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, Raúl G Barletta, Robert Powers
Author Information
Darrell D Marshall: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States.
Steven Halouska: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States.
Denise K Zinniel: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Robert J Fenton: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Katie Kenealy: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Harpreet K Chahal: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Govardhan Rathnaiah: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Raúl G Barletta: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
Robert Powers: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States. ORCID
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. smegmatisAlr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc155 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.
