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Isolation of auxotrophic mutants of Bacillus cereus Public Deposited

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  • Bacillus cereus excretes large amounts of penicillinase into growth media during incubation, hence the widely-used penicillin selection technique for isolating biochemical mutants of bacteria is not applicable to this species. Genetic transfer in B. cereus has not been reported; this is partly due to the lack of an effective isolation method for selection of nutritionally deficient mutants in this organism. The original purpose of this research was to work out an effective auxotroph isolation procedure which would be applicable to B. cereus; it was intended to take advantage of the difference in thermolability of spores and vegetative cells. Spores in which mutation has been induced by ultraviolet light irradiation do not germinate as readily as the normal spores in a germination medium; therefore, mutant spores can be concentrated selectively by heat inactivation to kill most germinated prototrophs. Spore suspensions were irradiated with UV light at a distance of 40.5 cm for three to four minutes which resulted in 99 percent kiII. The germinating broth medium contained adenosine and alanine in a pH 7.4 sodium phosphate buffer. Normally, germination was carried out in a 37°C water bath for 30 minutes. After heat-shocking at 65°C for 60 minutes, the culture was plated at appropriate diIutions onto minimal medium containing 0.2% nutrient broth-yeast extract medium (doubly-enriched minimal medium). Mutant cells formed only minute colonies on this medium in contrast to the normal cells which formed large colonies. The minute colonies were picked and characterized for nutritional requirements by replica plating onto various minimal media supplemented with different nutrients. The requirements of the mutants were later confirmed by growing the mutant cells in the presence and absence of the suspected nutrients. Results of this procedure indicated that nutritional mutants could not be readily obtained; the mutants isolated were found to be unstable in that they frequently reverted back to normal. Consequently, a second procedure, which was originally intended as a comparative method and involved using a chemical mutagen, was developed for selecting auxotrophs. Diethylsulfate (DES) in various concentrations was incorporated into a doubly-enriched minimal medium. The plates were dried at 25°C for four hours, and 1,000 spores were plated directly onto this medium. The sectored colonies were picked and assayed for mutants. DES in a final concentration of 4% (v/v) gave the following mutation frequencies (number of auxotrophs per number of total surviving colonies): 6464 cured, 4x10⁻³; 569R, 3x10⁻³; 6464A, 2x10⁻³, 569S, 1x10⁻³; 9139, 7x10⁻⁴; and 6464D, 1x10⁴. In the presence of 6% DES, however, 2x10⁻³ auxotrophs were obtained with 6464D. It was found that the mutagenic property of DES could be varied by changing the drying temperature of DES-containing agar plates. When the plates spread with 6464 cured were dried at 24°C for four hours, the auxotrophic frequency was 4x10⁻³; plates dried at 30°C and 37°C yielded mutation frequencies of 2x10⁻³ and 1x10⁻³ respectively. When different kinds of colonies picked were compared, the sectored colonies showed 4x10⁻³ auxotrophs in contrast to none obtained from small colonies at 24°C drying temperature. The mutagenic effect of DES on vegetative cells was also investigated. Results indicated that DES could induce mutations readily in cells which were in the exponential growth phase. Although mutations also occurred in cells in the stationary phase, the auxotrophic frequencies were much lower than that of cells growing exponentially. It was also found that the mutants obtained by DES treatment were far more stable than those obtained by UV irradiation. More than 70% of the mutants of 9139, 6464 cured and 569R were stable. Approximately 50% of all the mutants isolated were characterized. The majority of the mutants required one of the following amino acids: arginine, cysteine, glycine, histidine, leucine, methionine, phenyi-alanine and serine. Some others were found to require nicotinamide, adenine, hypoxanthlne, thiamine or uracil. Interestingly, mutants deficient in sulfur-containing amino acids (methionine, methionine and/or cysteine) were found to be more common than those requiring other nutrients.
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