A typical TLC plate with samples loaded in duplicate and lipid standards at the left and right edges of TLC plate is shown. becomes phenotypically tolerant to antibiotics, loses acid fastness and accumulates triacylglycerol (TAG)-containing lipid droplets for possible use as energy source during dormancy and reactivation[3][6]. The phenotypic drug tolerance of dormantMtbprolongs the treatment regimen required to cure TB, results in poor compliance and contributes to the emergence of multi-drug resistance[2]. Host-derived fatty acids are critically important as energy source duringMtbdormancy[4],[7],[8]but the metabolic pathways utilized byMtbfor activating host fatty acids destined for bacterial TAG accumulation as it enters dormancy remain unexplored. Fatty acid transport proteins (FATPs) appear to be critical players in the transport Ferrostatin-1 (Fer-1) of fatty acids across cell membranes[9][11]. FATPs are integral membrane proteins with two conserved domains: an ATP/AMP binding domain that is conserved Ferrostatin-1 (Fer-1) from bacteria to man and a fatty acid binding domain unique to FATPs[12],[13]. The long-chain acyl coenzyme A synthetase (ACSL) activity of the FATP, which mediates the activation of fatty acids, probably plays an important role in regulating the rate of fatty acid uptake and in channeling the imported Mmp11 fatty acids between various metabolic processes within the cell[13],[14]. Thus ACSLs are thought to act as metabolic sinks that drive fatty acid transport across membranes[15]. Furthermore, ACSLs are thought to be involved in the sequestration of fatty acids into distinct pools within the cell for diverse metabolic purposes[13],[16]. Mammalian FATP1 has been demonstrated to channel exogenous fatty acids into triacylglycerol biosynthesis[17]. Thus, it is possible that a mycobacterial ACSL may play a critical role in the sequestration of fatty acids for TAG synthesis withinMtb. We postulate that mycobacterial ACSL proteins may be involved in utilizing host lipid-derived fatty acids for the synthesis of TAG insideMtbduring dormancy. TheMtbgenome encodes 34 fatty acyl-CoA ligase [FACL]-like gene products and some of them synthesize acyl-AMP for use by polyketide synthases[18]. However, Rv1206 (FadD6/FACL6) is the only member of the family that belongs to a family of fatty acid transporters conserved across several species[12]. In this study, we show that the purified FACL6 protein displays acyl-coenzyme A synthetase activity and is able to indirectly stimulate fatty acid uptake inE. colicells. We found that the FACL6 protein level was higher inMtbcells in a dormant state than inMtbcells in exponential growth phase. We constructed anMtbmutant lacking FACL6 and found that the deletion of FACL6 resulted in a significant decrease in the accumulation of intracellular TAG inMtbunder dormancy-inducing conditionsin vitro. Complementation partially recovered the lost phenotype. == Materials and Methods == == Chemicals and reagents == [14C]Oleic acid (56 Ci mol1) was purchased from American Radiolabeled Chemicals, Inc. All other chemicals and media were purchased from Sigma (St. Louis, MO) and Thermo Fisher Scientific Inc. Nucleotide primers were synthesized by Integrated DNA Technologies, (Coralville, IA). == Bacterial strains and growth conditions == M. tuberculosis(Mtb) H37Rv,facl6(Rv1206) deletion mutant ofMtb(d-FACL6) andfacl6-complemented strain of d-FACL6 (C-FACL6) were grown in Middlebrook 7H9 broth (supplemented with 0.05% Tween 80, 10% oleic acid-albumin-dextrose-catalase enrichment, and 0.2% glycerol) up to OD600nm of 0.6. The strains were stored at 80C as stock cultures with 15% (v/v) glycerol. The d-FACL6 and C-FACL6 strains were grown in media containing hygromycin (Hyg, 75 g ml1) and Hyg (75 g ml1) plus kanamycin (Kan, 30 g ml1) antibiotics respectively. TheMtbstrains were subjected to multiple-stress conditions as we have described previously[19]. Briefly,Mtbcultures in log-phase were exposed to a Ferrostatin-1 (Fer-1) combination of stresses that are thought to be experienced by the pathogen in the human body (low [5%] oxygen, high [10%] carbon dioxide, low pH [5.0] and nutrient starvation [10% Dubos medium]). Luria-Bertani broth was used for allE. colicultures, and when required, antibiotics were added to the culture at the following concentrations: kanamycin, 50 g ml1; carbenicillin, 50 g ml1. == Multiple-sequence alignment == The amino acid sequence ofM. tuberculosisFACL6 (Rv1206; Gene ID: 13319785) was aligned with human FATP1 (HsFATP1; Gene ID: 376497), human FATP4 (HsFATP4; Gene ID: 10999) and yeast FATP (ScFAT; Gene ID: 852329) amino acid sequences using the ClustalW2 multiple sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The alignment was shaded using the BoxShade program (http://www.ch.embnet.org/software/BOX_form.html) and manually adjusted for optimal alignment. == Cloning and expression of recombinant FACL6 == Thefacl6 (Rv1206)ORF corresponding to the.