An attempt to classify fourteen strains of Clostridium sporogenes and thirty strains of Clostridium botulinum types A, B, C, D, E, and F by numerical taxonomy was made. All proteolytic strains of C. botulinum types A, B, and F and C. sporogenes were classified in the phenon I at a level of 86% S‐value. In the phenon I, these strains coexisted in a mixed‐up fashion, irrespective of conventional species or type of bacteria. A concentrated lysin solution, which was prepared from an induced lysate of a C. botulinum A190 culture, lysed vegetative cells of all proteolytic C. botulinum and C. sporogenes strains classified in the phenon I, but did not lyse the cells of the other Clostridia. However, cells of nonproteolytic C. botulinum F‐OSU and four out of five strains of Clostridium tetani were lysed by the concentrated lysin to a limited extent. The phenon II contained all nonproteolytic strains of C. botulinum B, C, D, E, and F, which were mutually linked with S values of more than 85%. Strains of Clostridium histolyticum used as a reference group formed the phenon III. Any strain grouped in one phenon was differentiated from strains grouped in the other phenons by low S‐values, that were less than 77%. A striking difference in the number of strains susceptible to mitomycin C was demonstrated between C. botulinum and C. sporogenes. In all thirty‐five cultures, except four substrains of C. botulinum B‐NIH, of proteolytic and nonproteolytic C. botulinum, bacterial lysis was consistently induced by the treatment of 1 μg per ml of mitomycin C, while only four out of thirty‐seven C. sporogenes strains were as sensitive to mitomycin C as C. botulinum. Toxigenic substrains of C. botulinum B‐NIH, NIH15 and NIH19, were sensitive to mitomycin C, but nontoxigenic strains, NIH5, and NIH‐NP, were not.
Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050‐fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate‐gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ‐toxin of Cl. perfringens.
A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.
When cells of the Clostridium botulinum A190 strain were subjected to treatment with mitomycin C or to irradiation with ultraviolet ray and then grown at 37 C, an induced lytic agent(s) was produced in the resultant lysate. Production of the lytic agent in the induced bacterial culture was inhibited in the presence of chloramphenicol. A crude preparation of the phage-free lytic agent readily lysed freezethawed or acetone-treated cells of C. botulinum B-NIFI19, and heated or chloroform-treated cells to a lesser extent at the optimal pH of 6.8. The lytic agent was stable at or below 37 C, but unstable above 37 C. A crude preparation of the lytic agent did not exhibit a detectable effect on the viability of vegetative cells.
Clostridium tetani excretes hemolysins of two size classes, a high-molecularweight hemolysin (HMH), which was eluted near void volume of a Sepharose 6B column, and conventional tetanolysin (molecular weight, approximately 50,000). The total hemolysin activity in the culture supernatant increased sharply with growth of bacteria and remained at a high level during autolysis. The content of HMH, however, decreased from 41% at 4 h of culture to 0.4% at the early stage of autolysis. The cell bodies also exhibited hemolytic activity, 70% of which could be solubilized and separated into HMH and the 50,000 Mr tetanolysin as extracellular hemolysins. The activity ratio of HMH to the total solubilized hemolysins was 0.45, on the average, at 6 h of culture but was 0.23 at the middle of logarithmic growth. Partially purified HMH from both sources appeared as broken pieces of cytoplasmic membranes under an electron microscope. The ratio of proteins to phospholipids in HMH was found to be 3.26, a value similar to that in cell membrane. The total cell hemolytic activity decreased by 90 or 75% upon addition of chloramphenicol or anti-tetanolysin serum, respectively, into a 6-h-old culture of bacteria. It is suggested that HMH is a complex of tetanolysin with a membrane fragment and releases the conventional tetanolysin during bacterial culture.
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