Honors Program


Date of this Version

Spring 3-12-2018

Document Type



Hoff, PL, Zinniel, DK, Raul, RG. (2018). Identification of D-alanine Transaminase in Mycobacterium smegmatis and Effects of Mutations in Peptidoglycan Enzymes on Canonical and Non-Canonical Tuberculosis Drugs. University of Nebraska - Lincoln

Mycobacteria, tuberculosis, peptidoglycan, D-cycloserine, L-cycloserine,

Beta-chloro-D-alanine, Beta-chloro-L-alanine, D-alanine transaminase, da


Copyright Philion L. Hoff 2018


Tuberculosis is a disease that is one of the leading ten causes of death worldwide and is believed to latently infect one-third of the human population. Tuberculosis is caused by Mycobacterium tuberculosis, which has become increasingly drug resistant. The model organism of M. tuberculosis is Mycobacterium smegmatis. Production of the mycobacterial cell wall is dependent on production of a molecule called peptidoglycan which requires different enantiomers of amino acids. In humans, D-amino acids are practically unavailable. In an effort to more effectively treat drug resistant tuberculosis, we investigated the complementary ability of a proposed mycobacterial enzyme (D-alanine transaminase) coded by Msmeg_5795 on deletion or insertion alr/murI or double mutant strains. We also investigated resistance or susceptibility of strains overexpressing or having null mutations in alr/murI to certain canonical and non-canonical inhibitory agents. We performed growth studies by creating different agar media plates supplemented with different combinations of amino acids or with different inhibitory agents. We then streaked or spotted live cells on these plates and imaged growth over a one or two week period. Growth was subjectively graded. We found evidence that supports the hypothesis that Msmeg_5795 does indeed code for a D-alanine transaminase-like enzyme and that this enzyme is likely multi-functional. Our results also suggest the existence of another D-alanine transaminase as well. We confirmed observations previously made regarding inhibitory agent resistance but found two non-canonical results: First, glutamate racemase (MurI) appears to be inhibited not only by Beta-chloro-D-alanine (BCDA) but also by D-cycloserine (DCS), and L-cycloserine (LCS). Previous research identified MurI as only being inhibited by BCDA. Second, alanine dehydrogenase (Ald) overproduction appears to impart susceptibility to LCS and have no effect on DCS. We confirmed previous research that identified the deletion of Ald as likely having resistance-imparting characteristics. Our non-canonical results indicate the need for further research.