Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of an L-Arginine/gamma -Aminobutyric Acid Permease in Mycobacterium bovis BCG

Department of Microbiology and Molecular Genetics and New Jersey Medical School National Tuberculosis Center, UMDNJ/New Jersey Medical School, Newark, New Jersey 17103, USA.
Journal of Bacteriology (Impact Factor: 2.81). 03/2000; 182(4). DOI: 10.1128/JB.182.4.919-927.2000
Source: PubMed Central


Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism
within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to
therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene
interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis
of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain
compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in
a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type
cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in 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 unactivated cultured murine macrophages compared with wild-type BCG.

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    • "The arginine transporter is the only inner-membrane amino acid transporter from mycobacteria characterized genetically and by transport experiments [133]. It was found that the Rv0522 gene product from Mycobacterium bovis BCG is an L-arginine and also a -aminobutyric acid permease [133]. "
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