PHYSIOLOGY OF THE SPORULATION PROCESS IN CLOSTRIDIUM BOTULINUM. I. CORRELATION OF MORPHOLOGICAL CHANGES WITH CATABOLIC ACTIVITIES, SYNTHESIS OF DIPICOLINIC ACID, AND DEVELOPMENT OF HEAT RESISTANCE.
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Day, Lawrence E. (Michigan State University, East Lansing), and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. I. Correlation of morphological changes with catabolic activities, synthesis of dipicolinic acid, and development of heat resistance. J. Bacteriol. 88:690-694. 1964.-A reasonable degree of synchrony in the sporulation of Clostridium botulinum 62-A was attained by using a large inoculum of a young culture into a medium containing 4% Trypticase and 1 ppm of thiamine. Sporulation was complete within 24 to 36 hr. Cells harvested at various intervals were studied for their fermentative activity with l-alanine and l-proline as substrates. The Q values (microliters of gas per hour per milligram of dry cells) were maximal at the time a large percentage of the cells had initiated sporulation as indicated by swelling. They declined to a plateau at about the same level as found in vegetative cells by the time 10% of the cells had completed sporulation, and finally to a much lower level when sporulation was completed. The rates of accumulation of volatile acids (acetic, valeric, and propionic acids) corresponded closely with the catabolic potential observed. However, in the case of acetic acid, there was a significant decrease in the total acid present as the number of mature spores increased to over 50% of the final number. The total acetic acid then increased at a slow rate. The production of basic compounds during growth and sporulation more than balanced the rate of acid production, because the hydrogen ion concentration decreased exponentially throughout the period as indicated by the steady increase in pH. The synthesis of dipicolinic acid coincided closely with the development of heat resistance. Refractility developed 3 to 5 hr in advance of heat resistance. (+info)
PHYSIOLOGY OF THE SPORULATION PROCESS IN CLOSTRIDIUM BOTULINUM. II. MATURATION OF FORESPORES.
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Day, Lawrence E., (Michigan State University, East Lansing) and Ralph N. Costilow. Physiology of the sporulation process in Clostridium botulinum. II. Maturation of forespores. J. Bacteriol. 88:695-701. 1964.-Clostridium botulinum, strain 62-A, did not sporulate endotrophically, but forespores matured to refractile, heat-resistant spores when replaced in solutions containing l-alanine and l-proline, l-isoleucine and l-proline, or l-alanine and l-arginine. Solutions of l-arginine or l-citrulline would not support the maturation process. Acetate, CO(2), and delta-amino valeric acid were produced during sporulation in a replacement solution of l-alanine and l-proline, indicating the operation of the Stickland reaction. There was no large uptake of either exogenous l-alanine or acetate during this process. Similarly, there was no apparent protein or nucleic acid synthesis, since high levels of chloramphenicol, 8-azaguanine, or mitomycin C failed to inhibit, and no significant amount of P(32) was incorporated into the spore nucleic acids. Dipicolinic acid (DPA) was synthesized during forespore maturation. It is believed that these final steps in sporulation of C. botulinum require only an exogenous source of energy which can be obtained via the Stickland reaction system, and that the synthesis of DPA and other unknown materials relies primarily on endogenous substrates. (+info)
CHEMICAL SENSITIZATION OF CLOSTRIDIUM BOTULINUM SPORES TO RADIATION IN MEAT.
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Beef ground round inoculated with 1,000,000 spores of Clostridium botulinum 33-A per gram and containing various additives was exposed to gamma radiation. Spores were inactivated in samples (irradiated at 2.0, 2.5, and 3.0 Mrad) which contained sodium nitrate (1,000 ppm) plus sodium chloride (2.5%). Similar results were obtained when sodium nitrite (200 ppm) was substituted for sodium nitrate, except that there was evidence of spore survival in 1 of 120 cans irradiated at 2.0 Mrad. Spore destruction was based upon the absence of spores and mouse-lethal toxin in meat subcultures made from cans incubated at 35 C for 120 days. Spores were not destroyed when exposed to 2.5 or 3.0 Mrad in the absence of sodium nitrate, sodium nitrite, or sodium chloride. Furthermore, the use of these chemicals individually, together with radiation, was ineffective. The additives alone in the absence of radiation also did not cause spore destruction. Radiation levels of 2.0, 2.5, and 3.0 Mrad, when used with sodium chloride at 1.5 or 2.0% and sodium nitrate at 500 ppm or sodium nitrite at 100 ppm, were ineffective. (+info)
RADIATION STERILIZATION OF BACON FOR MILITARY FEEDING.
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Sliced, cured bacon, packed in cans and seeded with 6 x 10(5) spores per can of Clostridium botulinum strains 33A or 41B, or with 3 x 10(6) spores per can of strains 36A, 12885A, 9B, or 53B, was irradiated to various dose levels with gamma radiation. Evidence provided by swelling, toxicity, and recoverable C. botulinum with 2,200 inoculated, irradiated cans demonstrated that: (i) 4.5 Mrad were more than adequate as a sterilization dose; (ii) the experimental minimal sterilizing dose was 2.0 Mrad, and the theoretical 12-log reduction dose was 2.65 or 2.87 Mrad depending on the method of calculation; (iii) some spoilage occurred at dose levels below 2.0 Mrad; (iv) all visible spoilage of irradiated bacon was due to strains 33A and 12885A only, whose D values were, respectively, 0.141 and 0.177 Mrad based on spoilage data, and 0.221 and 0.188 Mrad, respectively, when based on recovery data; (v) toxic cans did not always result in swelling, nor did swollen cans always produce toxic spoilage; and (vi) viable C. botulinum can exist for at least 8 months in storage at 30 C without producing visible or toxic spoilage at doses below 2.0 Mrad. (+info)
COMPARISION OF SOLUBLE REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE OXIDASES FROM CELLS AND SPORES OF CLOSTRIDIUM BOTULINUM.
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Green, J. H. (Michigan State University, East Lansing), and H. L. Sadoff. Comparison of soluble reduced nicotinamide adenine dinucleotide oxidases from cells and spores of Clostridium botulinum. J. Bacteriol. 89:1499-1505. 1965.-The properties of purified reduced nicotinamide adenine dinucleotide (NADH(2)) oxidases from cells and spores of Clostridium botulinum 62-A have been studied to determine whether they are the same or different proteins. The spore NADH(2) oxidase was very heat-stable, whereas the vegetative enzyme was readily denatured at 70 C. The spore oxidase exhibited less affinity for the substrate than did the vegetative protein, but possessed a tightly bound cofactor. Atabrine was a noncompetitive inhibitor for both enzymes, but was less inhibitory to the spore NADH(2) oxidase. The enzymes could be separated from each other by gel filtration or chromatography on a diethylaminoethyl-cellulose column. The molecular weight of the spore oxidase was estimated to be 200,000 or greater, whereas that of the vegetative enzyme was 100,000 or less. Neither NADH(2) oxidase would cross-react with its heterologous antibody in a precipitation reaction. The conclusion drawn from this investigation is that the two NADH(2) oxidases are distinctly different proteins. (+info)
MECHANISM OF TRYPTIC ACTIVATION OF CLOSTRIDIUM BOTULINUM TYPE E TOXIN.
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Gerwing, Julia (University of British Columbia, Vancouver, B.C., Canada), Claude E. Dolman, and Arthur Ko. Mechanism of tryptic activation of Clostridium botulinum type E toxin. J. Bacteriol. 89:1176-1179. 1965.-The toxic peptide of trypsin activated Clostridium botulinum type E toxin was purified by chromatography through columns packed with Sephadex G-75 and G-50. The molecular weight of the active peptide was estimated to lie between 10,000 and 12,000. Amino acid analyses indicated that the active peptide had lost at least 18 of the amino acid residues present in the original protein. The active peptide and the original protein were found to have different N-terminal amino acid residues. The mechanism of tryptic activation apparently involves chiefly the removal of amino acids from the N-terminus of the toxin molecule. (+info)
ISOLATION AND CHARACTERIZATION OF A TOXIC MOIETY OF LOW MOLECULAR WEIGHT FROM CLOSTRIDIUM BOTULINUM TYPE A.
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Gerwing, Julia (The University of British Columbia, Vancouver, B.C., Canada), Claude E. Dolman, and Hardial S. Bains. Isolation and characterization of a toxic moiety of low molecular weight from Clostridium botulinum type A. J. Bacteriol. 89:1383-1386. 1965.-A toxic moiety of low molecular weight has been isolated from a type A strain of Clostridium botulinum, by a method involving ammonium sulfate precipitation and elution through diethylaminoethyl cellulose at pH 5.6. By means of electrophoresis and ultracentrifugation, the toxic substance was shown to be homogeneous; a molecular weight of 12,200 was calculated. (+info)
SURVIVAL OF CLOSTRIDIUM BOTULINUM SPORES.
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Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a "tail" comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The "tail" was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation "inactivated" spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth. (+info)