Megaterium Spores Research Articles

Increased Response of AnoxicBacillus MegateriumSpores to Radiation at High Dose-rates

SummaryBacillus megaterium spores have been irradiated in aqueous suspension with 5 μs pulses of electrons. The sensitivity of anoxic spores (N2) increases with increasing dose-rate in the range 10–10 000 rads/μs. The response of oxic spores (O2) is unaffected by these changes in dose-rate. OH radical scavengers prevent part of the dose-rate effect. N2O sensitizes spores at low dose-rates but not at high dose-rates. Reduction in the pulse length over the range 5 to 0·5 μs causes a decrease in the response characteristic of high dose-rates. It is estimated that the average lifetime of the radicals effective in increasing the anoxic response is about 2 to 5 μs.

Measurement of heat resistance parameters for spores isolated from canned products.

ABSTRACT.Spores of Bacillus subtilis, B. cereus, B. megaterium and B. pumilus were isolated from canned products. Heat resistance parameters were determined for these spores in distilled water and in different juices and brines. D values for B. subtilis, B. cereus, B. megaterium and B. pumilus suspended in distilled water were 1·5, 0·8, 1·6, and 1·5 min at 212°F. The D value for B. stearothermophilus in water was 3μ4 min at 248°F. Both D and Z values were reduced when spores of B. megaterium were heated in orange and mango juices. Similarly, spores of B. stearothermophilus were destroyed in okra brine faster than in water. Sodium chloride increased the thermal resistance of B. megaterium spores and this effect was independent of NaCl concentration of 2–15%. Pectin also conferred some protection against heat inactivation of spores of B. megaterium but sucrose had no effect.

Biochemical Studies of Bacterial Sporulation and Germination: XIX. PHOSPHATE METABOLISM DURING SPORULATION

Abstract Acid-soluble phosphate compounds represent about 25% of the total phosphate of Bacillus megaterium vegetative cells, but only 7% of the total phosphate in dormant spores. In vegetative cells, 12 compounds make up 90% of the acid-soluble phosphate. These same compounds predominate throughout growth and early sporulation, although their relative amounts change during the process. One hour before refractile spores appear within sporangia, 3-phospho-d-glyceric acid (PGA) becomes a major component of the sporangial pool and, with the appearance of refractile spores, PGA becomes more difficult to extract. PGA is the largest component of the acid-soluble phosphate pool of B. megaterium spores produced in rich as well as minimal media, and also predominates in Bacillus cereus and Bacillus subtilis spores. Spore PGA appears to be synthesized in a compartment which is inaccessible to inorganic phosphate in the medium, although, at the time of synthesis, Pi from the medium does enter the sporangium. PGA in dormant spores is very firmly bound, and does not readily exchange with external PGA.

Effect of an azo dye on germination and outgrowth of Bacillus megaterium spores.

E dimostrato che il violetto di genziana non interferisce nella germinazione indotta con inosina di spore diBacillus megaterium, mentre ne inibisce la crescita alle concentrazioni di 1 o piu μg per ml di cultura. Quando impiegato a concentrazioni comprese fra 0,4 e 0,1 μg/ml e possibile mettere in evidenza non solo forme vegetative ma anche protoplasti. Quest'ultima osservazione viene messa in relazione alla attivita inibente la sintesi dei precursori del cell wall da parte del violetto di genziana.

Germination of single bacterial spores.

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.

The bioassay of aflatoxins and related substances with Bacillus megaterium spores and chick embryos.

AbstractThe “paper disc plate” method of antibiotic assay usingBacillus megaterium spores was tested for the assay of aflatoxins and related compounds. Significant inhibitions were obtained in the range of 1–4 µg/disc for aflatoxin B1 and 4–8 µg/disc for aflatoxin G1. Dicumarol was the most active of the compounds tested and inhibited at 0.1–1.0 µg/disc. Compounds were also tested using nine‐day‐old chick embryos. It was found that embryos incubated in egg cartons were much more sensitive to toxins than eggs incubated on cotton padding or in a commercial egg incubator. The LD100 of aflatoxin B1 to carton‐incubated embryos was 0.01 µg, while to embryos from the commercial incubator the LD100 was >5.0 µg. The results of the two bioassay procedures did not correlate completely with each other.

Free Radical Concentration in Irradiated Bacterial Spores and Vegetative Cells as Measured by the ESR Method

In a previous investigation, the effect of postirradiation heat treatment on the colony-forming ability of dry spores of Bacillus subtilis was demonstrated (1). When an anoxic heat treatment was applied to irradiated spores, the 37 % survival dose (D37) became almost two times as high as that of untreated spores. This effect was first found in Bacillus megaterium spores by Powers and co-workers (2, 3). They proposed that the ionizing radiation causes three different classes of damage in living cells, one of which is at least partially restorable by the heat treatment. They also observed the corresponding effect on the pattern of the electron spin resonance (ESR) spectrum (4). The heat effect, however, was not found in the transforming ability of DNA extracted from irradiated B. subtilis spores (1). If the damage produced in DNA is a primary cause of death of irradiated cells, why did the DNA activity not show the heat effect? It was suggested in the previous paper that the high total dose used for the irradiation of the DNA might hinder the heat effect. The present experiment was designed to investigate the intracellular mechanism of the heat effect from the physical aspect. The criterion for biological damage is the behavior of radiation-produced free radicals as measured by the electron spin resonance method. Preliminary results were reported previously (5). On the other hand, the previous investigation (1) showed no heat effect on vegetative B. subtilis and other biological materials as well. The second question is whether the heat effect is a unique phenomenon found only in spores. For investigating this problem, dry vegetative cells were subjected to the ESR measurement. The results obtained were compared with the results on spores.

ULTRASTRUCTURAL LOCALIZATION OF MINERAL MATTER IN BACTERIAL SPORES BY MICRONINCINERATION.

The fine localization of mineral matter in spores of Bacillus megaterium and Bacillus cereus was studied by the technique of microincineration adapted for use with the electron microscope. The specimens, which included intact and thin-sectioned spores as well as shed spore coats, were burned either in the conventional way at high temperature or by a new technique using electrically excited oxygen at nearly room temperature. The ash residues were examined by bright field, dark field, and diffraction in the electron microscope and also with the phase contrast microscope. In some cases, the specimen was previewed in both microscopes before incineration. The results do not support a previous report that the mineral elements of the spore are confined to a peripheral layer, but rather indicate that the spore core as well as the coat are mineral-rich. The cortex may be deficient in minerals, but the possibility of artifact prevents a clear decision on this point. Incinerated B. megaterium spores show a highly ordered fine structure displaying 100 A periodicity in the ash of the middle layer of the coat. The nature of this structure is discussed, as is the technique which demonstrated it. The fine definition of the ash patterns, particularly those obtained with the low-temperature, excited-oxygen technique, suggests that microincineration may be generally useful in the study of fine structure.

Conditions affecting Bacillus megaterium spore germination in glucose or various nitrogenous compounds.

Hyatt, Mildred T. (Quartermaster Research and Engineering Center, Natick, Mass.) and Hillel S. Levinson. Conditions affecting Bacillus megaterium spore germination in glucose or various nitrogenous compounds. J. Bacteriol. 83:1231-1237. 1962.-The possibility that there is more than one metabolic pathway for triggering germination of Bacillus megaterium spores was investigated. Spores were germinated in seven different "physiological germinants" under varying conditions of concentration, pH, combinations of germinants, temperature before and during germination, and chemical inhibition. l-Alanine and l-valine appear to induce germination via the same metabolic pathway (same inhibitors are effective, similar germination rate and temperature requirements); and glucose and glucosamine also appear to act similarly, but by a different pathway than l-alanine and l-valine. The other germinants, l-leucine, l-proline, and KNO(3), do not correspond in all respects either to the glucose-glucosamine or to the alanine-valine pair in response to the different germination conditions. It is concluded that B. megaterium spore germination occurs via more than one pathway.

Spore Germination and Emergence of Bacillus megaterium

Bacillus megaterium spores have a characteristic polar knob and equatorial ridge, or groove. During germination, the spore case appears to split along this ridge, and thus allows the new cell to emerge. Mechanically ground spores also split along this ridge, one part of the spore case being hinged to the other, the ridge being evident along a free edge. The equatorial ridge appears to be an area of suspectibility to mechanical pressures and, perhaps, in normal germination, to enzymic action as well.

THE PHOSPHORUS FRACTIONS OF BACILLUS CEREUS AND BACILLUS MEGATERIUM: I. A COMPARISON OF SPORES AND VEGETATIVE CELLS

The phosphorus fractions of the spores and young vegetative cells of B. cereus and B. megaterium were compared using the methods of Schneider and of Schmidt and Thannhauser. Differences were found, not only between the two types of cell, but also between the species studied. The spores of B. cereus and B. megaterium contained, on a dry weight basis, similar amounts of nucleic acid, yet the spores of B. megaterium contained twice as much total phosphorus as those of B. cereus. The excess phosphorus of B. megaterium spores was present as an acid and alkali insoluble residue and was made up of empty spore coats. While this same fraction accounted for only 4% of the total phosphorus of B. cereus spores it made up some 60% of that of B. megaterium. A similar fraction from vegetative cells was low in phosphorus. The spore coats of B. megaterium, in contrast to the cell walls were lysozyme-resistant. Cold acid-soluble and lipid phosphorus could be properly estimated only on disrupted spores. Disruption also proved essential for the ready extraction of spore nucleic acids with hot TCA, but did not greatly alter the solubility of the residue phosphorus. Ribosenucleic acid (RNA) comprised 50% of the total phosphorus in the spores of B. cereus (3–4% of the dry weight) but only 25% in B. megaterium spores. In both species the RNA of the vegetative forms accounted for a larger proportion of the total P of the cell. Ribonuclease digested the RNA of spores and vegetative cells to the same degree. The Schmidt and Thannhauser method was found more suitable than the Schneider method for the estimation of desoxyribosenucleic acid (DNA). The DNA content of B. cereus spores was about 1% of their dry weight; that of B. megaterium spores was slightly less. Some 12–20% of the phosphorus of B. cereus spores and 6% of that of the young vegetative cells was present as acid-labile (non-nucleic acid) phosphorus which exhibited some of the characteristics of polymetaphosphate.