The Effect of Incubation Temperature on the Recovery of Spores of Bacillus subtilis 8057

S ummary . The recovery of Bacillus subtilis spores was studied after different heat treatments at 95° and incubation at different temperatures in roll tubes in a gradient temperature incubator. Plate count agar and brain–heart infusion agar were used in the roll tubes. Unheated spores showed similar recoveries at 16–48° whereas heated spores had an optimum recovery temperature of c. 30.9. The rate of germination of untreated spores was greatest at c. 41° and ceased at 50°. Heated spores germinated at 52°5°, suggesting that recovery of heat-treated spores is not limited by their ability to germinate. Outgrowth of spores at different incubation temperatures was similar for germinated and ungerminated spores. Accordingly it is outgrowth rather than germination which is sensitive to temperature.     

Species-specific peptide ligands for the detection of Bacillus anthracis spores

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.     

Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions

Aerial dispersal of fungal spores is common, but the role of wind and air movement in dispersal of spores of arbuscular mycorrhizal (AM) fungi is largely unknown. Several studies have examined the possibility of AM fungal spores being moved by wind vectors without observing spores taken from the air environment. For the first time this study observed the presence of AM fungal spores in the air. The frequency of AM fungal spores in the air was determined in six North American biomes composed of 18 ecoregions. Multiple samples were taken from both the air and the soil at each location. AM fungal spores were found in high abundance in the soil (hundreds of spores per gram of soil), however, they were rarely found in the air (most samples contained no AM fungal spores). Furthermore, only the Glomus morphotype was found in the air, whereas spores in the soil were taxomomically more diverse (Glomus, Acaulospora, Gigaspora, Scutellospora morphotypes were observed). The proportion of Glomus spores in the air relative to Glomus spores in the soil was highest in more arid systems, indicating that AM fungi may be more likely to be dispersed in the air in such systems. Nonetheless, the results indicate that the air is not likely a dominant mode of dispersal for AM fungi.     

Mechanism of the inactivation of bacterial spores by reciprocal pressurization treatment

AIMS: The mechanism of the inactivation of Bacillus subtilis spores by reciprocal pressurization (RP) was unclear. Therefore, the mechanism was investigated. METHODS AND RESULTS: To investigate the effects of RP and continuous pressurization (CP) treatments on the inactivation and injury of B. subtilis spores, spores were treated at 25, 35, 45 and 55 degrees C under 200, 300 and 400 MPa. RP treatment was effective in injuring and inactivating spores. Scanning electron microscopy and transmission electron microscopy observation showed that spores treated by RP treatment were more morphologically and structurally changed than the ones treated by CP treatment. There were significant differences between the release of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by RP and CP treatments. From this result, it was concluded that the core fraction was released into the spore suspension. CONCLUSIONS: The mechanism of RP treatment is believed to work as follows: hydrostatic pressure treatment initiated germination of bacterial spores, and the repeated rapid decompression caused disruption, injury and inactivation of the germinated spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that the physical injury of bacterial spores was effective to inactivate the bacterial spores through the disruption of spores and leakage of their contents.     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Differential scanning calorimetric observations concerning the activation mechanism of spores of Phycomyces blakesleeanus.

Spores of Phycomyces were scanned in a Differential Scanning Calorimeter. The spectrum obtained was clearly influenced by previous activation of spores by heat or by acetate.When spores were allowed to return to dormancy the original spectrum of dormant spores was restored. The high temperature at which the difference in the spectrum between activated and dormant spores was found points to a protein denaturation. It is suggested therefore that the activation of spores is obtained through a conformational change of a protein.     

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.     

Characterizing and handling different kinds of AM fungal spores in the rhizosphere

Spores are important propagules as well as the most reliable species-distinguishing traits of arbuscular mycorrhizal (AM) fungi. During surveys of AM fungal communities, spore enumeration and spore identification are frequently conducted, but generally little attention is given to the age and viability of the spores. In this study, AM fungal spores in the rhizosphere were characterized as live or dead by vital staining and by performing a germination assay. A considerable proportion of the spores in the rhizosphere were dead despite their intact appearance. Furthermore, morphological and molecular analyses of spores to determine species identity revealed that both viable spores and dead spores with contents were identified. The accurate identification of spores at different developmental stages on the basis of morphology requires considerable experience. Our findings suggest that surveys of AM fungal communities based on spore enumeration and morphological and molecular identification are likely to be inaccurate, primarily because of the large proportion of dead spores in the rhizosphere. A viability check is recommended prior to spore molecular identification, and the use of trap cultures would give more reliable morphological identification results. We show that the abundance and activity of AM fungi in the rhizosphere can be determined by calculating the density of viable spores and the density of spores that could germinate. The adoption of these methods should provide a more reliable basis for further AM fungal community analysis.     

Glycoconjugates for the recognition of Bacillus spores

Carbohydrates act as ligands in many biological processes, including the folding and secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. Fluorescent-labeled disaccharide glycoconjugates have been applied to evaluate binding to bacterial spores assuming that the spore surface is covered with carbohydrates. This study has shown that specific recognition of bacterial spores is based on interactions between disaccharide glycoconjugates acting as ligands and monosaccharide units expressed on the exterior of bacterial spores. Using fluorophore-assisted carbohydrate electrophoresis (FACE), carbohydrates that are expressed on the exterior of the spores were enumerated. The findings have an impact on how to improve ligand selection, essential for sensor development. In addition, the findings provide new information for inhibition of bacterial spores, and in general, demonstrate how carbohydrates function as recognition signals in nature.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

A Note on the Effect of Hydrogen Chloride on the Morphology of Bacillus subtilis Spores

Gaseous hydrogen chloride, in the presence of a minute amount of water vapour, rapidly inactivated bacterial spores. Scanning electron microscopy showed that the treatment caused spores of Bacillus subtilis to collapse. Modern theories of spore structure and resistance suggest that it is likely that hydrogen chloride inactivates and causes collapse of spores by breaking disulphide bonds in coat protein and neutralizing, by protonation, peptidoglycan carboxyl groups in the underlying cortex.     

Biological characteristics of the invasive stage of Myxosoma cerebralis (Myxosporidia: Myxosomatidae)

The data concerning biological peculiarities of spores of Myxosoma cerebralis obtained by the author and other researchers are summarized. The life cycle of M. cerebralis is well adapted to the seasonal cycle of the host owing to the fact that the infectivity of the spores is attained only after 4 months of aging in water and that the spores are highly resistive to freezing and drying. No sexual process during 4 month aging was observed. It is supposed that the maturity of spores depends to a great extent on the ability of polar capsules for extrusion.     

Has the prehistoric ice-man contributed to the preservation of living fungal spores?

Abstract There are arguments against the conclusion drawn by Haselwandter and Ebner that fungal spores have survived for some 5300 years on hay padding in the leather boots of frozen body discovered in the Austrian Alps. According to cryobiological experience, long-term survival of fungal spores is very unlikely at temperatures fluctuating between zero and −40°C. It is quite possible that living spores of these common species have recently reached this substratum.     

The Nature of the Iodinophilous Vacuole of Myxosporidan Spores, and a Proposal to Synonymize the Genus Myxosoma Thélohan, 1892 with the Genus Myxobolus Bütschli, 1882

SYNOPSIS. The myxosporidan genera Myxobolus and Myxosoma differ only by the presence or absence of a so-called iodinophilous vacuole in their spores which has in the past been shown to contain glycogen. In the present study, spores of Myxobolus pseudodispar, M. cyprini, M. muelleri, Myxosoma heterospora and Agarella gracilis were tested for glycogen using Best's carmine, iodine, and the Bauer-Feulgen technic. These tests showed that (a) Glycogen was present in variable amounts in spores of each species. In some spores this took the form of a vacuole, while in others the distribution was diffuse or particulate; (b) In no species of Myxobolus did the majority of spores contain a vacuole; (c) In Myxosoma heterospora vacuoles were present in a small proportion of spores. It is concluded that the iodinophilous vacuole cannot be used as a taxonomic criterion in the Myxosporida, and therefore that the genus Myxosoma should be synonymised with the genus Myxobolus .     

Structural alteration of the plasma membrane in spores of the microsporidium Nosema algerae on germination

The fine structure of the plasma membrane in spores of the microsporidium Nosema algerae, a pathogen of mosquitoes, was examined in the resting condition and after the spores were stimulated to germinate in vitro. Slow penetration of resin caused collapse of the germinated spores. Thin sections of germinated spores showed peculiar membrane infoldings that were never found in ungerminated samples. Analogous germination-dependent configurations of the plasma membrane were observed in freeze-fractured preparations of spores either fixed and impregnated with glycerol prior to freezing, or rapidly frozen with liquid propane while in the process of germination. In every case, the replicas presented germinated spores with indentations in the protoplasmic face of the plasma membrane, and apparently complementary blunt spines on the external face, that were absent in ungerminated spores. It suggests that these alterations of the plasma membrane result from a structural adjustment to a spontaneous contraction of the spore case after germination. We discuss this interpretation with regard to conflicting views on the nature of such morphological features.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

Blue Light Interference in the Phytochrome-controlled Germination of the Spores of Cheilanthes farinosa

Short exposure of the spores of Cheilanthes farinosa to low intensity red light promotes their germination, which is not reversed by a subsequent exposure to far red light. Germination is, however, inhibited by blue light administered before or after red light. Inhibition of germination by blue light is annulled by exposure to a higher intensity of red light, and germination of the repromoted spores is inhibited by far red light. Mutual photoreversibility of germination is also observed in repromoted spores irradiated successively with far red and red light. Although germination appears to be basically under phytochrome control, it is postulated that the presence of a blue light-absorbing pigment interferes with phytochrome transformations in the spores.     

The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

S ummary : The density gradient centrifugation of a suspension of spores of B. subtilis 8057 on both sucrose and renografin gradients gave 2 distinct fractions. Germination evidence suggested that the heavier fraction consisted of dormant spores and the less dense fraction, germinated spores. It is concluded that density gradient centrifugation may provide a useful technique for the separation of germinated from nongerminated spores.