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Chemical and Morphological Studies of Bacterial Spore Formation

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Abstract

From the stage of a completed membranous forespore to that of a fully ripened free spore, synchronously sporulating cells of a variant Bacillus cereus were studied by cytological and chemical methods. Particular attention was paid to the development of the three spore layers—cortex, coat, and exosporium—in relation to the forespore membrane. First, the cortex is laid down between the recently described (5) double layers of the forespore membrane. Then when the cortex is ⅓ fully formed, the spore coat and exosporium are laid down peripheral to the outer membrane layer covering the cortex. As these latter layers appear, the spores, previously dense by dark phase contrast, gradually "whiten" or show an increase in refractive index. With this whitening, calcium uptake commences, closely followed by the synthesis of dipicolinic acid and the process is terminated, an hour later, with the formation of a fully refractile spore. In calcium-deficient media, final refractility is lessened and dipicolinic acid is formed only in amounts proportional to the available calcium. If calcium is withheld during the period of uptake beyond a critical point, sporulating cells lose the ability to assimilate calcium and to form normal amounts of dipicolinic acid. The resulting deficient spores are liberated from the sporangia but are unstable in water suspensions. Unlike ripe spores, they do not react violently to acid hydrolysis and, in thin sections, their cytoplasmic granules continue to stain with lead solutions.

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... Also the sporulation and germination are influenced by the concentration of divalent cations, including calcium. The fast uptake of calcium was observed in stage IV of sporulation (Young and Fitz-James, 1962) and may ultimately comprise up to 3% of the dry weight of the spores (Murrell and Warth, 1965). The concentration of cations remarkably influenced the physiological properties of spores, in particular a resistance to heat, radiation, enzymes, disinfectants and other deleterious agents (Warth, 1979). ...
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... The mutant was subcultured every 7 days. For sporulation, the medium and techniques were basically as described by Young and Fitz-James (1959) For personal use only. litres: 0.5 g proteose peptone No. 2,0.5 g proteose peptone No. 3, and 0.32 g nutrient broth, all obtained from Difco. ...
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... In contrast with exponentially growing cells of Bacilli Bronner et al., 1975) and Escherichia coli (Silver & Kralovic, 1969;Rosen & McClees, 1974;Silver, 1977), which are thought to maintain a low Ca2+ concentration in the cytoplasm by metabolically active efflux, sporulating Bacilli are found to accumulate Ca2+ from stage IV to VI of sporulation, concomitant with biosynthesis of dipicolinic acid (Young & Fitz-James, 1962;Eisenstadt & Silver, 1972). On a dry-weight basis, spores of most bacterial species contain 2-4% of Ca2+ and 5-15% dipicolinic acid in an approx. ...
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Quantification of viable spores is a time taking task due to the lack of rapid, efficient and accurate methods. This study presented a simple spectrophotometric method for the detection of viable spores based on spore's property of losing refractivity during the germination process. By comparison of the results obtained by both spectrophotometric method and colony counting method, a good linear correlation (R 2 = 0.99) was achieved between viable spore concentration and OD loss under appropriate conditions. To avoid interference from ungerminable spores and vegetative cells, a turbidity complementation strategy of keeping the initial concentration of spore suspensions at the same and relatively lower level was required. The calibration equation developed could be used to predict the viable spore yield produced in a series of fermentation experiments. The experimental results proved that this novel spectrophotometric method was sensitive, rapid, and easy to perform compared to conventional colony counting method.
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Article
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A triple fixation method using a sequential application of 15 or 30% formaldehyde, 6% glutaraldehyde, and 1% osmium tetroxide resulted in excellent fixation of mature spores of Clostridium botulinum.
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A quantitative assay employing (125)I-labeled antibody has been developed for Bacillus cereus T spore coat protein. Populations of antibody molecules with various affinities for inner or outer coat can be prepared by selective adsorption to and elution from different coat preparations. Adsorption to and elution from intact spores results in an antibody preparation at least 15 times more reactive to outer coat. This antibody is useful for measuring the time and extent of spore coat maturation, i.e., outer spore coat formation. Rifampin inhibits the increase in content of this coat antigen.
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Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.
Article
Moberly, Betty J. (The University of Michigan, Ann Arbor), F. Shafa, and Philipp Gerhardt. Structural details of anthrax spores during stages of transformation into vegetative cells. J. Bacteriol. 92:220-228. 1966.-Anthrax spores in stages of dormancy, activation, germination, and outgrowth into vegetative cells were examined in an electron microscope. The fine structure proved to be much like that observed in related species of Bacillus, except for a visible alteration after heat activation and clusters of vesicle-like bodies in the cytoplasm of vegetative cells.
Article
Stahly, D. P. (University of Illinois, Urbana), V. R. Srinivasan, and H. Orin Halvorson. Effect of 8-azaguanine on the transition from vegetative growth to presporulation in Bacillus cereus. J. Bacteriol. 91:1875-1882. 1966.-The guanine analogue, 8-azaguanine (azaG), was found to inhibit sporulation of Bacillus cereus strain T when added to proliferating cells, but not to inhibit when added after the transition to presporulation. When azaG was added to vegetative cells, the growth rate was reduced, but no immediate bactericidal effect was demonstrated. Azaguanine was shown to be incorporated solely into ribonucleic acid (RNA). All of the natural purine bases and nucleosides were found to prevent azaG inhibition by blocking incorporation of the analogue into the RNA. Addition of a subinhibitory level of C(14)-azaG to proliferating cells resulted in an increase in incorporation paralleling the increase in number of cells. At the time of transition from growth to presporulation, a rapid removal of the azaG label from the cells occurred in the absence of net RNA breakdown. If differentiation was inhibited by increasing the concentration of azaG, then no expulsion took place. Instead, at the end of growth, net incorporation ceased, and a steady-state condition was established in which incorporation equaled breakdown. No azaG degradative enzymes are present in presporulating cells. The possibility is discussed that an increase in the ratio of natural purines to azaG occurred at the time of transition, and that the natural purine derivatives then were reincorporated into RNA preferentially to azaG. The data are consistent with the hypothesis than an increased rate of RNA turnover occurs at the time of transition from vegetative growth to presporulation. Addition of phosphate buffer (pH 7.0, 0.1 m) to azaG-inhibited vegetative cells caused reversal of inhibition, the reversal being accompanied by expulsion of the azaG. At least a partial explanation of this effect is that phosphate causes a decrease in the azaG intracellular pool size.
Article
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Ellar, D. J. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J. Bacteriol. 92 1748–1764. 1966.—A study was made of the fine structure of sporulating cells of Bacillus cereus grown in a chemically defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hr. This time period was shortened when spore wall peptide components were added to the medium, but the addition had no effect upon fine structure except to thicken the cell wall. Sporulation could be separated into six morphological stages which generally agreed with those published for other sporulating bacteria. The initiation of the spore (forespore) septum takes the form of an inward folding of the cytoplasmic membrane toward the pole of the cell. The inward folding forms a characteristic Y-shaped membrane structure enclosing an area within which vesicles are found. These vesicles comprise the perisporal mesosome of the cell. The membranes on opposite sides of the cell progress toward the cell center where they fuse to form the double unit membrane of the spore septum. As the proliferation of the spore septum continues, the vesicular areas move towards the pole. The end result is a double forespore membrane which completely encloses a part of the vegetative cell's chromatin. Sporal mesosomes, as well as membrane vesicles, are involved in the proliferation of the forespore. Vesicles are generally bounded by a single unit membrane, whereas in the sporal mesosomes several unit membranes are arranged concentrically. The latter become associated with the segregation of a portion of the nuclear material into the forespore region of the cell.
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The physiology of spore formation was studied in Bacillus cereus and a temperature-sensitive asporogenic mutant. The parent organism sporulates when cultured in a minimal medium at either 28 °C or 37 °C while the mutant sporulates only at 28 °C. The blocking of sporulation at 37 °C has been referred to as "abortive" sporulation. Uptake of calcium and zinc was followed during growth and sporulation or "abortive" sporulation. Calcium and dipicolinic acid (DPA) levels in sporogenic cultures increased as the medium calcium was increased. The asporogenic mutant took up less calcium and synthesized little DPA. Heat resistance of spores increased as the calcium and DPA increased. Over 99% of Ca45 or Zn65 were released from labelled spores when autoclaved to release DPA. Chemical fractionations were made of cells labelled with Zn65 and Ca45 and harvested at different times during the culture cycle. Smaller percentages of calcium than of zinc were located in the cold trichloroacetic acid soluble fraction. The alcohol-soluble, ether-insoluble fraction of spores contained a greater percentage of calcium than was found in vegetative cells. Cells which had undergone "abortive" sporulation contained the same percentage of calcium in this fraction as homologous vegetative cells.
Article
Abstract—As sporulation progresses, there is an increased resistance to UV irradiation of the cells of Bacillus cereus var. alesti. This progressive increase is independent of post-irradiation treatment and appears to be a property of the stage of sporulation. In addition, the proportion of photoproducts formed is different for each stage of sporulation. Cells irradiated at Stage I (axial filament) of sporulation display relatively large amounts of spore photoproduct ‘c’ and less of photoproduct ‘b’. As sporulation proceeds, UV irradiation results in the production of more spore photoproduct ‘b’ and less ‘c’, suggesting a progressive change in configuration of the DNA within the sporulating cell. If irradiated early in the process (Stage II), large amounts of cyclobutane-type dimers are also produced which, with the ‘spore-specific’ photoproducts, may be retained in the resultant spore. Although no excision-repair was detectable during germination of these spores, both vegetative and ‘spore-specific’ damage is reduced during this period. The ‘spore-specific’ repair mechanism may be able to remove vegetative damage from germinating spores.
Article
The transcriptional activity of the two genomes of the sporangium during spore formation was determined by pulse-labeling bacteria with 3H-uracil at different times of sporulation and preparing them for high resolution autoradiography. The quantitative analysis of autoradiographs shows that uracile incorporation in the whole sporangium decreases considerably between stages II and IV. However, the variations of the transcriptional activity are not identical in the mother cell and in the forespore. The one of the mother cell decreases rapidly between stages II and III and then remains stable until the end of stage IV, whereas that of the forespore which is low at stage II increases as the forespore grows ovoid and then quickly diminishes. It is very weak at the beginning of stage IV and negligible at the end of this stage. Pulse-chase experiments made in the presence of rifampine indicate that about 30% of the uracile incorporated is located in stable RNA. This value is found at any stage of sporulation in both cellular compartments whatever their rate of uracile incorporation. A relationship can be made between the nuclear shape and the activity of the genetic material. This confirms observations made by several authors in other bacterial species and other physiological conditions that the condensed shape corresponds to a state of low transcriptional activity whereas the more irregular and dispersed shape corresponds to a state of high activity.
1.1. Incubation at 37°C seems to prevent the formation of the calcium accumulation system during sporulation in Bacillus megaterium.2.2. The temperature sensitive period for the formation of the calcium accumulation system was approximately at the end of growth.3.3. KCN inhibited not only the formation of the calcium accumulation system but also calcium uptake, whereas chloramphenicol inhibited the formation of the calcium accumulation system alone.
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Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.
Article
1.1. Calcium uptake was examined in mutant strains of Bacillus megaterium unable to sporulate because of the absence of dipicolinic acid synthesis.2.2. Calcium uptake in the mutant strains was independent of dipicolinic acid synthesis.3.3. Calcium accumulation in the mutants increased when dipicolinate was added.4.4. Calcium was released from the mutant cells after reaching a maximum level of its accumulation.
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Measurement of certain membrane-bound enzymic activities was used to study the orientation of the outer membrane of the double-membraned forespore of Bacillus megaterium KM. 2. Adenosine triphosphatase, NADH dehydrogenase and L-malate intact protoplasts, but were readily detected in intact stage II or IV forespores, consistent with reversed polarity of the outer forespore membrane relative to the mother-cell plasma membrane. 3. Measurement of NADH oxidase activity revealed that intact stage III forespores had the same high affinity for NADH as protoplast membrane preparations and protoplast lystates, consistent with ready access of NADH to oxidation sites on the outer forespores membrane. 4. Forespores and protoplasts showed osmometric behaviour in solutions of non-permanent solutes consistent with the presence of an intact permeability barrier in these structures.
Article
The kinetics of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis as well as protein breakdown during sporulation by Clostridium perfringens were determined. Maximum levels of DNA and net RNA synthesis occurred 3 and 2 h, respectively, after inoculation of sporulation medium. The rate of RNA synthesis decreased as sporulation progressed. Deoxyadenosine increased uptake of [14C]uracil and [14C]thymine but depressed the level of sporulation and the formation of heat-resistant spores when added at concentrations above 100 mug/ml. Unlike Bacillus species, net protein synthesis, which was sensitive to chloramphenicol inhibition, continued during sporulation. The rate of protein breakdown during vegetative growth was 1%/h. During sporulation this rate increased to 4.7%/h. When added to sporulation medium at 0 time chloramphenicol reduced protein breakdown to 1%/h. If added at 3 h the rate decreased to 2.1%/h. The role of proteases in this process is discussed.
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A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment and the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect "commitment" to sporulation.
Article
The thin-sectioned spore of Bacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium. Images
Article
It has been demonstrated, using immunocytochemical techniques, that individual spore antigens are synthesized in discrete compartments of the bacterial cell. An outer layer of bacterial spores, demonstrable ultrastructurally as a distinct exosporium in Bacillus cereus or as an outer tight-fitting sporecoat in B. subtilis, is synthesized in the cytoplasm of the mother cell. Conversely, the layers of the inner sporecoat antigens are synthesized in the forespore compartment and in association with the forespore membranes. Different layers of the sporecoat are thus synthesized in separate morphological areas and are presumably under different genetic control. Immunocytochemical techniques indicate that dipicolinic acid is found in association with the sporecore.
Article
The frequency of association of spore loci with the "old" and "new" ends of rod-shaped sporangia in batch cultures of Bacillus megaterium ATCC 19213 was estimated by phase contrast microscopy. The analysis was facilitated by (i) the association of most of the sporangia into chains of two to five sporangia and (ii) the occurrence of two types of cross wall distinguishable by their degree of splitting. It was concluded that a newly formed spore is located at the "old" end of a sporangium. By inference, the sporulation division septum locus is distal to the ultimate normal cell division septum, i.e., proximal to the "old" pole of the B. megaterium sporangium. This result is discussed in relation to deoxyribonucleic acid segregation during sporulation.
Article
Enzymatic analyses of Bacillus thuringiensis extracts suggest that a modified Krebs tricarboxylic acid cycle (without alpha-ketoglutarate dehydrogenase) can operate during sporulation in conjunction with the glyoxylic acid cycle and the gamma-aminobutyric acid pathway.
Chapter
Spore is considered one of the most complex structures formed by single-celled bacteria. Sporogenesis involves a special type of cell division and its further development involves an integrated sequence of new biochemical reactions under genetic control. These result in the production of enzymes, substances, and structures new or different from vegetative material. During spore formation, the enzymes involved in the synthesis of such substances as the coat proteins and dipicolinic acid must be formed. The completed spore, which takes several generation times to form and results from complex differentiation process, is remarkably resistant to heat, chemicals, and adverse environmental changes. It can also revert rapidly within minutes to a heat-labile state in response to a variety of chemical and physical treatments. This chapter focuses on the present chemical knowledge of the mature spore along with the biochemistry of its formation. It also presents definitions and cytological changes during sporogenesis that help in assisting the understanding of the biochemistry of the spore.
Article
Two glucosamine (GCA)-requiring mutants have been isolated which grow on glucose minimal or nutrient sporulation medium only in the presence of either GCA or acetyl-GCA. They lack the l-glutamine-d-fructose-6-phosphate aminotransferase (EC 2.6.1.13), which is repressible by GCA and whose activity in the standard strain decreases after cessation of growth. But the mutants can grow on GCA as sole carbon and ammonia source, because GCA induces the synthesis of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10). With respect to sporulation, the GCA-requiring mutants are in a serious dilemma, as GCA represses the onset of massive sporulation and yet a small amount of GCA-6-phosphate derivatives is necessary to allow sporulation. When GCA is continuously provided in small quantities, sporelike particles are produced which contain little or no spore cortex but a normal spore coat. Apparently, GCA derivatives are needed especially for cortex formation. Many of the sporelike particles can produce colonies after octanol, but not after heat treatment. When they are purified by treatment with lysozyme and sodium dodecylsulfate, they do not show the decrease in optical density at 600 nm typical of germination nor do they produce offspring.
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The sporulation process in Bacillus subtilis has been studied principally with KMnO4 fixation, but also, for the purpose of comparison, with OsO4 and mixtures of both fixatives. At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex. The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail. Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.
Article
1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.
The sporulation process in Bacillus subtilis has been studied principally with KMnO4 fixation, but also, for the purpose of comparison, with OsO4 and mixtures of both fixatives. At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex. The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail. Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.
Article
Experimental conditions were developed whereby a culture of Bacillus cereus formed spores with reasonable synchrony following a growth cycle of some 8 hours. The cytology of this metamorphosis was studied by dark phase contrast, bright-field microscopy and electron microscopy of thin sections. Particular attention has been paid to the changes in chromatin patterns and these have been correlated with quantitative chemical estimations of the nucleic acids. The cell commencing sporulation contains two compact chromatin bodies and twice the spore amount of deoxyribonucleic acid. Following fusion of the two chromatin bodies, one-half of this chromatin becomes located at a cell end. A transverse septum growing inwards from, and remaining attached to, the inner surface of the cell wall separates this end-piece of chromatin and some associated cytoplasm from the rest of the cell to form the primordial spore. Although the synthesis of deoxyribonucleic acid ceases during the segregation process, it recommences in this organism and continues at a linear rate as the spore develops. Tracer studies with radioactive phosphorus indicated that this further synthesis is confined to the non-spore portion of the sporangium. Although the net synthesis of ribonucleic acid ceased prior to the onset of sporogenesis, some evidence of a turnover of this fraction during the sporulation process was found.
Article
On sporulation the cells of the insect pathogen Bacillus thuringiensis contain at one end a spore and at the other end a diamond shaped crystal. A method has been developed for the separation of the alkali dispersible crystal material from spores, vegetative cell debris, and their alkali soluble components. Two methods are given for the preparation of suspensions of intact crystals free from other material. One method depends on the mechanical disruption of the spores, the other on germination and autolysis of the spore contents. After either method clean suspensions of crystals were obtained by washing and differential centrifugation. The purest preparations have yielded a substance precipitable with trichloracetic acid, possessing the ultraviolet absorption characteristics of protein, containing over 17% nitrogen and at least 17 amino acids, but no phosphorus. Examination of the crystals with the electron microscope has shown that they have a tetragonal form and possess some fine structures; no virus pa...
Article
Spores are formed when vegetative cells of sporing aerobes are shaken with distilled water at 37 degrees . These spores are derived from the small number of cells which survive lysis. The sporulation process involves increase and concentration of solid material in the cell, and is achieved at the expense of the products of lysis of 80 to 90 per cent of the resuspended cells.
Article
Washed vegetative cells of Bacillus mycoides obtained and treated under specified conditions have been found to sporulate when shaken in distilled water under specified conditions. Within limitations of the methods, a heat-resistant cell (spore) is produced for each heat-sensitive vegetative cell present initially. Several different experiments designed to detect massive lysis and cell growth during sporulation in distilled water yielded uniformly negative results. Evidence is furnished for the conclusion that a freshly formed spore (heat-resistant cell) weighs considerably less than its progenitor vegetative cell. The observed results are most satisfactorily explained as a direct conversion of a vegetative cell to a spore.
Article
When sprayed on the leaves of tobacco plants inoculated with tobacco mosaic virus 8-azaguanine caused a delay in the production of virus, and a delay in the development of systemic infection. From alkaline hydrolysates of the nucleic acid prepared from virus isolated from 8-azaguanine-treated plants, a compound was isolated which had the expected properties of 8-azaguanylic acid. This evidence together with analytical data showed that in tobacco mosaic virus from 14-day-old infections in 8-azaguanine-treated plants, about 3–4% of the guanine in the virus nucleic acid was replaced by 8-azaguanine. This incorporation of 8-azaguanine appeared to reduce the infectivity of the virus. Several other purine analogues were ineffective against tobacco mosaic virus. A serological-chromatographic method is described for the estimation of small amounts of tobacco mosaic virus in crude plant extracts.
Article
In experiments of 6 hours duration, no replacement of phosphorus or purine and pyrimidine carbon in DNA, nor flow of these atoms from RNA to DNA, could be detected in rapidly growing cultures of E. coli. The slow replacement that has been demonstrated for many substances in non-proliferating tissues of other organisms, though it may occur also in bacteria, is not greatly accelerated under conditions of rapid cellular growth, and therefore cannot be a characteristic feature of synthetic processes.
Article
The toxic principle in sporulated cultures of Bacillus sotto, which causes paralysis and death in Bombyx mori larvae, is associated with the crystalline inclusions produced by this microorganism. The toxin is not present as a typical exotoxin but is soluble either in silkworm gut juice or dilute alkali solutions.
Article
1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.
Article
WHEN bacteria, which have grown in a mineral salts medium, are transferred to fresh medium of the same composition a lag period may develop before growth is resumed1. Lag is reduced by addition of sterile culture filtrates or by certain compounds shown to be present in such filtrates2. We have recently observed changes within the cells themselves when they are transferred to phosphate buffer. Esch. coli was harvested from a growth medium containing 0.5 per cent peptone, 0.2 per cent glucose and mineral salts and the crop was divided into two equal parts. One half was disrupted immediately in the Hughes bacterial press3 without abrasive and the crushed cells extracted with phosphate buffer. The other half of the crop was suspended in the same volume of phosphate buffer as that of the medium in which the cells had grown. After incubation at 30° C. for 2 hr. the bacteria were harvested, crushed under the same conditions as those used for the first half of the original crop and the two extracts were adjusted to the same protein content by addition of buffer. The ultracentrifuge pattern for the extract from the cells crushed immediately after harvesting from their growth medium is shown in Fig. 1a. It agrees closely with patterns reported by previous authors4-6. When the cells are incubated in the absence of compounds necessary for growth, however, a dramatic modification occurred in the distribution of macromolecules in the extract. The leading boundary, which has an uncorrected sedimentation coefficient of 40S, was largely abolished while that sedimenting at 20S was somewhat augmented (Fig. 1b).
Article
Systems were investigated in which RNA could be synthesized in the absence of protein formation and in which protein synthesis could subsequently be induced.It has been shown that the preformed RNA thus obtained participates neither in general protein synthesis nor in induced enzyme (β-galactosidase) formation. The results obtained in the participates in the synthesis of the protein.
Article
The parasporal protein inclusion of Bacillus cereus var. alesti accounts for one third of the dry weight of the washed products of sporulation. Although the spore:crystal ratio is 1:1, the weight ratio is 1.5:1. Like other parasporal crystals, those of var. alesti are swollen but not dissolved by alkalinity up to a critical pH (11.5–11.8); above this pH, dissolution occurs. Toxicity tests on extracts made at increasing pH levels indicated that the crystal protein itself is the toxic agent and not the carrier of it. The protein of actively dividing cells and of spores was non-toxic. Crystal-forming cells contained a toxic protein which would dissolve at a lower pH than would the free crystal.
Article
1.1. Mercaptoethanol in adequate concentration (about 0.1 M) will block division of Dendraster excentricus eggs if applied at any time before metaphase. Since the time schedules of various echinoid eggs seem to be comparable, it may be useful to restate this finding in time units. In this case where the normal time from fertilization to first cleavage is 50 minutes, the mercaptoethanol block can be applied at any time from fertilization to about the fortieth minute.2.2. If the mercaptoethanol is applied after metaphase, it has no detectable effect on the division that is about to take place, but will block the next division. The discontinuity between the sensitive period and the insensitive period is very sharp.3.3. Concentrations of mercaptoethanol that are too low to block the first division may arrest cleavage at a subsequent division. In general, the lower the concentration, the more divisions will take place before blockage occurs.4.4. The effect of mercaptoethanol is completely reversible.5.5. Roughly, the time required for the blocked eggs to complete division when mercaptoethanol is removed is equivalent to the normal time required for the eggs to pass to division from the stage at which the mercaptoethanol was applied.
Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides.
The nucleoids of Escherichia coli, independently of the physiological state of the bacteria, are shown to be preserved as a fine-stranded fibrillar nucleoplasm by an OsO(4) fixation under defined conditions: acetate-veronal buffer pH 6, presence of Ca(++) and amino acids, stabilization with uranyl-acetate before dehydration. The same fixation procedure applied to the DNA of vegetative phage reveals a pool of homogeneous fibrillar structure very similar to the nucleoplasm. The "versene test," which produces a coarse coagulation of these plasms, emphasizes the similar behaviour of the pool and the nucleoids. The heads of mature phage are preserved in their true polyhedral shape by the standard fixation procedure, although they may be badly distorted when fixed under different conditions. Lanthanum nitrate and uranyl-acetate are shown to increase markedly the contrast of both phage and cytoplasm. The consequences of the fibrillar structure of the genetic material are discussed in relation to the probable division process.
Article
Spores of Bacillus cereus strain terminalis formed "endotrophically" by transferring granular vegetative cells to distilled water were found to be relatively susceptible to heat and deficient in dipicolinic acid. Calcium ions alone, added in low concentration shortly after the cells were placed in water, could completely relieve these abnormalities. Although the water-formed spores were sensitive to heat, they were as fully resistant as normal spores to gamma radiation or phenol.
Bacterial cells were fixed in OsO4, washed, dehydrated, and embedded in a methacrylate mixture. Ultrathin sections were cut on a Porter-Blum ultramicrotome and were examined in an RCA electron microscope, type EMU-2D. The sections revealed that the cytoplasmic membrane undergoes a centripetal growth to form a membrane septum. This septum is formed as a double structure. Constriction of the daughter cells and deposition of cell wall material lead to the separation of the daughter cells. The bacterial cytoplasm appears to consist largely of 200 A granules and occasionally reveals arrays of parallel dense lines.
The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques. During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process. During their formation both the spore and crystal incorporated methionine-35S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete. The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.
Article
A correlative study was made of some structural and functional features of a reasonably synchronized culture of Bacillus cereus strain terminalis during spore formation and maturation. The successive stages of development could be recognized by phase contrast microscopy and in electron micrographs of ultra-thin-sectioned cells. Attempts were made to correlate these changes with the acquisition of heat resistance and the synthesis of dipicolinic acid. The outer coat of the spores was observed to be formed first around the forespore; the exosporium, cortex, and inner coat then appeared sequentially and independently of existing sporangial membranes. Dipicolinic acid synthesis began in the early transitional stage, just after forespore formation, and reached one third of the maximum level before an increase of heat resistance in the population was detectable, indicating the possibility of a correlation only above a threshold level of the compound.
Article
IT has been shown that the enzyme beta-galactosidaso is inducible in Staphylococcus aureus if the organism is supplied with galactose and a mixture of amino-acids; enzyme formation in intact and disrupted cells is markedly stimulated by the presence of glucose and a mixture of purines and pyrimidines1,2.
Bacillus polymyxa sporangia and spores were prepared for examination in the electron microscope by methods whose critical features were apparently: judicious use of vacuum, to encourage complete penetration of the embedding medium; the use of epoxy resins as embedding media; and cutting of the thin sections with a diamond knife. Electron micrographs of material prepared in this manner exhibit undeformed sporangial sections. Some of the structures revealed have been shown before, though perhaps less distinctly; other structures are revealed here for the first time. While this single study does not pretend to elucidate all the complexities of sporulation in bacteria, these and similar images should make this possible, and some mention of the preparatory techniques that lead to them seems advisable at this time.
Article
Washed vegetative cells of various species of aerobic spore-forming bacteria sporulate abundantly when shaken in distilled water in air. The spores thus formed possess the same heat resistance as spores formed in a complete growth medium. Various factors influencing sporogenesis in water are described. Glucose in low concentration completely suppresses sporogenesis under these conditions and the suppression is relieved by the presence of ammonia as an exogenous source of nitrogen. Various amino acid and purine antimetabolite analogues inhibit sporogenesis and their inhibitory effects are completely reversed by much smaller amounts of the corresponding metabolites. Sporogenesis is thus regarded as a de novo synthesis of spore proteins from preexisting endogenous (enzyme) proteins. Cells low in protein fail to sporulate and the capacity of the cell to adaptively attack maltose and trehalose is strongly interfered with after the cell is irreversibly committed to sporulation, but not before that. Evidence is advanced supporting the hypothesis that sporogenesis is an endogenous process which commences when the supply of exogenous energy and carbon is depleted. It utilizes low molecular weight nitrogenous substances liberated by the degradation of preexisting enzyme proteins of the vegetative cell. Sporogenesis and adaptive enzyme formation are regarded as competitive synthetic processes, both utilizing endogenous enzyme proteins. The events of sporogenesis suggest that this process may be an adaptive protein synthesis, analogous to adaptive enzyme synthesis.
Spores of microorganisms. VIII. The synthesis of specific calcium-and cystinecontaining structures in sporulating cells of bacilli
  • V Vinter
VINTER, V., Spores of microorganisms. VIII. The synthesis of specific calcium-and cystinecontaining structures in sporulating cells of bacilli, Folia Microbiol., 1960, 5, 217.
Monochromatic ultraviolet microscopy of microorganisms: Preliminary observations on bacterial spores
HASHIMOTO, T., and GERHARDT, P., Monochromatic ultraviolet microscopy of microorganisms: Preliminary observations on bacterial spores, J. Biophysic. and Biochem. Cytol., 1960, 7, 195.