Minimal medium supplemented with sub-optimal levels of parental auxotrophic requirements increases recombinant recovery from matings of Nocardia erythropolis more than 500-fold when compared with nutrient agar matings. In early reports of recombination in Nocardia erythropolis (1, 4) some mated pairs interacted with such efficiency that recombinants comprised up to 1% of the cells in suspensions prepared from the mated populations. This high frequency recovery appeared to be partially strain

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... endent. Subsequent reports showed consistently lower fractions of recovered recombinants. Usually a 10-5 or less fraction of the mated population was recombinant (2, 5, 6, 8) under standard conditions in which mating was carried out on nutrient agar (NAg). Although chromosomally located mating factors, Ec and eC, control fertility between N. erythropolis strains (3, 7), it was not known if these loci, or episomes analogous to those in Escherichia coli, determined high efficiency mating and recombination. Mutations at loci affecting colonial morphology and associated with specific auxotrophic genes were reported to increase efficiency of recombinant recovery (9) in matings of strains containing these markers. Some evidence showed that nutritional conditions during mating could affect recombinant recovery efficiency (1, 4). The present experiments test some of these parameters with respect to strains hithertofore shown to produce recombinants at both, high and low efficiencies. We show that nutritional conditions effect recombinant recovery. Nutritional conditions during mating of N. erythropolis can be established, increasing recombinant recovery significantly over that found in standard NAg mating conditions. In keeping with procedures established for standard nocardial matings (2, 5, 9) , pairs to be mated were grown in mixed culture for a period of 3 days at 30 C. A variety of mating media was tested. Each parent was checked for reversion at selected loci by individual culture in parallel on identical media. After incubation, suspensions were prepared from the mated and control cultures. Appropriately diluted samples were placed on selective media, previously described (1, 2), and incubated 7 days for the detection of recombinant colonies. Estimates of the total viable populations were made by plating diluted suspension samples on Difco nutrient broth solidified with 1.5% agar (NAg) and incubating for 3 days. The recombinant recovery fraction is expressed as the number of recombinant colonies detected on selective media per milliliter of suspension, divided by the number of viable colony forming units per milliliter of suspension as estimated on NAg. All incubations were carried out at 30 C. In early experiments (1, 4) N. erythropolis auxotrophic strains JA2-13, mating type Ec, and JA3-3, mating type eC, produced prototrophic recombinants in frequencies as great as 1% of the sampled populations. Therefore, we reexamined these strains using stocks not more than two successive subcultures removed from those of our early experiments. Standard NAg matings using inocula directly from the stocks produced prototrophic recombinants at 10-6 or less fractions of the sampled populations (Table 1) . Matings using the predominant smooth colony types isolated from these stocks gave similar results. Isolates of flat colony variants. similar to those described earlier (9), were approximately 10-fold more fertile than the predominant soft colony type (Table 1) . We hypothesized that the disparity between these NAg matings and our early work was due to the loss, during prolonged culture storage, of a genetic factor(s) responsibile for high-frequency mating. To try to regain this hypothesized factor, induced variants were tested for enhanced fertility. Colony forming survivors of ultraviolet (UV) irradiation killing 99.99% of the initial population were tested for increased recombinant recovery. Colonies of purine-requiring mutants, after growth in media containing 20 ug of 646 on May 9, 2020 by guest