Isolation and mapping of Salmonella typhimurium mutants defective in the utilization of trehalose.

Interest in genetic markers mapping near the histidme (his) loci on the Salmonella typhimurium genetic map prompted a study of the "locus" for the utilization of trehalose as a carbon source. In colicin factor-mediated crosses of S. typhimurium LT2, which gives only weak or negative trehalose fermentation tests, to strongly fermenting S. typhimurium strains, the trehalose fermentation character segregated as though determined at a locus between trp (tryptophan requirement) and cysCD (cysteine requirement), i.e., near his (T. V. Subbaiah and B. A. D. Stocker, personal communication). Since strain LT2, despite its weak fermentation reaction on trehalose, utilized this sugar as sole carbon source, it was convenient to isolate mutants unable to utilize trehalose. For mutagen treatment, 0.1 ml of an overnight minimal-medium culture of LT2 wild-type bacteria was added to 5 ml of minimal salts solution containing several drops of diethyl sulfate (Fisher Scientific Co., Pittsburgh, Pa.) or 2 Ag (per ml) of N-methyl-N'-nitro-N-nitrosoguanidine (Aldrich Chemical Co., Inc., Milwaukee, Wis.). The tubes were incubated at 37 C for 30 min; samples were then transferred to tubes containing Difco Nutrient Broth and incubated overnight at 37 C. Overnight cultures were plated on minimal medium lacking citrate but containing 0.01% (w/v) sodium succinate, 0.4% trehalose (Pfanstiehl Chemical Corp., Waukegan, Ill.), and 0.015% Casamino Acids (Difco). Casamino Acids was added to allow growth of amino acid auxotrophs, and the succinate concentration was sufficient to enable limited growth of bacteria incapable of utilizing trehalose (Tre-). Small colonies were selected from plates incubated for 48 hr at 37 C. Master plates of these presumptive Tremutants were replicaplated on minimal medium containing 0.4% (w/v) trehalose as sole carbon source. Eleven Tremutants were found. None of the mutants grew on trehalose medium within 48 hr, whereas