title: The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.
abstract: D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).
authors.affiliation: Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, U.P., India, Pin: 226031.
authors.forename: Kumar Sachin
authors.initials: KS
authors.lastname: Singh
authors.affiliation: Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, U.P., India, Pin: 226031.
authors.forename: Sudheer Kumar
authors.initials: SK
authors.lastname: Singh
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D007651:Keto Acids / Q000378:metabolism; D008292:Malate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D011773:Pyruvates / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D007651:Keto Acids; D011773:Pyruvates; D011994:Recombinant Proteins; C017092:bromopyruvate; D003605:D-Amino-Acid Oxidase; D008292:Malate Synthase