Dipicolinic Acid Content Research Articles

Microcycle sporogenesis of Bacillus cereus in a chemically defined medium.

A chemically defined medium which allowed germination, outgrowth, and subsequent resporulation of Bacillus cereus T spores, without intervening cell division (microcycle sporogenesis), is described. No medium replacement was required. The second-stage spores were heat-stable and had similar germination characteristics and dipicolinic acid content to primary spores. Deoxyribonucleic acid (DNA) replication began soon after germination and there was a doubling in the DNA content of the cells within 2 hr.

芽胞形成菌におけるジピコリン酸生合成部位に関する研究

Intracellular distribution of dipicolinic acid (DPA) during sporulation was studied by using a strain of B. subtilis. In the initial stage of sporulation, DPA was detectable only in the soluble fraction and the content rapidly decreased with increase in the content of DPA in the residue fraction. In contrast, no DPA was detectable in the supernatant derived from the cells in the stage of maximal sporulation.When the matured spores were mechanically disrupted for a relatively short time, it was found that DPA could easily be released from the matured spores and the kinetic pattern of the release was not parallel with the release of protein and nucleic acid from the spores. On the distribution of DPA in the disintegrated spores, it was also found that this compound is mostly detected in the residual supernatant after ammonium sulfate fractionation.From these results, it was suggested that DPA may be mainly synthesized outside the forespore and then accumulated inside the spore coat in a free from or at least in a loosely bound form.

Regeneration of Mycelial Protoplasts of Fusarium culmorum

The sublethal dose of calcium hypochlorite (CH) of 0.2--0.3 mg/ml active chlorine did not cause, after 5 min, morphological changes in the spores of Bacillus anthracoides which could be detected by phase contrast microscopy, or a decrease in the content of dipicolinic acid (DPA) in the spores. Further cultivation of the spores treated with the sublethal dose of CH om MPA resulted in a delay of changes which were typical of normal germination process (swelling, loss of light refraction, decrease in DPA content). The action of the lethal dose of CH (0.2--0.3 mg/ml active chlorine during 1.5 hr or 5.6 mg/ml active chlorine during 1 hr) causes a decrease in light refraction, changes in the dimensions of spores, and a decrease in the content of DPA in the spores by a factor of 4--5. A sharp decrease in the content of DPA in the spores may characterize not only their germination but also their death caused by lethal doses of the chlorine containing disinfectant.

Latent effects of haemolytic agents.

The content of dipicolinic acid (DPA) was assayed in the spores of Bacillus anthracoides 96 during various stages of its growth. The content of DPA was ca. 10.7 per cent of the dry biomass weight in the seven-day-old culture containing 96 to 99 per cent of the spores in a "starvation" medium. The morphology of the culture was modified, and the content of DPA in the spores fell to 3.6 per cent half an hour after the inoculation into the medium favourable for the growth (MPA). During the following one to four hours of the germination, the refraction index of the spores and the content of DPA in them decreased (the content of DPA to 2 per cent).

Inhibitory Effect of Dipicolinic Acid on the Anaerobic Oxidation of Glucose by the Cell-free Extract from Vegetative Cells of B. subtilis

IT has been reported that the dipicolinic acid content of several species of bacterial spores ranges from 5 to 10 per cent of the dry weight of spores1,2. The biological actions of dipicolinic acid in spores have been discussed in a few reports. Harrell3 suggested that the release of dipicolinic acid from spores has some relation to the ability of the organism to oxidize glucose. Harrell, Doi and Halvorson4 have further shown that dipicolinic acid has a stimulatory effect on glucose oxidation of the cell-free extract of B. cereus spores. The relationships of dipicolinic acid content to the heat stability5 and to the stainability6 of spores have also been studied.

CHANGES IN THERMORESISTANCE OF CLOSTRIDIUM ROSEUM AS RELATED TO THE INTRACELLULAR CONTENT OF CALCIUM AND DIPICOLINIC ACID.

The cellular content of calcium and dipicolinic acid was determined quantitatively throughout the sporulation and germination processes of Clostridium, roseum. Cell counts were made throughout both processes to relate the changes in the thermoresistant population with the changes in cellular calcium and DPA. Calcium accumulation and DPA biosynthesis occurred early in sporogenesis and preceded the transition of the vegetative cells to thermoresistant spores. Calcium accumulation preceded both DPA biosynthesis and thermoresistance. During germination 80% of the cells became thermosensitive within 15 minutes after heat shock; however, the loss of calcium and DPA from the cells proceeded at a much slower rate. Approximately 2 hours of incubation were required for completion of the germination transition and the onset of cell division.

ULTRAVIOLET-ABSORPTION SPECTRA OF DRY BACTERIAL SPORES.

Bailey, Glen F. (Western Regional Research Laboratory, Albany, Calif.), Saima Karp, and L. E. Sacks. Ultraviolet-absorption spectra of dry bacterial spores. J. Bacteriol. 89:984-987. 1965.-The possibility of obtaining reasonably satisfactory ultraviolet-absorption spectra of dry spores embedded in KBr has been demonstrated. Such spectra show the three peaks characteristic of calcium dipicolinate. The dipicolinate spectra are more distinct when reference pellets containing appropriate amounts of the analogous spore coats are employed. These spectra are considered evidence that some type of calcium-dipicolinic acid chelate accounts for at least part of the calcium and dipicolinic acid content of the spore.

RESPONSES OF BACILLUS SUBTILIS SPORES TO IONIC ENVIRONMENTS DURING SPORULATION AND GERMINATION.

Fleming, H. P. (University of Illinois, Urbana), and Z. John Ordal. Responses of Bacillus subtilis spores to ionic environments during sporulation and germination. J. Bacteriol. 88:1529-1537. 1964.-The ionic environments of germination and sporulation menstrua had a prominent influence on characteristics of Bacillus subtilis spores. There was a synergistic effect for l-alanine and inorganic ions on spore germination. The maximal rate of germination in solutions of l-alanine was dependent on ionic concentration and species. Germination was negligible in l-alanine at low ionic strength but increased as the ionic strength was increased up to about 10(-1)m with a variety of salts. Phosphate was the most active ion tested, and divalent cations were the least active in supporting germination in l-alanine. Germination progressed slowly at 45 C in sodium chloride or sodium phosphate alone but not in CaCl(2) alone. Germination rates in l-alanine were retarded at high ionic strengths (mu in the range of 0.1 to 1.0). Inhibitory effects of high concentrations of certain divalent cations on germination were related to the binding abilities of these metals. High concentrations of NaCl (10(-1) to 1.0 m) in the sporulation medium resulted in lowered heat resistance and germination rate of the resulting spores. The addition of calcium (5 x 10(-2)m CaCl(2)) to the sporulation medium relieved the repression of NaCl on germination and caused the spores to have a greater heat resistance. Calcium and dipicolinic acid (DPA) contents of the spores were unaffected by NaCl in the sporulation medium. The calcium, but not the DPA, content of spores increased as a result of supplementing the sporulation medium with calcium. Possible roles of ions in the germination of spores are discussed.

SPORES OF MICROORGANISMS XVI. A CONTRIBUTION TO THE STUDY OF COMBINED RADIATION- AND HEAT-INACTIVATION OF BACTERIAL SPORES.

The heat inactivation of spores ofBacillus cereus irradiated and non-irradiated with X-rays was investigated with respect to their dipicolinic acid (DPA) content. Spores with a high DPA content (98 μg./mg. dry weight) displayed a biphasic heat inactivation curve. This biphasic character of the curve was preserved for the heat inactivation of previously irradiated spores even if the length of the initial lag of inactivation rate was reduced. Spores with a lower DPA content (24 μg./mg. dry weight) display a single-phase logarithmic order of dying from the beginning of heating. In both types of spores differing in the DPA content a pronounced sensitization of radiation-surviving spores toward subsequent heating was observed.

CHEMICALLY DEFINED, SYNTHETIC MEDIA FOR SPORULATION AND FOR GERMINATION AND GROWTH OF BACILLUS SUBTILIS.

Donnellan, J. Edward, Jr. (U.S. Army Natick Laboratories, Natick, Mass.), Ella H. Nags, and Hillel S. Levinson. Chemically defined, synthetic media for sporulation and for germination and growth of Bacillus subtilis. J. Bacteriol. 87:332-336. 1964.-From 90 to 130 mg (dry weight) of spores (about 1% dark forms) were obtained (per liter) from a chemically defined, synthetic medium, with a two-phase (polyethylene glycol-potassium phosphate) harvest procedure. Optimal sporulation occurred when glucose and glutamic acid were at a concentration of 10 mm in the medium. Ca(++) and Mn(++) were required for sporulation. Heat resistance, dipicolinic acid content, and properties of germination and postgerminative development of spores grown in different concentrations of Ca(++) were investigated. Heat shock did not increase germination of spores derived from the synthetic medium. A synthetic medium, in which spore germination, emergence, and first cell division approached synchrony, was devised.

GROWTH AND SPORULATION CHARACTERISTICS OF AN ORGANIC SULFUR-REQUIRING AUXOTROPH OF BACILLUS CEREUS.

Lundgren, D. G. (Syracuse University, Syracuse, N.Y.) and K. F. Bott. Growth and sporulation characteristics of an organic sulfur-requiring auxotroph of Bacillus cereus. J. Bacteriol. 86:462-472. 1963.-This paper reports investigations of several aspects of growth and sporulation of an organic sulfur-requiring auxotroph of Bacillus cereus ATCC 4342. The wild type and B. cereus T were also studied for comparative purposes. Growth of the mutant on minimal medium plus methionine was normal, but sporulation was completely inhibited. Some reaction involved in vegetative-cell maturity was probably blocked at a point just prior to the "triggering" of sporulation, since abnormally large amounts of poly-beta-hydroxybutyric acid (PHB) were formed. Growth of the mutant in the presence of cystine or cysteine was also accompanied by the build-up of large amounts of PHB, but some endospores were formed (approximately 5% by 72 hr). Results of dipicolinic acid (DPA), calcium, and heat-resistance studies revealed that the few spores formed by the auxotrophic mutant when grown on cysteine were somewhat below wild-type strains in their development of heat resistance, and considerably lower in content of Ca and DPA. This was not the case with spores formed in cells grown on cystine; heat resistance compared favorably with wild-type spores, but the Ca and DPA levels were lower.

SOME CHARACTERISTICS OF SPORES OF BACILLUS CEREUS PRODUCED BY A REPLACEMENT TECHNIQUE

A replacement technique, whereby spores of Bacillus cereus were produced by transferring vegetative cells into non-nutritive solutions, was employed for the investigation of the relationship between calcium, dipicolinic acid (DPA), and heat resistance in spores. Replacement solutions of distilled water, fresh medium, calcium, strontium, barium, nickel, and other metals were employed. Spores produced in distilled water were heat sensitive and contained low levels of calcium and DPA. Heat-resistant spores with a normal complement of calcium and DPA were formed only in fresh medium or in solutions containing suitable levels of calcium. Calcium and DPA were present in the spores in nearly equimolar quantities. Of the other metals tested, only strontium stimulated DPA synthesis above the level of the spores produced in water. Spores of intermediary heat resistance but low DPA content were formed in solutions of barium and nickel.

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, alpha, epsilon-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.

Germination properties of spores with low dipicolinic acid content.

Keynan, A. (University of Wisconsin, Madison), W. G. Murrell, and H. O. Halvorson. Germination properties of spores with low dipicolinic acid content. J. Bacteriol. 83:395-399. 1962.-When the dipicolinic acid content of spores of Bacillus cereus strain T is reduced from 7.5 to 2 or 3%, the spores germinate spontaneously after heat activation and are sluggish in their response to l-alanine and other germination agents. Only germination initiated by calcium dipicolinic acid is unaffected. l-Alanine-induced germination is stimulated by exogenous dipicolinic acid. These results support the hypothesis that endogenous dipicolinic acid regulates the l-alanine-triggered germination.

Dipicolinic Acid Content, Heat-Activation and the Concept of Dormancy in the Bacterial Endospore

TWO distinct meanings have been applied to the use of the term ‘dormancy’ as applied to the bacterial endospore. First, the expression ‘the dormant bacterial endospore’ usually describes the apparent metabolic inactivity of the spore stage as compared with the vegetative cell. Secondly, the same term has been used to describe the fact that spore suspensions respond poorly or not at all to germination agents under conditions that permit rapid germination of aged1 or heat-activated2 spores. It is in the latter sense that the term dormancy is applied in this communication.

Relation of dipicolinic acid content of anaerobic bacterial endospores to their heat resistance.

Relation of dipicolinic acid content of anaerobic bacterial endospores to their heat resistance.

Dependence of the heat resistance of bacterial endospores on their dipicolinic acid content.

IT is generally accepted that mature spores from aerobic or anaerobic bacteria contain large quantities (5–15 per cent of the dry weight) of dipicolinic acid, whereas it is absent in the non-resistant vegetative cells1,2. Biosynthesis of dipicolinic acid is usually considered to be indicative of the formation of heat-resistant spores. In Clostridium roseum3 sporulation involves the development first of a refractile spore-like body, which is followed by the synthesis of dipicolinic acid, and later of a heat-resistant spore. In the aerobic bacteria, however, a close correlation between these three processes makes difficult the isolation of the first stage in sporulation.

FURTHER STUDIES ON THE IONIZING RADIATION INACTIVATION OF BACTERIAL SPORES

Previous observations showing that there are two classes of radiation inactivation for bacterial spores are extended to germinating forms. Spores which inactivate in a multiple target fashion inactivate in a two target fashion upon germination, whereas spores which inactivate in a single target fashion inactivate in a single target fashion upon germination. Spores of the multiple target class seem to have a higher content of dipicolinic acid per g of dry spore material than do those of the single target class. (auth)