Mycobacterium smegmatis D-alanine racemase mutants are not dependent on D-alanine for growth

O. Chacon, Z. Feng, N. B. Harris, N. E. Cáceres, L. G. Adams, Raul G Barletta

Research output: Contribution to journalArticle

45 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)47-54
Number of pages8
JournalAntimicrobial Agents and Chemotherapy
Volume46
Issue number1
DOIs
StatePublished - Jan 15 2002

Fingerprint

Alanine Racemase
Mycobacterium smegmatis
Alanine
Cycloserine
Growth
Peptidoglycan
Mycobacterium
Cell Wall
Kanamycin Resistance
Kanamycin
Homologous Recombination
Drug Delivery Systems
Pharmaceutical Preparations
Permeability
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Mycobacterium smegmatis D-alanine racemase mutants are not dependent on D-alanine for growth. / Chacon, O.; Feng, Z.; Harris, N. B.; Cáceres, N. E.; Adams, L. G.; Barletta, Raul G.

In: Antimicrobial Agents and Chemotherapy, Vol. 46, No. 1, 15.01.2002, p. 47-54.

Research output: Contribution to journalArticle

Chacon, O. ; Feng, Z. ; Harris, N. B. ; Cáceres, N. E. ; Adams, L. G. ; Barletta, Raul G. / Mycobacterium smegmatis D-alanine racemase mutants are not dependent on D-alanine for growth. In: Antimicrobial Agents and Chemotherapy. 2002 ; Vol. 46, No. 1. pp. 47-54.
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