Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani

Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.     

Inducible Lysis in Clostridium tetani

Lysis was induced in seven strains of Clostridium tetani by exposure to mitomycin C. The search for a suitable indicator strain to detect bacteriophage in lysates has, so far, been unsuccessful. Inhibition studies on macromolecular synthesis during induction have shown that deoxyribonucleic acid, ribonucleic acid, and protein syntheses are all involved in the lysis induced by mitomycin C. In experiments comparing toxin and protein content in induced and uninduced cells of C. tetani, the toxin-protein ratio proved to be the same in both systems up to the point of lysis. Several possible hypotheses deduced from these results are discussed.     

Clostridium botulinum Type F in the Soil of Argentina

One strain of Clostridium botulinum belonging to type F has been isolated from a soil sample taken in Buenos Aires Province, Argentina. Comparative studies carried out with strain Langeland of C. botulinum type F have shown a few differences between the two strains.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

Taxonomy of Clostridium tetani and related species.

Clostridium tetani and its related species C. tetanomorphum, C. cochlearium and C. lentoputrescens were examined for DNA-DNA homology and biochemical properties. Two distinctly different groups were included under the name of C. tetanomorphum: one was identical with C. cochlearium and the name C. tetanomorphum was applied to the other group with some amendment of biochemical properties. Comparison of the type strain of C. lentoputrescens with wild strains obtained from horse faeces indicated that the name C. lentoputrescens should be abolished as a later synonym of C. cochlearium. Liquefaction of gelatin and spore shape, which have been used as the important criteria for differentiation of C. tetani-related species, were genetically insignificant.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

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.     

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.     

Identification and characterization of the surface-layer protein of Clostridium tetani

Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.