Broth cultures of Bacillus pumilus NRRL B-3275 (BpB1) grown at 25, 30, or 37 C contain 1 to 2% spontaneous auxotrophic mutants in both the exponential and stationary phases of growth. Of 70 such mutants isolated from cultures grown at 37 C, approximately two-thirds reverted at such a high frequency as to preclude their study. Of the remaining 22 mutants, 18 required a single amino acid, 1 required adenine, and 1 required uracil. Two of the auxotrophs each required two unrelated amino acids

more »

... ting from two independent mutations. All of the mutations reverted spontaneously. Enhanced reversion of approximately one-third of the mutations was obtained with nitrosoguanidine, ethyl methane sulfonate, or diethyl sulfate, or with more than one of these mutagens. The reversion of one mutation was enhanced by 2-aminopurine. The reversion of the remaining mutations was not enhanced by the above mutagens, nor by mutagens known to induce (and revert) frameshift mutations in other bacterial systems. Nine of 10 mutants examined did not show a selective growth advantage over the parents. All but three of the mutations could be linked by PBS1 transduction to one of the previously described auxotrophic markers in strain BpB1. No evidence was obtained for clustering of the mutations on the BpB1 genome. Six of the mutations conferred a requirement for serine. One linked by transduction to trp-2, three linked to argAl, and two (ser-2, -3) linked to argOl. Pigmented mutants (containing a carotenoid-like pigment), which occur spontaneously in BpB1 cultures at a frequency on the order of 1 to 5 mutants per 104 cells, link by transduction to ser-2, -3. Spontaneous mutants of strain BpB1 resistant to rifampin, streptomycin, erythromycin, 5-fluorouracil, or 5-methyltryptophan occur at a frequency similar to that of strains of B. pumilus which do not exhibit a high rate of spontaneous mutation to auxotrophy. It is suggested that certain sites or regions of the BpB1 genome exhibit a high rate of spontaneous mutation. Bacillus pumilus NRRL B-3275 (BpB1) has the ability to adapt to the "L-form" type of growth during cultivation in liquid media containing 7% sodium chloride (4). With respect to several other physiological properties, strain BpB1 behaves as a typical strain of B. pumilus (15) . The adaptation of strain BpB1 to the Lform state occurs without prior mutagenesis, and the L-forms revert to the bacillary form after a reduction of the sodium chloride concentration of the medium to 3% (Lovett, unpublished data). The reversion is prevented by including penicillin in the growth medium (unpublished data). Since several passages are required for both the "induction" of the Lform and its reversion to the bacillary form, it is not clear whether the medium containing a high salt concentration selects a class of spontaneous mutant that outgrows the bacillary form or whether the high salt concentration causes a phenotypic alteration of the cells. During the isolation of mutants from strain BpB1 for use in genetic studies (16, 17) , it became evident that a variety of spontaneous mutations arose in this strain at a frequency significantly higher than is observed in other strains of B. pumilus. The present study was undertaken to examine the properties of two 977 on May 9, 2020 by guest