Isoleucine-valine requiring mutants of Salmonella typhimurium. 3. Valine-sensitive strains.

Abstract

Bacterial strains: The mutant strains were derived from wild-type S. typhimurium LT2, using the mutagen N-methyl-N-nitro-N-nitrosoguanidine and the procedure described by ADELBERG, MANDEL and CHEN (1965). The strains sensitive to valine were detected by replica plating from nutrient agar master plates onto minimal medium (ARMSTRONG and WAGNER 1964) containing 4 pg L-valine per ml. The abbreviation VS (valine sensitive) was arbitrarily selected to designate those strains whose growth is inhibited by the supplementation of minimal medium with valine. Another characteristic shared by the VS strains is a slow rate of growth on minimal medium. Of the 15 strains isolated, four (VS-7, 9, 10, and 13) were selected for the analyses presented in this report. Other S. typhimurium strains utilized in this study include: LT2; ilvAil8 (threonine dehydratase deficient) ; iluC8, 42, and 66 (reductoisomerase deficient) ; and ilvD6, 18, and 47 (dihydroxyacid dehydratase deficient) (ARMSTRONG and WAGNER 1964; ELLIOTT and ARMSTRONG 1968). Cotransduction tests: The procedures utilized can be found in ARMSTRONG and WAGNER (1964). For these studies four VS and two iluC strains were used as donors. In crosses with the VS strains, transduction mixtures wcre plated on minimal medium and incubated for a total of 48 hr (fist 24 hr at 37°C followed by 24 hr at room temperature). Donor recombinants were identified by their small colony size. To avoid feeding of the donors by the background growth in crosses with iluA and C strains, phage and cells were mixed in 2.5 ml of molten minimal-medium agar (0.75%) and spread as an overlay on minimal medium plates. This procedure allowed for a more accurate determination of donor recombinants. In crosses with iIvC42 and 66 as donors, use was made of partial revertants of these strains that can grow suboptimally on a valine supplement. Transduction mixtures were spread on minimal medium plates supplemented with 4 pg of L-valine per ml. After 4 days of incubation at 37C, the wild-type and donor recombinants were easily identified by the difference in colony size. Growth studies: Duplicate assay tubes, each containing 3 ml of medium, were used. The supplementations to minimal medium are listed in Tables 3 and 4. Inocula were prepared from nutrient-broth cultures grown overnight at 37°C on a rotary shaker. These cultures were centri