Amino acid transport and metabolism in mycobacteria: Cloning, interruption, and characterization of an L-arginine/gamma-aminobutyric acid permease inMycobacterium bovis BCG

Genes encoding L-arginine biosynthetic and transport proteins have been sho wn in a number of pathogenic organisms to be important for metabolism withi n the host. In this study we describe the cloning of a gene (Rv0522) encodi ng an amino acid transporter from Mycobacterium bovis BCG and the effects o f its deletion on L-arginine transport and metabolism. The Rv0522 gene of B CG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mut ant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented wit h the wild-type gene in single copy. Transport analysis of these strains wa s conducted using C-14-labeled substrates. Greatly reduced uptake of L-argi nine and gamma-aminobutyric acid (GABA) but not of lysine, ornithine, proli ne, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved fo r 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-[C-14]arginine uptake by th e mutant but not the wild-type strain increased strongly. Exogenous L-argin ine but not GABA, lysine, ornithine, or alanine was shown to be toxic at co ncentrations of 20 mM and above to wild-type cells growing in optimal carbo n and nitrogen sources such as glycerol and ammonium, L-Arginine supplied i n the form of dipeptides showed no toxicity at concentrations as high as 30 mM, Finally, the permease mutant strain showed no defect in survival in un activated cultured murine macrophages compared with wild-type BCG.