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

Abstract

ABSTRACT 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.