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.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating 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.     

THE REVERSIBLE HEAT ACTIVATION INDUCING GERMINATION AND INCREASED RESPIRATION IN THE ASCOSPORES OF NEUROSPORA TETRASPERMA

The heat activation of Neurospora tetrasperma ascospores is a reversible process, since activated spores may be returned to secondary dormancy by preventing respiration, and these secondarily dormant spores may be induced to germinate by reheating. Activation of the spores brings about a large increase in respiration prior to the germination of the spores. As the spores are reversibly activated or deactivated the rate of respiration is increased or is decreased. By poisoning the cells with iodoacetamide it is possible to prevent all germination without greatly inhibiting this increase in respiration. Precisely with the beginning of germination a secondary rise in respiration occurs. The respiration of the spores is cyanide sensitive. The heat activation has a critical temperature at about 49 to 52°C.; and at a constant temperature within this range, the percentage of the spores activated as plotted against the time, follows an S-shaped population curve.     

Viability and physiological state transitions of <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> sporangiospores in tempe starter culture

The viability and various physiological characteristics of individual sporangiospores of Rhizopus oligosporus in tempe starter cultures that had been stored for 8, 10, 16 and 30 months were examined by flow cytometry in combination with fluorescent dyes. Besides live, dead, and dormant spores we distinguished a category of sublethally damaged spores. Results indicated that the shelf-life of tempe starters was not limited by the death of spores, but by sublethal damage to spores as well as by dormancy which can be overcome by resuscitation, respiratory activation. During storage, the number of dormant and sublethally damaged spores increased: the longer the starter cultures were stored, the less dormant spores could still be activated. In contrast, the transition from sublethally damaged (spores that are not able to transform cFDA and emit green fluorescence except by activation treatment) to activated spores did not decrease with longer storage. However, after very long (30 months) storage, sublethally damaged spores could still be activated but could not germinate anymore. The shelf-life of spores in tempe starter is related to the physiological state of spores being sublethally damaged; a mechanism of physiological state transitions of R. oligosporus sporangiospores is proposed.     

Study of heat resistance in a bacterial nucleosidase

The enzyme nucleosidase (EC. 3.2.2.1.) is present in the intact spores, germinated spores as well as vegetative cells of $\text{Bacillus cereus}$ T. In the intact spores the enzymeis resistant to heat and, in fact, has a high temperature optimum. Though the spores themselvesbecome sensitive to heat on germination, the enzyme retains its resistance to heat on germination as well as its high temperature optimum. The vegetative cell enzyme is sensitive to heat. The enzyme in all types of cells &; spores is resistant to octyl alcohol. There is a close correlation between the development of heat resistance in the sporulating cells and that of heat resistance of the enzyme.     

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.     

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.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

Heat inactivation of Mycobacterium avium-Mycobacterium intracellulare complex organisms in meat products.

The spores of four species of vesicular-arbuscular endophytes were L-dried at 22°C, and their viability was tested after heating at 80°C for up to 40 min. L-drying of spores in the soil in which they developed was a very effective method of preservation of all spore types examined. Slow L-drying of spores separated from soil and supported on glass fiber filters also gave high viability for spores of some species. A scheme for the long-term preservation of vesicular-arbuscular endophyte spores is proposed.     

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.     

Comparison of airborne spore concentrations and fungal allergen content

The exposure to spores causing health effects is usually assessed by determining the concentration of viable spores per cubic meter of air (CFU/m³).Since allergens might also be present in dead spores or smaller particles, the objective of this study was to investigate the correlation between the viable spores of Alternaria and Cladosporium at different indoor and outdoor sites and the corresponding allergen concentration detected with a specially developed ELISA (Enzyme Linked Immunosorbent Assay). In outdoor air, the results show a strong correlation between the different sampling techniques applied for viable spores (Slit-Sampler and Multistage Liquid Impinger) and between the viable spores and the allergen concentrations detected in the liquid samples of the impingers. Indoors, the number of viable spores and the allergen concentration do not correlate and the allergen load is underestimated if colony counting methods are used. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pressure Inactivation of Bacillus Endospores

The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure. We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B. amyloliquefaciens, and B. licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80°C) in mashed carrots. A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70°C for 4 min ranged from more than 6 log units to no reduction. The sporulation conditions further influenced their pressure resistance. The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores. Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores. These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium. The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B. subtilis CIP 76.26. Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination. At a pressure between 600 and 800 MPa and a temperature greater than 60°C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level. Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B. amyloliquefaciens, which form highly pressure-resistant spores.     

Parka decipiens and land plant spore evolution

The morphology and ultrastructure of the spores of the enigmatic Lower Devonian plant Parka decipiens are briefly described and previous interpretations of the plant are discussed. It is suggested that the alete spores of Parka could have been haploid or diploid. The possibility that some early plant spores were diploid may be viewed as supporting the interpolation theory for the origin of alternation of generations in land plants. The spores of Parka may lie (in morphological and developmental terms) somewhere between the oospores produced by algae and the triradiate haploid spores of the bryophytes.     

A method for the extraction and enumeration of resting spores of Plasmodiophora brassicae from infested soil

A technique is described for the extraction of the resting spores of Plasmodiophora brassicae from soil. After deflocculation, coarse mineral matter was filtered from soil samples and the remainder mixed with 40% sucrose. The mixture was allowed to stand for 2–5 days; this enabled most mineral particles to settle out leaving the spores in suspension. The spores were counted optically on a microscope using Nomarski interference contrast optics, although preliminary studies had indicated that electronic image analysis was a more satisfactory method. Good recovery of spores was achieved from artificially infested soils containing 10⁶ spores/g but the technique can be satisfactorily applied to less heavily infested soils. Clubroot symptoms developed on seedlings dipped in suspensions of spores recovered in this way.     

Spore morphology of pteridophytes from China Ⅲ. Thelypteridaceae 1. Cyclosorus Link.

Spore morphology of 51 species of Cyclosorus in the Thelypteridaceae from China was investigated using scanning electron microscopy (SEM). The spores are monolete, bilaterosymmetric, ellipsoidal in polar view, and kidney-shaped in equatorial view. On the basis of the variation in their surface ornamentation, the spores fall into three main types. Type I: The surface of spores is echinate or perforate with fimbriate wings. 19 species belong to this type. Type Ⅱ: The surface of spores is cristate. 11 species belong to this type. Type Ⅲ: The surface of spores is echinulate. 10 species belong to this type. The remaining 11 species have spores with mixed surface ornamentation of the above three types, which are considered as intermediate types. The results are valuable for a better understanding of the taxonomy and palynology of the genus Cyclosorus.     

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair‐like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH‐sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.