Assessment of metabolic changes in Mycobacterium smegmatis wild type and alr mutant strains: evidence for a new pathway of D-alanine biosynthesis.

Authors

Darrell D. Marshall, University of Nebraska - Lincoln
Steven Halouska, University of Nebraska - Lincoln
Denise K. Zinniel, University of Nebraska - LincolnFollow
Robert J. Fenton, University of Nebraska - LincolnFollow
Katie Kenealy, University of Nebraska - Lincoln
Harpreet K. Chahal, University of Nebraska - Lincoln
Govardhan Rathnaiah, University of Nebraska - Lincoln
Raul G. Barletta, University of Nebraska-LincolnFollow
Robert Powers, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

2017

Citation

J Proteome Res. 2017 March 03; 16(3): 1270–1279

Comments

HHS Public Access

doi:10.1021/acs.jproteome.6b00871

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.