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.     

Continuous Production of Clostridium tetani Toxin

The continuous production of Clostridium tetani toxin has been carried out in a 1-liter stirred culture vessel for as long as 65 days. Toxin production of approximately 120 flocculating units per ml was maintained with a dilution rate of 0.125 hr^-1, a temperature of 34 C, a pH of 7.4, and the addition to the medium of 0.1 g of potassium chloride per liter. The average minimal lethal intraperitoneal dose of the toxin in mice was approximately 10⁶ per ml.     

Isolation of Bacteriophage for Clostridium tetani

The purpose of this report is to note the isolation of a Clostridium tetani phage from soil and to specify the procedure used to obtain suitable lawns for plaque formation.     

The flagellar protein of Clostridium tetani.

Clostridium tetani ATCC 19406 was investigated with regard to the flagellar filaments produced by this anaerobic species. Flagellar filaments were removed from the cell bodies by hydrodynamic shear forces and purified by differential centrifugation. Exposure to acid was shown to result in disaggregation of the flagellar filaments into a preparation of flagellar protein containing 3.5% carbohydrate. The protein was judged homogeneous after examination by acrylamide gel electrophoresis in the presence of 4 M urea at several pH levels, and was shown to have a molecular weight of 35,000 daltons. Amino acid analyses indicated the absence of cysteine and tryptophan and a preponderance of acidic residues, epsilon-N-methyllysine was shown to be absent and the N-terminal amino acid was identified as alanine. Analysis of the C-terminal region indicated the sequence -Leu-Leu-Arg. These findings indicated that the obligate anaerobe C. tetani produced flagella filaments similar to previously studied filaments of aerobic and facultatively anaerobic bacteria.     

Phage-like particles produced by Clostridium tetani]

Two C. tetani strains used for toxin production spontaneously produce two varieties of phage-like particles with isometric heads. Type A has a contractile tail, whereas type B shows a non-contractile tail with a long, wavy tail fiber. No relationship between the presence of these particles and the amount of toxin produced was found.     

Novel actin-like filament structure from Clostridium tetani

Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.     

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.     

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.