title: 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.
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583-0905, USA.
authors.forename: Ofelia
authors.initials: O
authors.lastname: Chacon
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Feng
authors.forename: N Beth
authors.initials: NB
authors.lastname: Harris
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Cáceres
authors.forename: L Garry
authors.initials: LG
authors.lastname: Adams
authors.forename: Raúl G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism*; D000904:Antibiotics, Antitubercular / Q000494:pharmacology; D003523:Cycloserine / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D010641:Phenotype
chemical_list: D000904:Antibiotics, Antitubercular; D003523:Cycloserine; D000411:Alanine Racemase; D000409:Alanine