Fullertubes inhibit mycobacterial viability and prevent biofilm formation by disrupting the cell wall.

Mycobacterium tuberculosis and nontuberculous mycobacteria such as Mycobacterium abscessus cause diseases that are becoming increasingly difficult to treat due to emerging antibiotic resistance. The development of new antimicrobial molecules is vital for combating these pathogens. Carbon nanomaterials (CNMs) are a class of carbon-containing nanoparticles with promising antimicrobial effects. Fullertubes (C90 ) are novel carbon allotropes with a structure unique among CNMs. The effects of fullertubes on any living cell have not been studied. In this study, we demonstrate that pristine fullertube dispersions show antimicrobial effects on Mycobacterium smegmatis and M. abscessus. Using scanning electron microscopy, light microscopy, and molecular probes, we investigated the effects of these CNMs on mycobacterial cell viability, cellular integrity, and biofilm formation. C90 fullertubes at 1 µM inhibited mycobacterial viability by 97%. Scanning electron microscopy revealed that the cell wall structure of M. smegmatis and M. abscessus was severely damaged within 24 h of exposure to fullertubes. Additionally, exposure to fullertubes nearly abrogated the acid-fast staining property of M. smegmatis. Using SYTO-9 and propidium iodide, we show that exposure to the novel fullertubes compromises the integrity of the mycobacterial cell. We also show that the permeability of the mycobacterial cell wall was increased after exposure to fullertubes from our assays utilizing the molecular probe dichlorofluorescein and ethidium bromide transport. C90 fullertubes at 0.37 µM and C60 fullerenes at 0.56 µM inhibited pellicle biofilm formation by 70% and 90%, respectively. This is the first report on the antimycobacterial activities of fullertubes and fullerenes.

Stress-induced non-replicating Mycobacterium smegmatis incorporates exogenous fatty acids into glycopeptidolipids.

Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is critically linked to biofilm formation by NTM which also includes opportunistic pathogens such as Mycobacterium abscessus. Although GPLs have been investigated in many earlier studies, the biosynthesis of GPLs using exogenous fatty acids in M. smegmatis subjected to stresses encountered by mycobacteria during infection of the human body has not been studied. Therefore, we subjected M. smegmatis to different combinations of the three stresses of hypoxia, acidic pH and nutrient starvation and report here that the metabolic incorporation of radiolabeled long-chain fatty acids into alkali-stable GPLs was significantly increased under these stress conditions. Endogenously synthesized fatty acids were not preferred for GPL biosynthesis by M. smegmatis subjected to the triple stress combination. Our observations indicate that GPLs may play important roles in cell surface modifications associated with the non-replicating state of M. smegmatis. Our experimental model reported here would be useful in the further study of GPL biosynthesis from exogenous fatty acid sources in M. smegmatis subjected to hypoxia, nutrient starvation and acidic stress conditions and help in the screening of candidate drugs that target this biochemical pathway in pathogenic NTM.

The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function.

Rv2477c is an antibiotic-sensitive manganese-dependent ABC-F ATPase in Mycobacterium tuberculosis.

The Rv2477c protein of Mycobacterium tuberculosis (Mtb) belongs to the ATP-binding cassette (ABC) subfamily F that contains proteins with tandem nucleotide-binding domains but lacking transmembrane domains. ABC-F subfamily proteins have been implicated in diverse cellular processes such as translation, antibiotic resistance, cell growth and nutrient sensing. In order to investigate the biochemical characteristics of Rv2477c, we expressed it in Escherichia coli, purified it and characterized its enzymatic functions. We show that Rv2477c displays strong ATPase activity (Vmax = 45.5 nmol/mg/min; Km = 90.5 µM) that is sensitive to orthovanadate. The ATPase activity was maximal in the presence of Mn2+ at pH 5.2. The Rv2477c protein was also able to hydrolyze GTP, TTP and CTP but at lower rates. Glutamate to glutamine substitutions at amino acid residues 185 and 468 in the two Walker B motifs of Rv2477c severely inhibited its ATPase activity. The antibiotics tetracycline and erythromycin, which target protein translation, were able to inhibit the ATPase activity of Rv2477c. We postulate that Rv2477c could be involved in mycobacterial protein translation and in resistance to tetracyclines and macrolides. This is the first report of the biochemical characterization of an ABC-F subfamily protein in Mtb.

The mycobacterial Rv1551 glycerol-3-phosphate acyltransferase enhances phospholipid biosynthesis in cell lysates of Escherichia coli.

Latent tuberculosis is caused by dormant Mycobacterium tuberculosis (Mtb) that is phenotypically tolerant to antibiotics. Dormant Mtb accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. The Rv1551 (PlsB1) gene is annotated as a putative glycerol-3-phosphate acyltransferase (GPAT) in the Mtb genome. GPAT catalyzes the first step of the glycerophospholipid biosynthetic pathway that synthesizes the lipid precursors for triacylglycerol biosynthesis. Although triacylglycerol biosynthesis is associated with Mtb dormancy, the functionality of the Rv1551 acyltransferase has not been investigated. We cloned the open reading frame of the Rv1551 acyltransferase and expressed it in Escherichia coli to study its function. We observed that E. coli cell lysates expressing Rv1551 displayed increased synthesis of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin from radiolabeled glycerol-3-phosphate and fatty acyl-coenzyme A precursors. When cultured in medium supplemented with long-chain fatty acids, E. coli expressing Rv1551 exhibited significantly higher viable cell counts during the exponential and stationary phases. These results suggest that Rv1551 displays function as a GPAT by enhancing the synthesis of phospholipids from exogenously provided fatty acids in E. coli cell lysates. This is the first report showing that Rv1551 is a functional GPAT that catalyzes the initial step of glycerophospholipid biosynthesis in the mycobacterial cell.

The perilipin-like PPE15 protein in Mycobacterium tuberculosis is required for triacylglycerol accumulation under dormancy-inducing conditions.

Mycobacterium tuberculosis (Mtb) causes latent tuberculosis infection in one-third of the world population and remains quiescent in the human body for decades. The dormant pathogen accumulates lipid droplets containing triacylglycerol (TAG). In mammals, perilipin regulates lipid droplet homeostasis but no such protein has been identified in Mtb. We identified an Mtb protein (PPE15) that showed weak amino acid sequence identities with mammalian perilipin-1 and was upregulated in Mtb dormancy. We generated a ppe15 gene-disrupted mutant of Mtb and examined its ability to metabolically incorporate radiolabeled oleic acid into TAG, accumulate lipid droplets containing TAG and develop phenotypic tolerance to rifampicin in two in vitro models of dormancy including a three-dimensional human granuloma model. The mutant showed a significant decrease in the biosynthesis and accumulation of lipid droplets containing TAG and in its tolerance of rifampicin. Complementation of the mutant with a wild-type copy of the ppe15 gene restored the lost phenotypes. We designate PPE15 as mycobacterial perilipin-1 (MPER1). Our findings suggest that the MPER1 protein plays a critical role in the homeostasis of TAG -containing lipid droplets in Mtb and influences the entry of the pathogen into a dormant state.

An acyl-CoA synthetase in Mycobacterium tuberculosis involved in triacylglycerol accumulation during dormancy.

Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.

Expression of fungal cutinase and swollenin in tobacco chloroplasts reveals novel enzyme functions and/or substrates.

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

Wax ester synthesis is required for Mycobacterium tuberculosis to enter in vitro dormancy.

Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of( 14)C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of (14)C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages.

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

A novel in vitro multiple-stress dormancy model for Mycobacterium tuberculosis generates a lipid-loaded, drug-tolerant, dormant pathogen.

BACKGROUND: Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen. METHODOLOGY/PRINCIPAL FINDINGS: We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO(2) (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells. CONCLUSIONS/SIGNIFICANCE: The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

AccD6, a member of the Fas II locus, is a functional carboxyltransferase subunit of the acyl-coenzyme A carboxylase in Mycobacterium tuberculosis.

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (alpha3) and AccD6 (beta6) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the alpha3-beta6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the alpha3-beta6 complex was inhibited by the epsilon subunit. The alpha3-beta6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the beta6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.

Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress.

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C(26) was a major fatty acid in this TG, indicating that the TGS1 gene product uses C(26) fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C(26 : 0)-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis.

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K(m) of 7.57 mM and V(max) of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.

Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent acyl-CoA Carboxylase in Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa alpha3-subunit (AccA3, Rv3285), the 59-kDa beta5-subunit (AccD5, Rv3280), and the 56-kDa beta4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the beta5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered epsilon-subunits of Streptomyces coelicolor, suggesting that it might be an epsilon-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted alpha3-beta5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the epsilon-subunit stimulated the carboxylation by 3.2- and 6.3-fold, respectively. The alpha3-beta4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This epsilon-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of epsilon-protein was highest during the log phase and decreased during the stationary phase.

Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture.

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.