Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains

Evidence of a New Pathway of d -Alanine Biosynthesis

Darrell D. Marshall, Steven Halouska, Denise K. Zinniel, Robert J. Fenton, Katie Kenealy, Harpreet K. Chahal, Govardhan Rathnaiah, Raul G Barletta, Robert Powers

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc2155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc2155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.

Original languageEnglish (US)
Pages (from-to)1270-1279
Number of pages10
JournalJournal of proteome research
Volume16
Issue number3
DOIs
StatePublished - Mar 3 2017

Fingerprint

Mycobacterium smegmatis
Biosynthesis
Alanine
Alanine Racemase
Peptidoglycan
Metabolomics
Mycobacterium
Transaminases
Mycobacterium tuberculosis
Metabolism
Carbon
Switches
Nuclear magnetic resonance

Keywords

  • Mycobacterium smegmatis
  • Mycobacterium tuberculosis
  • NMR metabolomics
  • alanine racemase
  • d -alanine biosynthesis

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains : Evidence of a New Pathway of d -Alanine Biosynthesis. / Marshall, Darrell D.; Halouska, Steven; Zinniel, Denise K.; Fenton, Robert J.; Kenealy, Katie; Chahal, Harpreet K.; Rathnaiah, Govardhan; Barletta, Raul G; Powers, Robert.

In: Journal of proteome research, Vol. 16, No. 3, 03.03.2017, p. 1270-1279.

Research output: Contribution to journalArticle

Marshall, Darrell D. ; Halouska, Steven ; Zinniel, Denise K. ; Fenton, Robert J. ; Kenealy, Katie ; Chahal, Harpreet K. ; Rathnaiah, Govardhan ; Barletta, Raul G ; Powers, Robert. / Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains : Evidence of a New Pathway of d -Alanine Biosynthesis. In: Journal of proteome research. 2017 ; Vol. 16, No. 3. pp. 1270-1279.
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abstract = "In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc2155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc2155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.",
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