Refractile Spores Research Articles

Course of sporulation of Bacillus popilliae in liquid medium containing activated carbon

Refractile spores of Bacillus popilliae, the causative agent of a milky disease of Japanese beetle ( Popillia japonica) larvae, appear in liquid culture in the presence of activated carbon during the early part of the 2nd week of incubation. Forms resistant to drying occur during the 1st week when no refractile spores are seen. They usually number no more than a few hundred per milliliter and they vary in their resistance from day to day. During the 2nd week when refractile spores are present, the number of resistant forms rises to a peak in the vicinity of 100,000 per ml and then gradually decreases throughout the rest of a 3-week observation period. Refractile spores often disappear toward the end of the 3rd week. Thus, the terminal resistant forms may consist largely of slowly germinating spores.

ニトロフラン誘導体の食品防腐剤としての研究-XIV

The effect of furylfuramide (FF) on germination and outgrowth of two bacterial spores was studied in a slide culture, in order to define the point at which FF acts to prevent the developmental process, from a morphological point of view. Concentrations of FF higher than minimal inhibitory concentration (MIC) did not interfere with the reduction of refractility. However, the developmental process stopped there, and the germinated spores eventually lysed before elongation occurred, shedding the empty spore coats. At less than MIC, many distorted cells appeared and gradually lysed. The bacteriostatic action of FF was shown as a result of the prolongation of their outgrowth. FF acted to arrest the development of bacterial spores not at germination, but after the stage from “swelling” to “emergence”. A discrepancy between the two organisms existed in that many refractile spores of B. coagulans, isolated from spoiled fish sausage, still remained on the culture after 35 hours at 37°C, but not in the case of B. subtilis.

Interference contrast and phase contrast microscopy of sporulation and germination of Bacillus megaterium.

The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium. The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies.

Biochemical Studies of Bacterial Sporulation and Germination: VIII. PATTERNS OF ENZYME DEVELOPMENT DURING GROWTH AND SPORULATION OF BACILLUS SUBTILIS

The levels of eight enzymes from several different metabolic pathways were determined during growth and sporulation, as well as in the mature spore, of Bacillus subtilis. Three different patterns of development were observed. In one, the levels of DNA polymerase, adenylate kinase, and nucleoside phosphorylase closely followed that of total protein throughout growth and sporulation. In a second pattern, arginase, malic dehydrogenase, and ornithine transcarbamylase were undetectable in log phase cells and appeared only after the end of this period. In the third, aspartic transcarbamylase and IMP dehydrogenase were present during exponential phase but disappeared by the time of appearance of refractile spores. The pattern of development of several of these enzymes was different in an asporogenous, protease-negative strain. The susceptibility of certain of these enzymes to protease activity in extracts was observed, suggesting a possible role for protease elaborated during sporulation. In most instances, the specific activities of the enzymes in the spore were similar to their levels in stationary phase cells at the time of forespore formation. Certain enzymes were absent from spores or were present at very low levels. The results are discussed in relation to possible mechanisms for the origin of proteins found in the spore core.

The Relationship of Dipicolinate and Lysine Biosynthesis in Bacillus megaterium

Abstract A mutant of Bacillus megaterium has been obtained which lacks the enzyme that condenses pyruvate and aspartic semialdehyde to form dihydrodipicolinate. The effect of the mutation is pleiotropic. In addition to manifesting a requirement for diaminopimelate and lysine, the organism is unable to make dipicolinate and cannot sporulate. Dipicolinate added to cultures of the mutant restores the ability to produce heat-stable refractile spores. It is concluded that the same isomer of dihydrodipicolinate participates in both dipicolinate and lysine biosynthesis. Further, the enzyme that produces dihydrodipicolinate during vegetative growth also functions as the source of this compound for dipicolinate synthesis during sporulation.

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus.

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.

REVERSIBLE ACTIVATION FOR GERMINATION AND SUBSEQUENT CHANGES IN BACTERIAL SPORES

Lee, W. H. (University of Illinois, Urbana) and Z. John Ordal. Reversible activation for germination and subsequent changes in bacterial spores. J. Bacteriol. 85:207-217. 1963.-It was possible to isolate refractile spores of Bacillus megaterium, from a calcium dipicolinate germination solution, that were activated and would germinate spontaneously in distilled water. Some of the characteristics of the initial phases of bacterial spore germination were determined by studying these unstable activated spores. Activated spores of B. megaterium were resistant to stains and possessed a heat resistance intermediate between that of dormant and of germinated spores. The spontaneous germination of activated spores was inhibited by copper, iron, silver, or mercury salts, saturated o-phenanthroline, or solutions having a low pH value, but not by many common inhibitors. These inhibitions could be partially or completely reversed by the addition of sodium dipicolinate. The activated spores could be deactivated and made similar to dormant spores by treatment with acid. Analyses of the exudates from the variously treated spore suspensions revealed that whatever inhibited the germination of activated spores also inhibited the release of spore material. The composition of the germination exudates was different than that of extracts of dormant spores. Although heavy suspensions of activated spores gradually became swollen and dark when suspended in solutions of o-phenanthroline or at pH 4, the materials released resembled those found in extracts of dormant spores rather than those of normal germination exudates.

CHEMICAL AND MORPHOLOGICAL STUDIES OF BACTERIAL SPORE FORMATION

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 (1/3) 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.

Changes in refractility and permeability accompanying germination of heat-resistant spores of Micromonospora vulgaris.

Summary: Progressive stages in the germination at 60 of the highly refractile spores of Micromonospora vulgaris were observed by means of phase-contrast microscopy. The development of individual spores was followed in slide cultures. Mass cultures were also prepared in liquid and solid media from spore suspensions in phosphate buffer which had been heated at 100 for periods of 1–20 min. and 30 min.-4 hr. The liquid cultures derived from heated and unheated spores were sampled at frequent intervals and examined in wet preparations containing dilute toluidine blue. Throughout, loss of refractility preceded germination, which occurred in 1–3 hr. Germinating spores appeared as dense opaque bodies by phase contrast and were then capable of absorbing the dyestuff to which non-germinating spores were impermeable. Heat activation at 100 for 1 min. enhanced the initial germination rate within the first 3 hr. period. No differences were observed between heated and unheated spores during the processes of germination. Indications of an eccentric nucleus were occasionally found in phase-contrast examination of very lightly stained fixed spores from material heated for short periods, or in electron micrographs of unheated growths. Heating at 100 for the shorter periods (1–10 min.) resulted in a higher proportion of the spores becoming darkened than was the case with the longer exposures. Artificial darkening of the spores in the manner successfully used for eubacterial spores was not accomplished. Sectored coloniesthe sectors showing loss of aerial mycelium and impaired viabilityfrequently developed from spores which had been subjected for more than 1 hr. to a temperature of 100.