Studies on the Bacterial Spore Coat

Properties of alkali-soluble components from the spore coat of Bacillus megaterium were examined by physicochemical methods. They were composed of acidic polypeptides of various molecular weights with small amounts of phosphorus and sugar. They were allowed to dissociate to unit components by incubation with SDS. The major component was partially purified by gel filtration, and shown to have a mean molecular weight of about 11,000.     

Cytology of Spore Formation in Clostridium perfringens

The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits.     

Coat and enterotoxin-related proteins in Clostridium perfringens spores

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.     

The Clostridium perfringens Germinant Receptor Protein GerKC Is Located in the Spore Inner Membrane and Is Crucial for Spore Germination

The Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens causes a variety of diseases in both humans and animals, and spore germination is thought to be the first stage of C. perfringens infection. Previous studies have indicated that the germinant receptor (GR) proteins encoded by the bicistronic gerKA-gerKC operon as well as the proteins encoded by the gerKB and gerAA genes are required for normal germination of C. perfringens spores. We now report the individual role of these GR proteins by analyzing the germination of strains carrying mutations in gerKA, gerKC, or both gerKB and gerAA. Western blot analysis was also used to determine the location and numbers of GerKC proteins in spores. Conclusions from this work include the following: (i) gerKC mutant spores germinate extremely poorly with KCl, l-asparagine, a mixture of asparagine and KCl, or NaP_i; (ii) gerKC spores germinate significantly more slowly than wild-type and other GR mutant spores with a 1:1 chelate of Ca²⁺ and dipicolinic acid and very slightly more slowly with dodecylamine; (iii) the germination defects in gerKC spores are largely restored by expressing the wild-type gerKA-gerKC operon in trans; (iv) GerKC is required for the spores'' viability, almost certainly because of the gerKC spores'' poor germination; and (v) GerKC is located in the spores'' inner membrane, with ∼250 molecules/spore. Collectively, these results indicate that GerKC is the main GR protein required for nutrient and nonnutrient germination of spores of C. perfringens food-poisoning isolates.     

Immunogenic properties of Clostridium perfringens type A anatheta-hemolysin

Guinea-pigs were immunized with anatheta-hemolysin preparations adsorbed on aluminum hydroxide, as well as with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated. Anatheta hemolysin preparations were obtained with the use of homogeneous theta hemolysin, as well theta hemolysin of various purification degrees. As a result, antatheta hemolytic guinea-pig sera capable of neutralizing 2,000-8,000 HU of theta-hemolysin were obtained. Tests made to establish the degree of protection in the immunized guinea-pigs did not show that the animals immunized with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated, had any advantages in the degree of protection over the animals immunized with the toxoid alone. But there is no doubt that this component plays a positive role under the conditions of natural gas gangrene when the hemolytic action of Cl. perfringens toxin becomes considerably pronounced.     

Spore coat protein and enterotoxin synthesis in Clostridium perfringens.

Polyacrylamide gel profiles of Clostridium perfringens spore coat protein revealed four and occasionally five components. Pulse-chase experiments indicated that synthesis of coat protein polypeptide and enterotoxin was an early sporulation event. However, maximum synthesis occurred coincident with the onset of heat resistance.     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

枯草芽孢杆菌芽孢中的芽孢衣

芽孢衣是赋予芽孢对有机溶剂和溶菌酶的抗性,以及对外界出芽诱导物的感应能力的保护性结构。该主要介绍由枯草芽孢杆菌(bacillus subtilis)所形成的芽孢的芽孢衣中已鉴定的主要蛋白质．及其基因表达调控和应用等方面的研究进展。     

Haemagglutinin production by enterotoxigenic strains of Clostridium perfringens type A

Thirty-nine enterotoxigenic cultures of Clostridium perfringens type A were studied for enterotoxin and haemagglutinin production. Enterotoxin was quantitated by sandwich ELISA and DOT-ELISA techniques and haemagglutinin titres were determined using sheep and human erythrocytes. Haemagglutinins from only six cultures reacted against both sheep and human erythrocytes; a further 13 reacted only against human erythrocytes, and another five only against sheep cells.The authors are with the Department of Veterinary Public Health and Epidemiology, Ranchi Veterinary College, Birsa Agricultural University, Ranchi-834007 (Bihar), India.