A Mutant of Mycobacterium smegmatis Defective in Dipeptide Transport

Mycobacteria are characterized by long-term survival within vacuoles in the macrophage. There is little detail concerning the supply of nutrients available to the intravacuolar parasite. Peptides might serve as an excellent nutrient source for intracellular mycobacteria. We have undertaken studies with the nonpathogenic species of mycobacteria Mycobacterium smegmatis in order to begin to understand oligopeptide metabolism in the genus. The substrate used was the dipeptide b-alanyl-Lhistidine, or carnosine. In the present study, a genetic screen was developed to isolate a mutant of M. smegmatis deficient in the utilization of carnosine as a sole source of carbon and nitrogen. Small peptides can be used as a sole carbon and/or nitrogen source by M. smegmatis (18). To isolate a mutant defective in the utilization of the dipeptide carnosine, the following scheme was devised. First, wild-type cells were plated on basal salts agar (1) with reduced concentrations of glycerol and ammonium chloride as carbon and nitrogen sources, respectively. Very small colonies (,1 mm) were visible on minimal medium agar when glycerol and ammonium chloride were reduced to 0.05 and 0.01%, respectively. When this limiting medium was supplemented with 1.0% carnosine, wild-type cells used carnosine as a source of both carbon and nitrogen and thus produced larger colonies (4 mm). Mutants unable to use carnosine as a carbon or nitrogen source would exhibit small-colony morphology on limiting medium plus carnosine. A population of mutagenized cells (N-methyl-N-nitroso-N-nitroguanidine [1]) was plated on limiting medium plus carnosine and