Comparisons of Cells, Refractile Bodies, and Spores of Bacillus popilliae

Spores of Bacillus popilliae from infected larvae and refractile bodies produced in a Trypticase-barbiturate medium were similar but distinct from vegetative cells of this organism in protein, nucleic acid, and enzyme composition. The spores and refractile bodies were found to have catalase activity, some of which was heat-resistant. This enzyme was not found in the vegetative cells. The spores contained dipicolinic acid, but the refractile bodies did not. The latter were similar to cells in having considerably higher levels of phosphate extractable with cold trichloroacetic acid and of poly-beta-hydroxybutyrate than had the spores. Electron microscopy demonstrated conclusively that the refractile bodies are distinctly different from either cells or spores of B. popilliae. The possibility that these bodies are formed as a result of an aborted sporulation process is discussed.     

Zygnematalean zygospores: Morphological features and use in species identification

Suitable morphological characteristics for identification of zygnematalean algae were determined using a combination of classical light microscopy (LM) techniques, fluorescence microscopy (with blue and green excitation), scanning electron microscopy (SEM) and specialized culture methods. Characteristics of spore germination, growth and reproduction under culture conditions identified Zygnema chalybeo-spermum in a mixture of zygnematalean spores collected from a small fishpond in Czechia. Reproduction in general, particularly aplanospore formation and lateral conjugation was more frequent and occurred earlier in a nutrient poor medium than in a nutrient rich medium, where vegetative growth was more vigorous. Variability in spore size was wide and influenced by the origin (sexual and/or asexual) of the spores. Asexual spores, particularly partenospores were rounded and significantly smaller than sexual ones. Thus spore morphology alone (size and shape) is not a particularly helpful characteristic for species identification. The mesospore of mature spores contained lipids and a sporopolenin-like material (algaenan), which showed green autofluorescence with blue excitation. The mesospore ornamentation, the only characteristic found that is suitable for identification purposes, can be observed easily in LM and SEM after exospore removal by KOH treatment. Detailed LM and SEM observations of the zygospores of all Zygnema species thus could provide basic data necessary for the preparation of an atlas and key for species identification which, after completion with molecular methods, brings clarification into the genetic relationships between morphospecies.     

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

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

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.     

The effect of growth medium on B. anthracis Sterne spore carbohydrate content

The expressed characteristics of biothreat agents may be impacted by variations in the culture environment, including growth medium formulation. The carbohydrate composition of B. anthracis spores has been well studied, particularly for the exosporium, which is the outermost spore structure. The carbohydrate composition of the exosporium has been demonstrated to be distinct from the vegetative form containing unique monosaccharides. We have investigated the carbohydrate composition of B. anthracis Sterne spores produced using four different medium types formulated with different sources of medium components. The amount of rhamnose, 3-O-methyl rhamnose and galactosamine was found to vary significantly between spores cultured using different medium formulations. The relative abundance of these monosaccharides compared to other monosaccharides such as mannosamine was also found to vary with medium type. Specific medium components were also found to impact the carbohydrate profile. Xylose has not been previously described in B. anthracis spores but was detected at low levels in two media. This may represent residual material from the brewery yeast extract used to formulate these two media. These results illustrate the utility of this method to capture the impact of growth medium on carbohydrate variation in spores. Detecting carbohydrate profiles in B. anthracis evidentiary material may provide useful forensic information on the growth medium used for sporulation.     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth.Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.     

Preparation of Refractile Spores of Clostridium thermosaccharolyticum Involves a Solventogenic Phase

Conversion of vegetative cells of Clostridium thermosaccharolyticum to refractile endospores was achieved by sequential transfer and dilution at each generation, with a final dilution into a sporulation medium that contained xylan supplemented with excess calcium. The subsequent growth was synchronous and resulted in elongated, solventogenic cells that were then shifted to 35°C to permit further differentiation without cell division. The synchronized cells grown in xylan medium supplemented with Ca gluconate produced total solvents that reached 9.63% (vol/vol). One hundred percent of these elongated solventogenic cells (4.84 × 10⁹ cells per ml) entered the sporangial stage and continued to differentiate into refractile spores. Only cells sequentially transferred and diluted at a critical time of the growth cycle are synchronized, induced to elongate (≥fourfold), become highly solventogenic in the presence of excess calcium, and are converted to a homogeneous population of refractile spores.     

Endospores of Sporosarcina halophila: characteristics and ultrastructure

Sporosarcina halophila forms endospores. Electron micrographs revealed ultrastructural similarity to spores of S. ureae. Spore germination indicated by loss of refractility, darkening, swelling and formation of new vegetative cells was followed by phase contrast light microscopy. To induce spore germination, the endospores needed to be heat avtivated. After activation, they were inoculated into nutrient broth medium supplemented with sea-water. Double concentrated sea-water was found to be optimal for germination. Similar to other bacterial endospores, the spores were found to be resistant to heat and ethanol. An ultraviolet absorbing substance was isolated from suspensions of free spores; it was identified to be pyridine-2,6-dicarboxylic acid (DPA) usually present in bacterial spores. DPA was detected in amounts ranging from 5–7% of the spore dry weight; it was not detected in extracts of vegetative cells.Abbreviation DPA 2,6-pyridine-dicarboxylic acid     

Sporulation in Bacillus subtilis is independent of membrane fatty acid composition.

Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined. The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains. The fatty acid composition of cellular lipids depended on the compound used to support growth. Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids. The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells. Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids. The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

The Ultraviolet Photochemistry and Photobiology of Vegetative Cells and Spores of Bacillus megaterium

The ultraviolet (UV) photochemistry and photobiology of spores and vegetative cells of Bacillus megaterium have been studied. The response of vegetative cells of B. megaterium appears qualitatively similar to those of Escherichia coli, Micrococcus radiodurans, and Bacillus subtilis with respect to photoproduct formation and repair mechanisms. UV irradiation, however, does not produce cyclobutane-type thymine dimers in the DNA of spores, although other thymine photo-products are produced. The photoproducts do not disappear after photoreactivation, but they are eliminated from the DNA by a dark-repair mechanism different from that found for dimers in vegetative cells. Irradiations performed at three wavelengths produce the same amounts of spore photoproduct and give the same survival curves. Variation of the sporulation medium before irradiation results in comparable alterations in the rate of spore photoproduct production and in survival.     

Germination of Yeast Spores Lacking Mitochondrial Deoxyribonucleic Acid

A population of petite ascospores (mitochondrial deoxyribonucleic acid [mtDNA]-less), produced by brief ethidium bromide (EthBr) mutagenesis prior to transfer to sporulation medium, was used to examine the role of the mitochondrial genetic system on germination and outgrowth in Saccharomyces cerevisiae. Petite ascospores, which are morphologically indistinguishable by phase-contrast microscopy from wild-type spores, germinate and proceed through outgrowth at a rate and extent only slightly less than that of wild-type spores. Both developmental processes occurred in the absence of mtDNA synthesis and measurable cytochrome oxidase activity. These results indicate that neither respiration nor a functional mitochondrial genome are required for germination and outgrowth. The properties of the petite clones were typical of petites formed during vegetative growth. Individual sporal clones differed markedly from each other in suppressiveness. Petite sporal clones which exhibited a high degree of supressiveness also contained a reduced but detectable amount of mtDNA of altered buoyant density. One clone contained a unique mtDNA with a buoyant density higher than that of wild-type mtDNA.     

Selection and viability after ingestion of vegetative cells, resting spores and resting cells of the marine diatom, Chaetoceros pseudocurvisetus, by two copepods

Feeding response of two copepods Neocalanus flemingeri and Calanus sinicus on cultures of three life-forms; vegetative cells, resting spores and resting cells, of Chaetoceros pseudocurvisetus was investigated. N. flemingeri fed heavily on the vegetative cells but scarcely responded to feed on the resting spores. C. sinicus showed significantly higher filtering rate on the vegetative cells and resting cells than on the resting spores. Survival of the three life-forms of C. pseudocurvisetus after gut passage of the copepods was also studied. The resting spores could germinate from fecal pellets of both N. flemingeri and C. sinicus; however, both the vegetative cells and the resting cells could not survive ingestion by the copepods. These results suggest that resting spore forming diatoms, such as C. pseudocurvisetus form spores which have a low nutritional value and during gut passage are largely indigestible due to the heavily silicified frustules and thus minimize the effects of grazing by copepods.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

Production,long term preservation and synchronous germination of aerial spores of Streptomyces

Vigorous vegetative growth of various Streptomyces species (S. auroefaciens, S. collinus and S. granaticolor) was achieved in a new semisynthetic liquid medium. Unlike the media commonly used for the cultivation of the submerged mycelia of different streptomycetes, this one does not contain insoluble material which enables direct and reliable measurement of net production of biomass. The medium was formulated to meet the nutritional requirements of all the three species. Is also supported production of antibiotic in each of the strains. A method for bulk preparation of Streptomyces aerial spores, involving cultivation on agar plates covered with cellophane, was developed. Advantage of this method lies in higher yields of spores, their higher purity and easier harvesting. The spores were activated by amild treatment with an Ultra-Turrax homogenizer resulting in the breakage of fibrous sheath, suspended in 20% glycerol, and stored at ?60°C. Thus, treated spores germinated synchronously even after several months of the storage. Hence, such spore material may be used for precise inoculation in a large series of experiments implying synchronous germination, and the inoculations can be carried out from the same batch over a long period.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.