Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

Isolation and characterization of Clostridium botulinum type E from soil of Gwalior, India.

A strain of Clostridium botulinum type E has been isolated from soil samples of Gwalior, India. The isolated strain shows curved vegetative cells with oval, bulging, and terminal spores. The production of toxin was detected by immunodiffusion test, symptomatic death of mice and mouse protection assay with trivalent antitoxin (A+B+E). The culture supernatant gave 10(3) MLD (minimum lethal dose) per ml without any protease treatment. Group specific and serotype specific primers amplified the DNA fragments of 260 bp and 445 bp, respectively, indicating Clostridium botulinum type 'E.'

In situ characterization of Clostridium botulinum neurotoxin synthesis and export.

A monoclonal antitoxin/colloidal gold probe and sequential centrifugation were used to study synthesis, translocation and export of Clostridium botulinum strain 62A neurotoxin (NT). Exponential growth occurred after 5 h of anaerobic incubation of spores and continued for 15-16 h. NT was detected at 15 h using the probe and transmission electron microscopy (TEM), 2 h earlier than the first detection by the mouse bioassay. During exponential growth, the probe localized NT primarily in the cytoplasm, on the inner side of the cytoplasmic membrane and in the cell wall. During stationary and death phases, the NT was located within the cytoplasm, cell wall and extracellularly. NT was released from the cell during cell wall exfoliation. Cells retained NT after repeated gelatin-phosphate washes and sequential centrifugations, consistent with the TEM observation that the NT is bound to the cell wall. These observations indicate that the process of Cl. botulinum type A NT production follows a sequence of synthesis, translocation across the cytoplasmic membrane and export through the cell wall.

Characterization, self-assembly and reattachment of S layer from Clostridium botulinum type E saroma.

S layer of Clostridium botulinum type E Saroma and its subunits were isolated and characterized for their chemical and morphological properties. The S layer was composed of a number of subunits with apparent molecular weights ranging from about 10 to 150 kDa. The isolated S layer subunits possess the ability to assemble into recrystallized flat sheets in the absence of any supporting layer and to reattach to the cell wall from which they have been removed. Immunoblot analysis using an antiserum against whole cells of the organism showed that 60 kDa and 90 kDa subunits of the S layer were major somatic antigens of the organism. Immunogold-labeling using monospecific antiserum raised to the individual 60 and 90 kDa proteins revealed that both subunits were exposed evenly over the entire cell surface. The amino acid compositions of both subunits showed that aspartate and glutamate were predominant whereas cystine and methionine were poor. The amino acid composition and acidic property of the two subunits of the S layer agree well with the results obtained from the S layers of other bacterial species as well as other pathogenic clostridia.

Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A.

An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.

Germination of spores from Clostridium botulinum B-aphis and Ba410.

The germination of spores from Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-1 in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germinated at rates of 0.16% min-1 in CTB and 0.04% min-1 in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanine- and L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains.

Isolation of nontoxigenic variants associated with enhanced sporulation and alteration in the cell wall from Clostridium botulinum type a 190L by treatment with detergents.

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage-like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.

Ultrastructure of a hexagonal array in exosporium of a highly sporogenic mutant of Clostridium botulinum type A revealed by electron microscopy using optical diffraction and filtration.

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing and equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. the adjacent globular subunits appeared to be interconnected by delicate linkers.

High and low toxin production by a non-toxigenic strain of Clostridium botulinum type C following infection with type C phages of different passage history.

Toxin production in Clostridium botulinum types C and D is governed by specific bacteriophages. Prior passages of a phage controlling type C toxin production caused subsequently lysogenized bacteria to become variably toxigenic. This appears to be one of the causes of the decrease in toxigenicity which is common in some type C and D strains. The morphology of bacteria was also changed from rod-shaped to filamentous by infection with a successively propagated phage.

[Characteristics of spore formation in Clostridium botulinum type C]. / Osobennosti sporoobrazovaniia u C1. botulinum tipa C.

Electron microscope study of C1. botulinum, tyep C, showed that microbial cells were surrounded with a five-layer wall. Structures characteristic of sporulating cells and phage particles whose intracellular development led to reduction and lysis of the cytoplasm were revelaed in the area of the cytoplasm. Mature spores were encountered rarely. Formation of prespore, cortex was observed, but the elements of the spore membrane were chaotically dispersed in the whole cytoplasm. Such disturbances could be connected with the presence of phage in the culture.

The effect of rifampicin on the developmental phases of germinating spores of Clostridum sp., MSp+.

The effect of rifampicin on the developmental phases of germinating spores of Clostridium botulinum, MSp+, has been studied. At sublethal concentrations of rifampicin (0.05 ng/ml) the time periods required for outgrowth and vegetative growth was significantly prolonged because of the inhibition of RNA and protein synthesis. However, rifampicin had essentially no effect on DNA synthesis or on subsequent spore formation. Chemical analyses showed that the amount of protein present in vegetative cells of the rifampicin-treated cultures was twice as great as in the untreated cultures but the total protein content of endospores was the same in both cases. It was revealed in ultrastructural studies of rifampicin (0.1 ng/ml) treated cultures, examined after 22 h, that septum formation and normal cell division of the emerging cell was blocked and a few cells showed constriction which produced one normal and one protoplast-like daughter cell.

The effect of sublethal doses of rifampin on the sporulation of Clostridium botulinum.

Sublethal doses of rifampin (0-005 mug/ml), added to vegetatively growing cultures of a sporogenic mutant of Clostridium botulinum at inoculation time or after 4 h, resulted in a decrease of growth and in blockage of spore formation. But when rifampin was added 6 to 24 h after inoculation, normal growth and sporulation occurred, indicating that the time of addition was critical and that rifampin was most effective on rapidly dividing, exponential-phase cells. Ultrastructural studies showed that when rifampin was added at the time of inoculation, endospore development was blocked at stage III. During subsequent incubation (greater than 10 h) the cells lost their rigidity, and lysis of the mother cell was followed by that of the forespore. When the cultures were treated with rifampin at 4 h, about 40% of the cells were blocked at stage III and about 60% reached stages IV and V. Some showed excessive elongation and contained developing spores at each pole. They appeared to be derived from two daughter cells unable to form a division septum because of a specific inhibitory effect of rifampin on division. It would seem, therefore, that two daughter cells which are genetically coded to form endospores will do so irrespective of the development of a division septum, and the spores are formed at the 'old' polar regions.

[Use of a synthetic medium for cultivating pathogenic anaerobes]. / Primenenie sinteticheskoi sredy dlia kul'tivirovaniia patogennykh anaerobov

It was shown that a synthetic medium suggested by the authors earlier was useful for the growth and toxin formation of Cl. tetani, Cl. botulinum and Cl. perfringens, types B and E. A study of the character of growth and toxinogensis, microscopic examination of morphology of culture cells and results of passages showed the suggested synthetic medium to be of value; a possibility of its application for studying the nutrient requirements and the role of individual components of the nutrient media in the process of growth and toxinogenesis was also demonstrated.