Oxidation of Manganese and Formation of Mn3O4 (Hausmannite) by Spore Coats of a Marine Bacillus sp

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl₂ at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl₂ solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.     

Fine Structure of Five Species of Myxomycetes with Clustered Spores*

SYNOPSIS. Electronmicroscopic studies were made of 5 species of Myxomycetes with clustered spores to determine the exact mechanism holding the spores together. In Dianema corticatum the neighboring spore walls were closely appressed and appeared to be fused at numerous points. In Trichia synsporum there was a substance between the outer spore walls of adjacent spores which seemed to cement them together. In Badhamia nitens the spore walls were completely fused along their closely appressed surfaces. The adjacent spores of Badhamia versicolor appeared to be free, or if any fusion point was present it was only at the tips of the ornamentation. In Physarum bogoriense such tips were in contact with those of neighboring spores and were occasionally fused at these points.     

THE COMPOSITION AND STRUCTURE OF BACTERIAL SPORES

The composition of the insoluble "integuments" and soluble "contents" fractions of spores of four Bacillus species of widely differing heat resistance were compared. Electron microscopy of thin sections was also used to determine and compare the morphological structures in the integument preparations. The soluble fractions of the thermophiles, B. coagulans and B. stearothermophilus, had a higher content of hexose and dipicolinic acid. The hexose content of both fractions of the four species was related to heat resistance. Integument fractions consisted chiefly of protein together with variable amounts of the mucopeptide constituents, α, ε-diaminopimelic acid (DAP) and hexosamine. In the thermophiles the DAP and hexosamine were found chiefly in the insoluble integuments fractions, while in B. cereus and B. subtilis most of this material was soluble. Integument preparations, containing mainly protein with little mucopeptide, consisted chiefly of outer and inner spore coats, while preparations having more mucopeptide contained also residual cortical material and a cortical membrane (possibly the germ cell wall). The results suggest that spore integuments consist of mainly proteinaceous outer and inner coats together with variable amounts of residual cortex and cortical membrane which contain the mucopeptide material.     

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     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Biochemical genetics of bacterial sporulation

Summary The morphological and biochemical characters of twenty nine sporulation mutants were compared. Some of the predictions made on the basis of unidirectional pleïotropic interactions were confirmed, namely that the latest proteolytic enzymes, like elastase, are related to late morphological stages. From the cytological point of view, mutants blocked at various stages were described. Among the late mutants, both coatless mutants with normal but incomplete cortex and cortexless mutants with flexible spore coats were found. Particularly interesting is the class of abnormal late sporulation mutants which form normal mature heat-resistant spores at high frequencies, but, in addition, present various anomalies in the structure of the spore coats and various sporangial inclusions such as a spongy fibrous material, resembling the cortex, and either onion-like or rod-shaped inclusions, probably formed by spore coat components. The presence of these structures is related to the derepression of elastase activity and may reflect overproduction of spore components. Several mutants also contain abnormal, large, dark, membrane-bound mesosomes, either compact or loose, whose presence is related to the lack of oxidation of tetrazolium dyes. The morphological heterogeneity of mutant populations is also noted. These findings are discussed in relation to the theory of sequential gene activation.     

A New Toxic Substance,Teleocidin, Produced by Streptomyces

The production and isolation of a new toxic substance, Teleocidin, and its biological properties were previously reported^1,2). Thereafter it has been found that an other strain of Streptomyces produced such specific toxic substance as Teleocidin in its cultured mycellium. Comparative tests of these two purified crystalline powders showed the new toxic substance resembles Teleocidin closely though differs in certain chemical properties. Therefore, the original Teleocidin is designated Teleocidin A, whereas that produced by a new strain of Streptomyces is named Teleocidin B, which had been tentatively called as the SK-toxic substance.

From the results of the chemical studies of Teleocidin B and its hydrogenated derivative, which was easily obtained as a crystalline form by the catalytic hydrogenation of Teleocidin B with Adam’s catalyst, molecular formula, C₂₈H_39~41N₃O₂ was postulated for Teleocidin B.

It was also recognized that an alcoholic hydroxyl, a lactam ring and a heterocyclic ring like indole or pyrrole structure existed as the functional groups of Teleocidin B.     

The fine structure of frozen-etched bacillus cereus spores

Summary The fine structure of Bacillus cereus spores was studied using the freeze-etching technique and compared with that already described in chemically fixed cells. Although the basic structures did not appear different, the freeze-etching technique permitted the examination of membrane surfaces and their involvement in sporulation. In this respect, some membranes were found to be covered with small 120 Å particles believed to represent multi-enzyme complexes, while other membranes appeared relatively smooth.One feature revealed by the freeze-etching technique and not demonstrated in chemically fixed cells, was the presence of relatively large (500 Å) vesicular structures. In many instances these vesicles were closely associated with different layers or membranes in the developing spore. While the role of these vesicular structures is as yet undetermined, their possible connection with the lytic process which liberates the spore from the sporangium, is discussed.     

Chemical Nature and Mode of Stabilization of Egg-shell in Helicometra pulchella (Rudolphi, 1819) (Digenea: Opecoelidae)

The chemical nature and mode of stabilization of egg-shell protein in digenetic trematode Helicometra pulchella (Rudolphi, 1819) have been investigated using histochemical techniques. It was found that the egg-shell is stabilized by quinone-tanning together with dityrosine. Other structural proteins (elastin, collagen and keratin-like proteins), glycogen and acid mucopolysaccharides were absent in egg-shell. Tyrosine was present in vitelline cells and immature egg-shell indicating that the proteins involved in quinone-tanning were tyrosine rich and tyrosyl residues are modified to form dityrosine in mature egg-shell.     

Bacillus subtilis spore coats: complexity and purification of a unique polypeptide component.

Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecular-weight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.     

Fine structure of the mesosome and nucleoid in frozen-etchedBacillus subtilis

Summary An electron microscopical investigation ofBacillus subtilis prepared by freeze-etching revealed the fine structural changes that occur in the cell prior to spore formation. The initiation of growth from lyophilized cells was characterized by the appearance of numerous vesicular structures embedded in and attached to the plasma membrane. As growth continued, the number of vesicular structures decreased and lamellar membrane structures began to appear. Prior to spore formation, a fine, fibrillar material was found in the central portion of the cell and was believed to be the DNA.     

苏芸金杆菌伴孢晶体和芽孢孢衣的蛋白质组分及其与毒力的关系

研究了苏芸金杆菌(Bacillus thuringiensis)3个变种6个菌株的提纯伴孢晶体和芽孢对大蜡螟(Galleria mellonella)和大菜粉蝶(Pieris brassicae)的毒力、晶体的蛋白质和多肽成分及芽孢衣中的类晶体蛋白质成分.生物测定表明,晶体毒力高于芽孢,在总数量相同的情况下,晶体和芽孢近1:1的混合物的毒力高于单独的晶体或芽孢.芽孢衣中存在一种类似晶体蛋白质的成分,无晶体突变株及无效野生株的芽孢则无此种蛋白质且对两种昆虫无毒.变种wuhanensis和变种galleriae的晶体含MW138000的主要蛋白质和63000的次要蛋白质,经碱性缓冲液溶解后,上清液含MW138000的蛋白质,沉淀中含MW63000的蛋白质;变种aizawai的晶体中仅含138000的蛋白质.对大蜡螟无毒的HD-11(var.aizawai)晶体的蛋白质成分有别于上述晶体,其胰蛋白酶消化物的SDS凝胶电泳图型显示少2条多肽带,但对大菜粉蝶仍有效.结果表明,苏芸金杆菌的芽孢在昆虫病理中有重要作用,伴孢晶体的蛋白质和多肽成分与它们对昆虫的毒力特性之间有密切关系.     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Inhibition of Spore Germination in the Cellular Slime Moulds

The phenomenon of auto-inhibition of spore germination has beendemonstrated in the slime moulds Dictyostelium purpureum, strainno. 2; Dictyostelium discoideum, strain no. 1; Dictyosteliummucoroides, strain no. 2; and Polysphondy-Iium violeceum, strainno. 6. A water-soluble substance present in spores was shownto cause this self-inhibition. The substance was isolated fromeach species studied. Evidence as presented indicates that thesame substance occurs in each species of Dictyostelium, butthat it differs from that found in Pv6. An attempt was alsomade to relate this inhibitory substance to the phenomenon of'differential inhibition' in Dp2, Dd, Dm2, and Pu6. An explanationbased on evidence for differential sensitivity to a common inhibitorof spore germination in Dictyostelium and an inhibition in otherthan the spore stage in Pu6 is presented. Apparent differentialproduction of a common inhibitor in Dictyostelium is also reported.     

Nutritional requirements of keratinophilic fungi and dermatophytes for conidial germination

Germination of spores of Chrysosporium crassitunicatum, Nannizzia fulva (+), Nannizzia fulva (–) and Trichophyton equinum was studied in the presence of various carbon and nitrogen sources. Effect of different temperatures on spore germination was also determined. Maximum spore germination within 24 hours was recorded when glucose was used as a carbon source for all the test fungi. Except sodium nitrate all the inorganic nitrogen sources enhanced the spore germination at 0.05% concentration. Most of the organic nitrogen sources used were found to be stimulatory for the spore germination of test fungi. Optimum temperature i.e. 28 °C supported maximum spore germination of all the test fungi within 24 hours. C. Crassitunicatum, N. fulva(+), N. fulva(–) could germinate upto 35 °C but beyond that no spore germination was noticed in these fungi. T. equinum could germinate at a higher temperature of 40 °C but the percentage of germination was very low.     

Role of esterase gp70 and its influence on growth and development of Dictyostelium discoideum

Gp70 is an esterase originally called crystal protein because of its presence in crystalline structures in aggregation-competent Dictyostelium discoideum cells. Although postulated to break down spore coats, the function of gp70 in vivo was incompletely investigated. Our immunolocalization and biochemical studies of vegetative D. discoideum amoebae show that gp70 was recruited to phagosomes and found in lysosomes. Purified gp70 was effective at hydrolyzing naphthyl substrates with acyl chains typical of lipids and lipopolysaccharides, indicating that the gp70 was involved in digesting endocytosed molecules. The activity of purified gp70 was inhibited by reductants that retarded its electrophoretic mobility and verified the presence of intramolecular disulfide bonds predicted by its amino acid sequence. Compared to wild-type cells, cells overexpressing gp70 were more phagocytically active, had shorter generation times, and produced more fruiting bodies per unit area, while cells lacking gp70 were phagocytically less active with longer doubling times, developed more slowly, and had significantly fewer fruiting bodies per unit area. Consistent with the phenotype of a disrupted metabolism, one-third of the gp70-minus cells were large and multinucleated. Together, these results indicated that despite its crystalline appearance, gp70 was an active esterase involved in both the growth and the development of D. discoideum.     

Changes in the hydrophobic characteristics of Clostridium perfringens spores and spore coats by heat

The hydrophobic characteristics of Clostridium perfringens NCTC 8679 spores were demonstrated by adherence to toluene in a toluene-aqueous partition system. Spores and spore coat preparations were hydrophobic. Vegetative cells and spores extracted with a dithiothreitol-sodium dodecyl sulfate treatment known to remove spore coats were not hydrophobic. A heat activation treatment (75 degrees C for 20 min) which promotes more rapid spore germination increased the hydrophobicity of intact spores and decreased that of isolated spore coats. The hydrophobic changes were reversed by washing and stabilized by 0.5% glutaraldehyde. Heat-induced hydrophobic changes were observed in spore coats prepared from spores that were preheated and washed before rupturing in a buffer containing glutaraldehyde. These results suggest the occurrence of a heat-induced change in the spore coat (possibly in the conformation of a macromolecule) which was stable only within the architectural confines of the intact spore.