Isolation and characterization of two antigens of Corynebacterium hofmannii.

Banach, T. M. (University of Manitoba, Winnipeg, Canada), and R. Z. Hawirko. Isolation and characterization of two antigens of Corynebacterium hofmannii. J. Bacteriol. 92:1304-1310. 1966.-A serologically active substance, extracted from sonically treated cells of Corynebacterium hofmannii with hot HCl, produced two precipitin lines by immunodiffusion tests with a hyperimmune homologous serum. Extracts of other species failed to precipitate with the hofmannii antiserum. The active fraction was eluted from a diethylaminoethyl cellulose column in the third adsorption peak at a linear concentration of 0.5 m KCl, and produced two precipitin lines which corresponded in identity to those formed by the acid extract. Separation of the antigens was achieved by rechromatography on a Sephadex G-200 column; the major antigen was designated A; the minor, B. The homogeneity and purity of each antigen was established by immunoelectrophoresis and, in addition, that of antigen A by disc electrophoresis. Biochemical analyses showed that both antigens were composed of a major protein component with polysaccharide and nucleic acid present in an approximate ratio of 17:3:1, respectively. Glutamic acid, aspartic acid, alanine, glycine, valine, and leucine were the main amino acids present. Antigen A contained 17% less protein and 3.5% less carbohydrate than antigen B. The principal sugars of antigen A were identified as arabinose and glucose. The molecular weight, estimated by gradient centrifugation, was 16,500 for antigen A and 21,000 for antigen B.

Effect of cyclic AMP on catabolite repressed bacterial sporogenesis of an anaerobe.

The sporulation of a high frequency sporogenic mutant of Clostridium botulinum was reduced to less than 30% in a medium containing 270 mM glucose. The repression was reversed from 30 to greater than 80% sporulation by the addition of 10(-5) or 10(-4) M cyclic 3',5'-adenosine monophosphate (cAMP) or monobutyrul cyclic AMP (B-cAMP). No difference was observed in amount of growth with the addition of either the cAMP or B-cAMP. Glucose consumption was enhanced by the addition of either of the cyclic nucleotides and corresponding changes in pH were observed. The catabolite repression by glucose was reversed by ATP or ADP. Except for GTP, guanine nucleotides were not effective. The intracellular cyclic AMP levels were high in vegetative, sporulating and derepressed cells, but low in glucose-repressed cells. The findings suggest that the sporulation of the anaerobe was sensitive to catabolite repression which was specifically reversed by cyclic AMP.

Poly-beta-hydroxybutyrate metabolism during growth and sporulation of Clostridium botulinum.

The granules observed in the cytoplasm of cells of sporogenic and asporogenic strains of Clostridium botulinum type E were isolated at various developmental stages of growth and sporulation. Electron microscopy of thin sections showed that most of the granules were dispersed throughout the cytoplasm. Chemical analysis and electron microscopy showed that the granules were poly-beta-hydroxybutyrate (PHB). The polymer began to accumulate after 8 h of growth, reaching 9 and 13% of the cell dry weight in the sporogenic and asporogenic strains, respectively, during early stationary phase. (14)C-acetate was readily incorporated into PHB. The rate of assimilation paralleled the production and utilization of PHB, indicating that the acetate served as its precursor. (14)C-butyric acid was not utilized to any significant extent. Most of the PHB which had accumulated in the sporogenic strain was catabolized during the development of the spore, but in the asporogenic mutant it remained essentially unchanged. The findings suggest that the PHB provides endogenous carbon and energy for the synthesis of spore-specific components required for spore maturation.

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.

Outgrowth and sporulation studies on Clostridium botulinum type E: influence of isoleucine.

A defined medium (CDM) is described which supported growth and sporulation of type E strains of Clostridium botulinum, but not sporulation of other serotypes of C. botulinum or C. sporogenes. As compared to growth in complex medium, spore outgrowth was delayed and both the growth rate and the cell yield was reduced. However, efficiency of sporulation of the type E MSpt strain in a chemically defined medium (CDM) was the same as that in complex medium and, in fact, sporulation was nearly synchronous and completed within 3 h of the first appearance of phase-bright endospores, compared with completion in 9 h in TPGY. Growth studies with CDM, from which single amino acids were omitted, showed that isoleucine was essential for outgrowth of heat-activated spores of the MSp+ strain, whereas valine was required for that of the Ts-25 mutant. Radioactive isoleucine was incorporated by germinating MSp+ spores at an earlier stage and at a more rapid rate than labelled methionine or mixed amino acids. Uptake studies showed that isoleucine accumulated in a prominent acid-soluble pool during outgrowth, a period when its incorporation into protein was not evident. The results suggest that the isoleucine may be required for a purpose other than protein synthesis during outgrowth.

Comparison of butyric type of fermentation in sporogenic and asporogenic mutants of Clostridium botulinum.

Glucose-adapted cells of a sporogenic mutant. MSp(+), and an asporogenic mutant, RSpoIIIa, of Clostridium botulinum type E rapidly fermented glucose, fructose, maltose, and sucrose, resulting in cytoplasmic granulation, heavy growth, a pH of <6.0, and sporulation of the MSp(+) mutant ranging from 60 to 80%. In Trypticase peptone glucose broth, the MSp(+) mutant formed >80% refractile endospores in 25 h, whereas the RSpoIIIa mutant which was blocked at early forespore stage had commenced to lyse. Both mutants accumulated acetate and intracellular granules, reaching maximal levels at early stationary phase of growth. In MSp(+), as the levels of acetokinase, phosphotransacetylase, and butyryl-coenzyme A dehydrogenase reached a maximum, butyrate accumulation continued concurrently with an increase of endospore formation, whereas the levels of poly-beta-hydroxybutyrate decreased simultaneously with its precursor, acetate. Butyrate biosynthesis was blocked in the asporogenic mutant. As shown by isotopic assays, butyrate and acetate serve as precursors of spore lipids. beta-Phenethyl alcohol, fluoroacetic acid, and 2-picolinic acid inhibited anaerobic sporogenesis almost completely, butyrate biosynthesis by >87%, and acetate accumulation by 50 to 62%, showing a direct relationship between butyric type of fermentation and anaerobic sporulation.

Structural changes associated with extraction of group E spore antigen of Clostridium botulinum.

Spores of type E Beluga strain of Clostridium botulinum, which were hydrolyzed with HCl to remove the spore-specific antigen, showed a spore coat which was thinner and projected into the exosporium with multiple protuberances. The changes observed suggest that the antigen is a component of the spore coat connected with the maintenance of rigidity.

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.