasmic reduced glutathione with the spin label maleimido-Tempo. Erythrocyte populations with different Hb concentrations in isosmotic conditions were obtained through incomplete lysis, followed by cell resealing, and discontinuous density gradient separation. This procedure maintains normal cell shape and volume. Microviscosity of membrane-free Hb solutions was measured by addition of spin labeled glutathione. It was found that microviscosity values are similar for the erythrocyte cytoplasm and for Hb solutions of equivalent concentrations, showing that the erythrocyte membrane does not have any influence on internal microviscosity. The dependence of the microviscosity on the concentration of Hb solutions was compared with results of macroscopic viscosity obtained by other authors. It is concluded that microviscosity is sensitive to individual properties of the Hb molecule (intrinsic viscosity), but that it is not sensitive to intermolecular interactions. As the microviscosity behavior as a function of Hb concentration is the same in Hb solutions as in the erythrocyte cytoplasm, the inferences regarding macroscopic viscosity in Hb solutions could be translated to the rheological properties of the erythrocyte cytoplasm. Thus, these properties could be predicted from the values of the mean corpuscular Hb concentration.     

High-resolution cell surface dynamics of germinating Aspergillus fumigatus conidia

We used real-time atomic force microscopy with a temperature-controlled stage (37°C) to probe the structural and physicochemical dynamics of single Aspergillus fumigatus conidia during germination. Nanoscale topographic images of dormant spores revealed the presence of a layer of rodlets made of hydrophobins, in agreement with earlier electron microscopy observations. Within the 3-h germination period, progressive disruption of the rodlet layer was observed, revealing hydrophilic inner cell wall structures. Using adhesion force mapping with hydrophobic tips, these ultrastructural changes were shown to correlate with major differences in cell surface hydrophobicity. That is, the rodlet surface was uniformly hydrophobic due to the presence of hydrophobins, whereas the cell wall material appearing upon germination was purely hydrophilic. This study illustrates the potential of real-time atomic force microscopy imaging and force spectroscopy for tracking cell-surface dynamics.     

Conversion of furfural to furoic acid and furfuryl alcohol by Neurospora ascospores

Summary The metabolism of furfural was studied with regard to possible mechanisms by which the chemical induces germination in ascospores. Incubation of ascospores in furfural resulted in the uptake of a small percent of the furfural, and the conversion of the bulk of it to furoic acid which was in turn converted to furfuryl alcohol. Conversion also occurred in Neurospora mycelium and conidia with the order being furfural to furfuryl alcohol to furoic acid. Conversion appears to be a noninducible enzymatic process localized on the outer surface of the cell. Conversion was completely inhibited without preventing germination indicating that conversion is not involved in the breaking of dormancy in Neurospora ascospores.     

Physiology of the cell surface of neurospora ascospores

Summary Acids like hydrogen fluoride, hydrazoic and fluoroacetic have been shown to prevent the germination of ascospores of N. tetrasperma when dormant spores are treated. On the other hand, propionate, cysteine and others are ineffective when used in this way. When activated ascospores were treated, much lower concentrations of the acids were sufficient to poison the spores. As in other systems, these substances are most effective at a p_H below their pK_a.The kinetics of uptake of fluoride by dormant ascospores were studied and shown to be very different from those reported for cations. However, P³² was not absorbed by dormant ascospores, even at p_H 1.5.Respiratory inhibition by azide and fluoroacetate occurred immediately after the spores were activated, but in the case of 5-nitro-2-furfuryl methyl ether no effect was observed until just before germination occurred.These results suggest that a permeability barrier exists in the dormant ascospore which disappears upon germination. Moreover, the dormant spore seems to be permeable to acids of small size but impermeable to those possessing more than 3 methylene groups or of equivalent size.This work was made possible by a grant from the Michigan-Memorial Phoenix Project of the University of Michigan to whom the authors would like to express their gratitude.     

Regulation and glutamic acid decarboxylase during Neurospora crassa conidial germination.

Glutamic acid decarboxylase (GAD) from Neurospora crassa was assayed in dormant and germinating conidia that had been permeabilized by toluene and methanol. N. crassa conidia contained 10 times the GAD activity found in vegetativemycelia. During conidial germination, GAD activity rapidly decreased to low levels before germ tubes appeared. GAD activity in germinating conidia closely followed the decreasing rate of glutamic acid metabolism. Inhibiting protein synthesis partially blocked the decrease in GAD activity, but eliminating exogenous carbon sources did not alter the initial rate of decrease in this enzyme. However, when conidia were incubated for more than 3 h in distilled water, GAD activity began to increase and eventually reached levels comparable to those in dormant conidia. Either GAD was reversibly inactivated or this enzyme could be synthesized from endogenous storage compounds when conidia were incubated in distilled water. These results are consistent with the hypothesis that GAD is a developmentally regulated enzyme that is responsible for catalyzing the first step in the metabolism of the large pool of free glutamic acid during conidial germination.     

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.     

Changes in the permeability of ascospores of Neurospora tetra-sperma during germination

The respiration and germination of activated ascospores of Neurospora tetrasperma have been shown to be almost completely inhibited by concentrations of ethylene diaminetetraacetic acid (EDTA) as low as 0.0035 M. In contrast, however, dormant ascospores are insensitive to this chelating agent. At any time up to about 150 minutes after activation Ca(++) or Mg(++) can completely reverse this toxicity but Cu(++), Co(++), and Mn(++) only partially reverse it. After this time, the minerals of the Neurospora "minimal" medium taken singly, or in various combinations cannot reverse this effect. Adding EDTA at 120 minutes after activation eliminates the lag period associated with its effect upon respiration. Inhibition occurs even though the cells seem to be impermeable to EDTA. Cationic exchange resins, as another example of a non-penetrating metal-binding agent, gave effects similar to those noted with EDTA. Of the resins used the H(+) form of IR-120 and the Na(+) and K(+) forms of amberlite IRC-50 were the most toxic to activated ascospores. On the other hand, dormant ascospores were entirely unaffected by the resins. The release of Ca(++) from activated ascospores coincided with the period of maximum sensitivity to EDTA. More than 60 per cent of the cell's content of K(+) is released by EDTA-inhibited ascospores. A low pH decreased the effectiveness of EDTA as a poison. The data are consistent with the possibility that non-penetrating metal-binding agents are toxic because of the irreversible removal of essential cations from the cell. The kinetic data for the inhibitory effects, and for the release of Ca(++) establish that the permeability of germinating ascospores to minerals changes drastically as a result of activation.     

Induction of Enzymes in Dormant Spores of Aspergillus oryzae

In dormant conidia of Aspergillus oryzae, alpha-amylase, invertase, and glucose dehydrogenase were induced by their respective inducers. Neither germination nor swelling occurred during this period.     

Epidemiology and control of powdery mildew (Sphaerotheca pannosa) on roses

In examinations between September 1966 and December 1968 of 741 specimens of rose species and cultivars, cleistocarps of Sphaerotheca pannosa were found on thirty-two cultivars, mostly ramblers and old shrub roses, and on nine of these they were found in two or three successive seasons. On stem pieces placed on soil in each of the winters 1966-7, 1967-8 and 1968-9 the number of cleistocarps with asci and ascospores decreased during November and December and rose slightly in January, but none showed dehiscence. Cleistocarps on rose bushes examined during the winters of 1967-8 and 1968-9 showed a progressive degeneration of ascospores, and by December none was found. Perennation of S. pannosa in buds was demonstrated by field observations, by inducing bursting of dormant buds on surface-sterilized shoots and by dissection of dormant apical buds. In field studies in 1968 of the development of mildew, infected buds were noted on 22 March but secondary infections did not appear until 17 April, though viable conidia and susceptible leaf tissue were present during this period. Low temperatures appeared to be partly responsible for this lag. On detached leaves in the laboratory the fungus developed from germination to sporulation in 4 days at 20°, 7 days at 15°, 11 days at 10° and 28 days at 3°. Keeping inoculated detached leaves at 0° for 10 days apparently did not affect the viability of the conidia. In both 1967 and 1968 there were two host growth periods, each culminating in flowering, between May and September; mildew did not develop on the shoots until the second growth phase, then the disease increased logarithmically on shoots and blooms during August and September. The disease on the shoots was effectively controlled in the field during 1968 by applications of ‘Benlate’ (benomyl) or dinocap, but not by methy-rimol; these fungicides were less effective in controlling mildew on pedicels. Laboratory tests showed that ‘Benlate’ inhibited sporulation of S. pannosa by deforming the conidiophores.     

Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.     

Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola

Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.     

Phenethyl alcohol induced germination of ascospores of Neurospora

Summary Phenethyl alcohol (PEA) can activate dormant ascospores of Neurospora. Between 4 to 8×10^-8 M PEA solutions induce germination of ascospores of N. crassa and N. tetrasperma equal to or higher than that of those activated by heat. This PEA effect is significantly greater than that of related chemicals and of other compounds known to induce fermentative metabolism.Influences of duration of exposure, sporal age, respiratory inhibitors and alcohol dehydrogenase activity have been studied in their relationship with PEA activation of dormant ascospores.     

Spermiogenesis in the bullfrog (Rana catesbeiana): a study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The process by which spermatid cytoplasmic volume is reduced and cytoplasm eliminated during spermiogenesis was investigated in the bullfrog Rana catesbeiana. At early phases of spermiogenesis, newly formed, rounded spermatids were found within spermatocysts. As acrosomal development, nuclear elongation, and chromatin condensation occurred, spermatid nuclei became eccentric within the cell. A cytoplasmic lobe formed from the caudal spermatid head and flagellum and extended toward the seminiferous tubule lumen. The cytoplasmic lobe underwent progressive condensation whereby most of its cytoplasm became extremely electron dense and contrasted sharply with numerous electron-translucent vesicles contained therein. At the completion of spermiogenesis, many spermatids with their highly condensed cytoplasm still attached were released from their Sertoli cell into the lumen of the seminiferous tubule. There was no evidence of the phagocytosis of residual bodies by Sertoli cells. Because spermatozoa are normally retained in the testis in winter and are not released until the following breeding season, sperm were induced to traverse the duct system with a single injection of hCG. Some spermatids remained attached to their cytoplasm during the sojourn through the testicular and kidney ducts; however, by the time the sperm reached the Wolffian duct, separation had occurred. The discarded cytoplasmic lobe (residual body) appeared to be degraded with the epithelium of the Wolffian duct. It was determined that the volume of the spermatid was reduced by 87% during spermiogenesis through a nuclear volume decrease of 76% and cytoplasmic volume decrease of 95.3%.     

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.