Mycobacterium smegmatis d-Alanine Racemase Mutants Are Not Dependent on d-Alanine for Growth

Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the d-alanine racemase gene (alrA), which is involved in the synthesis of d-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM d-alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without d-alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of d-alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for d-alanine, suggesting the existence of a new pathway of d-alanine biosynthesis in M. smegmatis. Biochemical analysis demonstrated the absence of any detectable d-alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d-cycloserine. The MIC of d-cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d-cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of d-alanine racemase as a target of d-cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.