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

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 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.

FIG. 1.

Specific activities of d-alanine racemase and LDH in M. smegmatis cell extracts. Mean specific activities (in micromoles) of substrate consumed minute⁻¹ milligram⁻¹, of d-alanine racemase (solid bars) and LDH (striped bars) from wild-type and mutant strains were determined in cell extracts prepared as described in Materials and Methods. Cells were grown to exponential phase in medium with or without d-alanine (50 mM) as indicated at the bottom of the figure. Extracts were prepared from three independent cultures for each strain and medium and assayed in triplicate. Combined extracts of mc²155 and TAM23 (ca. 1:24 [wt/wt] protein mixture ratio, with TAM23 extract as the predominant component) were also assayed in triplicate. A mock assay was also carried out with bovine serum albumin (BSA) in place of equivalent amounts of the corresponding cell extracts. N.D., not determined. Error bars, standard deviations.

FIG. 2.

Bactericidal action of DCS on M. smegmatis wild-type and alr mutant strains. Cells were grown in MADC broth without d-alanine to an OD₆₀₀ of ca. 0.4. At this time (time zero), cultures for each strain were split in two, and DCS was added to one of these subcultures at a concentration of 50 times the MIC for the corresponding strain (see Materials and Methods). ODs (A) and CFU per milliliter (means ± standard deviations [error bars] of triplicate measurements) (B) were determined for mc²155 in the subcultures with (open circles) or without (closed circles) DCS. Identical measurements were performed for the corresponding subcultures of TAM23 with (open squares) or without (closed squares) DCS.

FIG. 3.

Uptake of d-alanine and DCS by M. smegmatis wild-type and alr mutant strains. Uptake assays for DCS (A) or d-alanine (B) for strains mc²155 (closed circles) and TAM23 (open circles) were carried out in as described in Materials and Methods. Values are means ± standard deviations (error bars) of triplicate measurements.