Autolytic Enzyme System of Clostridium botulinum

Isolated cell walls of Clostridium botulinum type A strain 190L released an autolysin during autolysis of the cell walls. The autolysin was isolated from the cell walls, and partially purified 18.6-fold by ammonium sulfate precipitation, chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The purified preparation of the autolysin showed 2 major and 2 minor protein bands on Polyacrylamide gel electrophoresis. Some properties of the autolysin were examined using SDS-treated cell walls of the organisms as a substrate. The autolysin was active over a pH range of 6 to 8, with a maximum near pH 6.8. The lytic activity was stimulated by 10^?4 M each of Co⁺⁺, Mg⁺⁺ and Ca⁺⁺ in the order, whereas it was inhibited markedly by Cu⁺⁺. Mercaptoethanol (10^?4–10^?3 M) significantly activated the lytic action. Trypsin and nagarse (10 μg/ml) also stimulated the lytic activity. The lytic spectrum of the autolysin toward the SDS-treated cell walls obtained from various types of C. botulinum and C. perfringens indicated a relatively high specificity. After treatment with hot formamide the cell walls of C. botulinum increased in susceptibility to the autolysin.     

Activation of Clostridium botulinum Type B Toxin by an Endogenous Enzyme

It was previously postulated, based on indirect evidence, that Clostridium botulinum type B produces neurotoxin which is initially of low toxicity but which then becomes activated to highly toxic form by the action of an endogenous enzyme(s). The first direct in vitro experimental evidence in support of this hypothesis is presented here. The mildly active toxin (progenitor toxin) produced by C. botulinum type B (Lamanna) was isolated from the filtrate of a 24-hr culture and partially purified chromatographically. An enzyme that activates the progenitor toxin was also isolated from the filtrate of a 96-hr culture and purified 200-fold. The enzyme hydrolyzes synthetic substrates of trypsin but not of chymotrypsin.     

Autolytic Activity and Butanol Tolerance of Clostridium acetobutylicum

The effects of acetone and butanol on the growth of vegetative cells and the stability of swollen-phase bright-stationary-phase cells (clostridial forms) of Clostridium acetobutylicum P262 and an autolytic deficient mutant (lyt-1) were investigated. There was little difference in the sensitivity of strain P262 and the lyt-1 mutant vegetative cells and clostridial forms to acetone. The stability of the different morphological stages was unaffected by acetone concentrations far in excess of those encountered in factory fermentations. Butanol concentrations between 7 and 16 g/liter, which are within the range obtained in industrial fermentations, increased the degeneration of strain P262 clostridial forms but had no effect on the stability of lyt-1 clostridial forms which never underwent autolysis. Vegetative cells of the lyt-1 mutant were able to grow in higher concentrations of butanol than strain P262 vegetative cells. It was concluded that there is a relationship between butanol tolerance and autolytic activity.     

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.     

Survival of Clostridium botulinum Spores

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.     

Distribution of Clostridium botulinum.

The distribution of Clostridium botulinum in the natural environments of Denmark, The Faroe Islands, Iceland, Greenland, and Bangladesh was examined. A total of 684 samples were tested. Type E was found in 90% of samples from the aquatic environment of Denmark, including sediments from young artificial lakes, and in 86% of samples from the marine environment of Greenland. Type E was not found in Danish cultivated soil and woodlands, including cultivated soil from reclaimed sea beds, but type B was frequently demonstrated in these environments. C. botulinum types A, B, or E were found in 2.6% of samples from the environments of the Faroe Islands and Iceland, whereas types C or D were demonstrated in 42% of samples from Bangladesh. The incidence of type E in aquatic sediments was not related to general industrial pollution or a high content of rotting vegetation. Fish or a rich aquatic fauna, on the other hand, appeared to contribute to a high incidence of type E. Based on these findings, it is suggested that type E is a true aquatic organism, because this environment offers the best conditions for survival of the spore in nature. It is further suggested that its presence in aquatic bottom deposits is based on sedimentation after proliferation in the carrion of the aquatic fauna and dissemination by water currents and migrating fish.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Autolytic Enzyme System of Streptococcus faecalis IV. Electron Microscopic Observations of Autolysin and Lysozyme Action

Cell walls (LOG walls) were isolated from cultures of Streptococcus faecalis ATCC 9790 in the exponential phase of growth. These walls were either allowed to undergo autolytic dissolution (in the presence or absence of trypsin) or wall autolysis was inactivated with sodium dodecylsulfate (SDS walls). Inactivated walls were treated either with lysozyme or with isolated, partially purified S. faecalis autolysin. During wall lysis, samples were removed, negatively stained with phosphotungstate, and examined in the electron microscope. Both lysozyme and isolated autolysin appeared to act over the entire surface of SDS walls. After partial dissolution, a fibrous network over the surface was revealed. Lysozyme digestion revealed the presence of prominent, highly-contrasted equatorial and subequatorial bands around the walls. After trichloroacetic acid extraction, the bands were seen less frequently and less distinctly in the partially lysozyme digested walls, suggesting that the bands contained nonpeptidoglycan polymers. In the absence of trypsin (which activates a latent form of the autolysin), autolysis of LOG walls appeared to start at the equatorial bands and to proceed back towards the apex of the coccus. Ribbons of wall material coming off the wide edge of the nearly hemispherical wall fragments were observed. Activation of latent autolysis resulted in lytic action over the entire wall surface. The results are consistent with the previously postulated location of active autolysin at the areas of new wall synthesis and the random location of latent autolysin in LOG walls.     

Flagellar glycosylation in Clostridium botulinum

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.     

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.     

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.