title: Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis.
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.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States.
authors.forename: Darrell D
authors.initials: DD
authors.lastname: Marshall
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Halouska
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
authors.forename: Denise K
authors.initials: DK
authors.lastname: Zinniel
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fenton
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
authors.forename: Katie
authors.initials: K
authors.lastname: Kenealy
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
authors.forename: Harpreet K
authors.initials: HK
authors.lastname: Chahal
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.
authors.forename: Govardhan
authors.initials: G
authors.lastname: Rathnaiah
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln , Lincoln, Nebraska 68583-0905, United States.|Center for Redox Biology, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0662, United States.
authors.forename: Raúl G
authors.initials: RG
authors.lastname: Barletta
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0304, United States.|Center for Redox Biology, University of Nebraska-Lincoln , Lincoln, Nebraska 68588-0662, United States.
authors.forename: Robert
authors.identifier: 0000-0001-9948-6837
authors.initials: R
authors.lastname: Powers
mesh_terms: D000409:Alanine / Q000096:biosynthesis* / Q000378:metabolism; D000411:Alanine Racemase / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis; D000637:Transaminases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D000637:Transaminases; D000411:Alanine Racemase; D000409:Alanine
keywords: Mycobacterium smegmatis; Mycobacterium tuberculosis; NMR metabolomics; alanine racemase; d-alanine biosynthesis