Spore formation and germination in Clostridium pasteurianum.

An experimental system is described in which rapid and extensive sporulation of the obligately anaerobic bacterium Clostridium pasteurianum ATCC 6013 was reproducibly obtained in batch culture using a simple, defined medium containing only salts, vitamins, glucose and ammonium ions. Electron microscopy was used to follow the progress of cytological changes during sporulation and to relate these to the time course of Incubation in the sporulation medium. The production of a refractile spore from a vegetative cell took 9 to 10 h at 37°C with the approximate times taken for the completion of each recognizable stage being as follows:- from septation to the completion of engulfment, 2 h; from engulfment to deposition of coat material, 3 h and from the first appearance of spore coat material to deposition of spore cortes, 2.5 h. Approximately 80% of the organisms produced mature, refractile spores and the degree of synchrony with which sporulation was accomplished compared very favourably with the best reported for aerobic sporulating bacteria. This C. pasteurianum system is therefore eminently suitable for use in studies of the biochemical changes associated with sporulation of an obligate anaerobe. A preliminary Investigation was made of some of the biochemical events associated with sporulation of C. pasteurlanum. Glucose was not a repressor of sporulation. in fact, high (3-4% w/v) initial concentrations were required for optimal sporulation in the defined minimal medium. A striking and characteristic early event of sporulation was swelling of the cell brought about by the extensive deposition of granulose (an amylo- pectin-like polymer) in the sporangial cytoplasm. Even so, glucose uptake and the production of acetate and butyrate proceeded continuously throughout sporulation, suggesting that the energy required for sporulation was derived from continuous fermentation of exogenous glucose. A somewhat surprising finding was that sporulation proceeded without the production of detectable levels of extracellular proteinase, though very low intracellular proteolytic activity was present during both growth and sporulation. Furthermore, the extensive protein turnover which is associated with sporulation in aerobic bacteria was not detected in C. pasteurianum. Another departure from the pattern of events normally associated with sporulation of Bacillus species was that C. pasteurianum apparently produces no antibiotic during spore formation. The exosporium of C. pasteurianum, discovered during the electron microscopic examination of the sporulation process proved to possess several interesting features, some of which were investigated in more detail. The presence of a large opening at its base - not a usual feature of exosporia, was confirmed by demonstrating that ferritin molecules or bacteriophage particles could gain entrance to the peri-sporal space of autolytically liberated spores. When the three-dimensional structure of the spore was examined using the critical-point drying technique, it was found that the spore was slung within its exosporium by a system of 'suspensors' which were not readily detectable by other methods of specimen preparation. A more detailed investigation of the nature of the exosporium showed it to possess a simple, homogeneous structure consisting of superposed sheets of identical, possibly doughnut-shaped, subunits which were arranged in a regular, close packed hexagonal lattice with a nearest neighbor separation of 5 nm. In surface view a subordinate, hexagonal pattern of 'holes' placed 10 nm apart was also discernible. The apparently paracrystalline structure of the exosporium suggested that during sporulation its synthesis might proceed by a process of spontaneous self-assembly. The presence of an opening in the exosporium posed the question of whether this might facilitate exit of the emergent germling cell. Examination of spore germination and outgrowth by electron microscopy revealed that whereas both spore coat and spore cortex underwent lytic digestion during this process, the exosporium remained essentially intire. However, escape of the germling cell was not impeded, since this invariably took place via the open end of the exosporium.