diavalent metal composition of spores

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JOURNAL OF BACTERIOLOGY, Mar., 1966

Copyright 1966 American Society for Microbiology

Vol. 91, No. 3 Printed in U.S.A.

Endotrophic Calcium, Strontium, and Barium Spores of Bacillus megaterium and Bacillus cereus1HAROLD F. FOERSTER2 AND J. W. FOSTER The University of Texas, Austin, TexasReceived for publication 15 November 1965

ABSTRACT

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 CaCl2, SrCl2, or BaC12. 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.More suitable designations are "strong electrolytes" and "weak electrolytes" (alanine, inosine) or "nonelectrolytes" (glucose), and the new terms will be used in this paper.] A determination of how spores are affected by modifications of the above may help to elucidate the role of metal ions in these unique cells. This paper deals with the substitution of strontium and barium for calcium in spore formation, and the properties, particularly germinative, of the variously constituted spores. To ensure that the effects obtained are concerned with spore formation per se, and not merely with the capacity of I Based on a thesis submitted by H. F. Foerster to vegetative cells to sporulate subsequently, the The University of Texas, 1963, in partial fulfillment endotrophic sporulation technique was employed of requirements for the Ph.D. degree. 2Present address: Department of Biology, Sam (20, 33). Strontium and barium spores have been preHouston State College, Huntsville, Tex.1333

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An important sector of spore science has emerged from the special significance of ions in these resistant cells. Bacterial spores are enriched in metallic ions, notably calcium (6); the heat resistance of spores is dependent on a relatively high metallic ion content, notably calcium (3, 6, 48); and exogenous strong electrolytes are essential for physiological germination of the great majority of spore strains and for maximal germination of the remaining minority (12, 43, 44, 45). [In earlier papers (43, 44, 45), germination compounds were classified as ionic (various salts) or nonionic (L-alanine, inosine, glucose).

1334

FOERSTER AND FOSTER

J. BACTERIOL.

initially to a reading of approximately 100 Klett units. Electroni microscopy. Spores were fixed for 2 hr in 2% aqueous, buffered KMnO4, and then were cenMATERIALS AND METHODS washed three times Bacteria. Bacillus megateriulm QM Bi551 and B. trifuged andby successive 10-min in water. They were dehydrated exposures to 30, cereus T were studied comparatively because their 70, 95, and 100%o ethyl alcohol. This was followed 50, by spores differ anatomically (37) and germinatively. a 15-min exposure to 100%7 propylene oxide. The The former can be germinated with inorganic ions or fixed spores were embedded for 1 to 2 hr in 25' oxide, glucose, or both, the latter with strong electrolytes in 506X0, epoxy resin dissolved in propylene (l, then and a mixture of alanine and inosine (21, 43, 45). Growth spores. The following medium was used: then overnight in 75% epoxy resin, followed by 100% glucose, 1.0 g; KH2PO4, 5.0 g; (NH4)2HP04, 1.0 g; epoxy resin. After 6 hr, the samples were embedded 5 gelatin MgSO4.7H20, 0.2 g; NaCl, 0.1 g; CaCI2, 5.0 mg; in no. Sectioning capsules and alloweda to harden at 60 C. was performed Porter-Blum MnSO4 5H20, 7.0 mg; ZnSO4 .7H20, 10 mg; FeSO4- microtome at thicknesses ranging with 300 to 600 A. from 5H20, 10 mg; sodium L-glutamate, 1.33 g; yeast Sections were floated on grids and examined and autolysate (Basamine, Anheuser-Busch, St. Louis, photographed with an RCA model EMU 3-D electron Mo.), 0.5 g; Difco agar, 20 g; water, 1 liter. Deion- microscope. ized distilled water was used throughout this work. A Preparation of spore bacterial suspension (0.1 ml) from a 12-hr nutrient disintegrator cylinder wascoat fractionzs.10The Nossal loaded with no. 12 agar slant was spread uniformly over the dry agar Ballotini beads and approximately 1 g g ofwashed of surface in petri plates. After incubation for 3 days at spores in 8 ml of water. The cap of the cylinder was 30 C, the spores were rinsed off the agar, washed six the to eight times by centrifugation in 25 volumes of cold sealed withanmasking tape, and bath container was between the acetone-Dry Ice water, and finally suspended in water. They remained chilled in repeated 25-sec shaking periods. These were continued stable indefinitely at 4 C. until microscopic Enidotrophic spores. Vegetative cells were grown in to contain only examination showed the suspension broken spores. The turbid the following medium which contained no added containing the coat fragments was decanted,liquid and calcium: glucose, 2.0 g; sodium L-glutamate, 5.2 g; bead washings were added to it. It was chilled and yeast autolysate (Basamine), 0.5 g; NaH2PO4, 1.0 g; centrifuged at 18,000 X g for 30 min at 0 C. The MgSO4.7H20, 0.2 g; NaCI, 5.0 mg; CuSO4.5H20, 0.1 pellet was suspended in 30 ml mg; MnSO4 *5H20, 10 mg; ZnSO4 5H20, 10 mg; centrifugation procedure was of cold water, and the repeated eight times FeSO4, 10 mg; water, 1 liter. A 3-liter Fernbach before lyophilization and storage over P205. Approxi1 liter of medium was inoculated mately 50% of the initial spore dry weight was reflask containing with 1 ml of an 8-hr nutrient broth culture and incu- covered as the coat fraction. Membranes probably bated at 30 C with continuous shaking. At the early were included, but light and maximal stationary phase of growth (12 to 14 hr), the examination indicated that theelectron microscopic cells were centrifuged, washed once in 50 ml of sterile consisted predominantly of pieces material recovered of coats. water, and then suspended in 500 ml of sterile 1.0 To determine metals, mM CaC12, SrCl2, or BaCI2. These suspensions were were ashed in porcelain samples of spore suspensions electric shaken at 30 C until sporulation was maximal, furnace at 650 to 700 Cmicrocrucibles in anwas disfor 1 hr. The ash usually for 24 to 48 hr. The final spore suspensions solved in a hot mixture of 0.05 ml of concentrated were prepared as described above. Determination of viability. Colony-forming capacity HCl and 0.5 ml of 30% H202, and made to 10.0 ml with washings of spores was determined with Difco nutrient agar in a sample byand water. Strontium was determined the method supplemented with 0.1% soluble starch (14), by (4) by use of a Beckman DU of Chow and Thompson flame spectrophotometer means of conventional spread plate procedures. Transmission Heat resistance. Deionized water suspensions con- equipped with a photomultiplier. 460 and 454 and were measured at mA, taining approximately 2,000 viable spores per milliliter background with the use of a slit width of 0.03. Calrespectively, were dispensed in sterile test tubes. One suspension cium was determined in a similar fashion but with was heated at each desired temperature and plated transmission measured at 422.7 m,u and background for viability. (5, 8). Heat activationz. The spore suspensions were heated at 418 m,u (24). Barium was measured gravimetrically BaSO4 for 30 min at 60 C prior to each germination test (10, as Dipicolinic acid (DPA) was determined spectro43). Optical denisity (OD) reduction. Germination was photometrically (30) or colorimetrically (22) in followed by Powell's (35) method. Suspensions of centrifuged extracts made by autoclaving spores for spores which microscopically have all lost their 15 min at 120 C (38). Barium ions interfered with the refractility display a 50 to 80% reduction in OD (41, assay; the barium spore extracts were treated with 42). Intermediate OD levels reflect the proportion of excess (NH4)2SO4, and the resulting BaSO4 was fully germinated (nonrefractile) versus ungerminated removed by filtration. Isotope counting techniques. Samples of the speci(refractile) spores in the suspension. A Klett-Summerson and a Bausch & Lomb photoelectric colorim- mens were evaporated to dryness in planchets. Radioeter were used. The spore suspensions were adjusted activity of the infinitely thin samples was measured

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