Hypertonic signal promotes stability of Dictyostelium spores via a PKA-independent pathway

Differentiation of Dictyostelium spores initiates with rapid encapsulation of prespore cells under the control of cAMP-dependent protein kinase (PKA), followed by further maturation processes involving cytoskeletal reorganization. Constitutive activation of PKA induces precocious formation of viable spores in development and confers the ability to encapsulate under specific submerged conditions. In this study, we show that the stability of these spores depends upon conditions of high osmotic strength during spore differentiation, indicating that a hypertonic signal is required in addition to PKA to induce maturation to stable spores. The formation of stable spores under hypertonic conditions requires high cell density, suggesting the involvement of additional cellular signaling.     

Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes

Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell-chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.     

Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation

Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (~5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.     

Increase in Si:N drawdown ratio due to resting spore formation by spring bloom-forming diatoms under Fe- and N-limited conditions in the Oyashio region

Resting spore formation and Si:N drawdown ratios were investigated under iron (Fe)- and nitrogen (N)-limited conditions using a unialgal culture of Thalassiosira nordenskioeldii and natural phytoplankton assemblages during the spring bloom in the Oyashio region. In the unialgal culture of T. nordenskioeldii, 20% and 100% of the cells formed resting spores under Fe- and N-limited conditions, respectively. The Si:N drawdown ratios were 2- and 14-fold higher in Fe- and N-limited conditions, respectively, compared to Fe- and N-sufficient conditions. At the start of the natural phytoplankton incubation, 18 among 47 identified diatom species were known resting spore-forming species. Approximately 15 common diatom species formed resting spores under Fe- and N-limited conditions. During the natural phytoplankton incubation, the percentage of the resting spores increased with time under both Fe- and N-limited conditions, reaching 25% and 40% of total diatom abundance, respectively. The Si:N drawdown ratios significantly increased with an increase in the contribution of resting spores in both the unialgal culture and natural phytoplankton incubations. These results suggest that if the bloom dominated by neritic, resting spore-forming diatom species decline by either Fe- or N-depletion, Si may be utilized preferentially to N in the upper mixed layer due to the formation of heavily silicified resting spores.     

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

Summary The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.     

Photodynamic inactivation of Bacillus subtitis spores

The effect of strong visible light on spores of Bacillus subtilis has been studied. With the illumination conditions used, spores were killed exponentially and viability decreased to less than 13% survivors after 24 h. This decrease did not take place when the cells were kept under strictly anaerobic conditions. The killing is, therefore, attributable to a photodynamic effect. The presence in the medium of exogenous photosensitizers did not increase the effect of light and this is attributable to the impermeability of spores to these agents. Once germinated, the spores were killed at a much faster rate, even when outgrowth was stopped. The highest sensitivity was reached 90 min after onset of outgrowth.     

Independence of Bacillus subtilis spore outgrowth from DNA synthesis.

The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidoxic acid and 6-(p-hydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhbition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors.     

A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Use of a new chemotaxis assay with Candida albicans as gradient source.

The locomotion of human blood neutrophil leucocytes was observed and analysed by time-lapse cinematography (1) under conditions where chemokinetic locomotion was stimulated, i.e. in a uniform concentration of casein; (2) in response to chemotactic gradients generated at a point-like source, namely blastospores of the pathogenic yeast Candida albicans in normal human plasma, and (3) in response to soluble chemotactic factors diffusing from Sephadex beads. Neutrophils moving in purely chemokinetic conditions tended to persist in straight paths and showed a preference for narrow angles of turn suggesting a “persistent random walk” type of locomotion rather than a pure random walk. Cells responding to Candida spores showed near straight-line locomotion to the gradient source over short distances (ca 50 μm) and brief time periods. They phagocytosed the spores on arrival and were usually immediately able to respond to a new gradient. Colchicine treatment caused the cells to turn through wider angles, but they were still able to home onto and phagocytose the spores. Colchicine-treated cells showed bizarre and fluctuating shapes but were nonetheless usually polarized towards the gradient source. Gradients from large sources, such as Sephadex beads containing soluble chemotactic factors, were more easily disturbed than those from Candida spores and directional locomotion of cells towards the beads was only seen in certain sectors. The angles of turn made by moving cells under these conditions were an important determinant of chemotaxis since paths of those cells reaching beads showed longer straight segments and narrower angles of turn than those which failed to show a directional response.     

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

Cell contact mediated differentiation in dictyostelium.

Major biochemical and ultrastructural changes occur in Dictyostelium discoideum plasma membranes following aggregation of the amoebae. The effects of cyclic AMP, Concanavalin A (Con A), and disruption of cell contacts on membrane particle synthesis and the subsequent differentiation of prespores and mature spores were determined. The results indicated that prespore cell differentiation always failed under conditions in which large particle formation was inhibited or cells bearing particles were restricted in their contacts. Although prespore cells exposed to Con A formed mature spores devoid of prespore vacuoles, the cell walls were defective. The research suggests that the interactions between membrane particles of apposing amoebae may initiate differentiation of prespores and mature spores.     

Survival of microbial films in the microwave oven.

Air-dried films of Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis spores on membrane filters, exposed to 10 min full power (650 W, 2450 MHz) irradiation in a Toshiba model ER776BT microwave oven, showed a 5-, 2-, and 0-log reduction of viable organisms respectively. Suspensions of cells or spores in phosphate buffer treated under similar conditions showed 8 logs of killing within 30 s (S. cerevisiae), 45 s(E. coli), and by 10 min (B. subtilis spores) of exposure.     

Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of Phanerochaete chrysosporium

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.     

产乳酸凝结芽胞杆菌N001的抗逆性研究

目的研究前期经初筛的产乳酸凝结芽胞杆菌N001芽胞的抗逆性。方法在模拟饲料制粒条件下和动物消化道内逆境条件下的存活能力,测定N001芽胞的抗热、抗酸、耐胆盐性能和对抗生素的敏感性。结果凝结芽胞杆菌N001芽胞具有很强的耐高温、耐酸、耐胆盐能力;同时N001芽胞对营养体敏感的抗生素也有良好的耐受性。结论凝结芽胞杆菌N001芽胞具有很强的抗逆性,可以作为益生菌制剂的良好菌种。     

Facile Method for Monitoring Inhibition of Anaerobic Spore Outgrowth

A device is presented for the laboratory monitoring of spore outgrowth under controlled temperature and anaerobic conditions. Alterations in pH, redox potential, headspace composition, and optical density are followed as the activated spores grow out into vegetative cells. An interlock system allows the addition of test solutions or the removal of medium under anaerobic conditions. The device may also be used for rapid (<4 h) chemical inhibition studies or adapted for temperature injury studies of aerobic or anaerobic cells. Data on outgrowth of Clostridium sporogenes and inhibition by nitrite solutions are presented.     

Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions

Classical enumeration methods for anaerobes are time-consuming and require special conditions. Solid phase cytometry (SPC) is a recent laser scanning technique for the quantitative detection of fluorescently labelled bacteria on a membrane filter that eliminates the need for a growth phase. Fluorescent labelling of cells results from the cleavage by intracellular esterases of a fluorescein type ester to yield a free fluorescein derivative, which is retained only in cells with an intact cytoplasmic membrane. However, as the standard labelling procedure is carried out under the conditions of aerobiosis, labelling of anaerobic bacteria does not appear to be obvious. We have labelled eight strains of vegetative anaerobic bacteria (i.e. Bacteroides thetaiotaomicron, Clostridium bifermentans, C. butyricum, C. perfringens, Fusobacterium nucleatum, Porphyromonas canoris, P. gingivalis, Propionibacterium acnes) and two strains of spores (C. butyricum, C. perfringens,) within 4 h under aerobic conditions. However, anaerobiosis remained necessary for spores of C. sordellii, C. sporogenes, C. tyrobutyricum. For vegetative cells of all strains, plots of SPC versus plate counts were linear with slopes exceeding 1.0, indicating that SPC consistently yielded higher numbers of bacteria.     

Experimental evidence for a persistent spore bank in Sphagnum

Spore capsules of four Sphagnum species were buried at different depths in peat on a bog. Spore viability was determined after 0, 1, 2 and 3 yr. Viability generally declined with time, but viable spores were still found at all depths after 3 yr. The light-coloured spores of S. balticum and S. tenellum retained their viability better than the darker spores of S. fuscum and S. lindbergii . Survival was highest under wet but aerobic conditions, but was also high under humid or periodically desiccated conditions. By contrast, most spores stored under wet, anaerobic conditions died within 2–3 yr. These results, and predictions from them, are not consistent with earlier results for spores of long-lived and dominant bryophytes, or for seeds of phanerogams of undisturbed wetlands and forests. There was no correlation between spore size and longevity across species, but the small spores from small capsules of S. balticum and S. tenellum generally showed higher viability than those from the medium-sized and large capsules of the same species. This suggests a positive intraspecific relationship between longevity and dispersal distance. There was an indication of conditional dormancy, controlled by weather, in Sphagnum spores. The experiments indicate that Sphagnum spores can form a long-term persistent spore bank under suitable conditions, with a half-life of between 1 and 20 yr (mean across species of 2.6 and 5.0 yr at two depths studied), and with potential values in individual spore capsules of several decades, or even of centuries. Sphagnum spores kept refrigerated showed 15–35% viable spores after 13 yr. The capacity to form a persistent spore bank that can be activated whenever favourable conditions occur might help explain the wide geographical distribution of many Sphagnum species in the boreal and temperate zones, where they have managed to colonize almost every suitable patch of acidic, nutrient-poor wetland.