Elective selection of proline-requiring mutants.

We recently discovered a method whereby proline-requiring (pro) mutants of some strains of Escherichia coli K-12 can be selected at high frequency (Table 1). The method seems to involve the general principle of differential growth rates. Only pro E. coli K-12 mutant strains grew in Penassay Broth (Difco) containing 1 to 3 ,ug/ml of 4-nitropyridine-N-oxide (4NPO), 10 ,ug/ml of 4-nitrosopyridine-N-oxide, or 50 ,ug/ml of 4-hydroxyl-aminopyridine-N-oxide, whereas strains not requiring proline (pro') were inhibited. The method can be illustrated by the results obtained with a pro+ E. coli K-12 strain, W3630. A single colony of this strain was inoculated into 10 ml of Penassay Broth and incubated at 37 C overnight, during which time growth usually reached a level of about 5 x 108 cells per milliliter. The cell suspension was diluted 10-3, and 0.2 ml was added to 100 rml of Penassay Broth along with 0.2 to 0.5 ml of a solution of 1 mg of 4NPO/ml. This mixture was incubated on a reciprocal shaker at 37 C for 20 to 26 hr, and 0.1 ml of the culture was diluted and plated on eosin-methylene blueglucose-agar medium (J. Lederberg, Methods Med. Res. 3:55, 1950). Colonies appearing on this medium then were replica-plated onto Davis's minimal (DM) agar with or without 40 ,ug/ml of L-proline. The percentage of pro mutants present in 4NPO-treated cultures is shown in Table 1. Pretreatment of bacteria with a mutagen, such as ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine, increased the frequency of appearance of pro mutants. A preliminary analysis of the method has been made with the following results: pro mutants are the only ones selected by this method at a detectable frequency; pro mutants of independent origin grow on DM agar containing the metabolic precursors of proline, glutamic-'y-semialdehyde or A1-pyrroline-5-carboxylic acid, but they do not grow on DM agar containing glutamic acid (presumably the mutants have a metabolic block between glutamic acid and glutamic-'y-semialdehyde); the mutants produced are stable; conjugation experiments indicate that the pro marker is located on the chromosome between the arabinose (ara) and lactose (lbc) loci, close to lac. Re-