Lecithinase activity of mutant and wild strains of Clostridium perfringens.

In a preliminary survey of 30 strains of type A Clostridium perfringens freshly isolated from human feces, soil, and foods, a wide variation in their lecithinase activities was observed (J. A. Schulze, M.S. Thesis, Univ. of Montana, Missoula, Montana, 1968). Other investigators also have noted such variations (1, 2, 5). Variation in lecithinase production by strains of C. perfringens may be the result ofmutant strains whose capacity to produce lecithinase has been altered. We investigated the possibility that changes in the lecithinase activity of C. perfringens may be induced by the mutagenic agent, N-methyl-N'nitro-N-nitrosoguanidine. The mutagenic properties of nitrosoguanidine were first demonstrated by Mandell and Greenberg (4) and it appears to be the most powerful chemical mutagen yet discovered. The mechanism of action of nitrosoguanidine upon the genetic system of the cell is yet unknown. In view of the fact that Gilham (3) has shown that nitrosoguanidine induces both chromosomal and nonchromosomal mutations, there is a high probability that every cell will be mutated at more than one site. Three strains of C. perfringens (UMJS-12, isolated from human feces; UMJS-29, isolated from meat; and UMJS-39, isolated from soil) were used in this study. Ten ml of thioglycollate broth (Difco) was inoculated with 1.0 ml of an 18to 24hr thioglycollate broth culture of C. perfringens. After 5 hr of incubation at 46 C, the cells were washed by centrifugation in saline and then resuspended in saline to obtain a cell suspension that yielded approximately 10 to 50 colonies per plate. This was done to avoid crowding of the colonies on the plates, which might influence colony size. Nitrosoguanidine (Aldrich Chemical Co., Milwaukee, Wis.) was added to the cell suspensions to yield a final concentration of 100 ,ug of nitrosoguanidine per ml. After the tubes were incubated for 20 min at room temperature, permitting the mutagen to act upon the cells, the cell suspensions were diluted in 0.1% peptone water, plated on 5.0% rabbit blood-agar, and incubated anaerobically for 24 hr. Mutants were selected on the basis of colonial morphology. The criteria that were used were (i) colonies with diameters which were greater or lesser than those of the parent colonies, and (ii) colonies with irregular margins. Because this marker was used to isolate mutants, colonies which appeared normal may also have exhibited a change in lecithinase activity. The mutant colonies produced hemolytic patterns similar to those of the wild type. The diameter of each mutant colony was measured by means of a vernier caliper. The mutant colonies were subcultured and maintained in thioglycollate broth. Lecithinase activity of each mutant strain was determined according to the method of Nakamura and Cross (5). In order to compensate for the differences in the cell densities ofthe cultures, the amount of lecithinase produced was calculated per mg of dry cell weight. The mutants that were isolated possessed lecithinase activity levels which were less than, as well as greater than, the levels produced by the parent strains (Table 1). Mutants with large colony diameters produced more lecithinase than the wild type. Mutants possessing small colonies and mutants with irregular colonial margins produced less lecithinase than the wild type. Only one mutant, UMJS-12D, failed to produce detectable amounts of lecithinase. The biochemical reactions of the mutants varied only slightly from those of the wild type. Three large-colony mutants produced 70 to 200% more lecithinase than the wild type. It is possible that multiple enzyme systems, rather than a single enzyme, are involved in the synthesis of lecithinase. This could account for the different amounts of lecithinase produced by the largecolony mutants. Another explanation is that in any population of a mutant there is a normal distribution of cells with different capabilities to produce lecithinase. Until recently, the genetic composition of C. perfringens has been neglected. There is a paucity of genetic studies dealing with anaerobic bacteria. Paquette and Fredette (6) reported that mutants of C. perfringens obtained by euflavine treatment lost their capacity to synthesize lecithinase as well as their ability to invade the animal body. They