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 microg 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 microg/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.