The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the ␤-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase ␣-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and ␣-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.

The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(⌬PE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY mar , in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY mar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY mar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(⌬PE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(⌬PE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(⌬PE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the ␤-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.Mycobacterium tuberculosis has a unique cell wall structure that accounts for the ability of the bacterium to grow in several contrasting environments and which is responsible for its low membrane permeability, contributing to its resistance to common chemotherapeutic agents (1). The cell wall has been implicated as a direct modulator of interactions between mycobacteria and the environment (2). This envelope, characterized by its high lipid content, comprises an inner membrane barrier composed of mycolic acids anchored to arabinogalactan, linked to peptidoglycan. Mycolic acids are a hallmark of the mycobacterial waxy coat: they represent key virulence factors required for intracellular survival (3, 4) and contribute to the physiopathology of tuberculosis. They consist of very long chains of ␣-branched ␤-hydroxy fatty acids (C 60 -C 90 ), whose biosynthesis is controlled by two elongation systems, the eukaryotic-type fatty acid synthase (FAS-I) 3 and the prokaryotic-like FAS-II (5, 6). FAS-I consists of a single multifunctional polypeptide, catalyzing de novo synthesis of medium length acyl-CoA chains (C 16 -C 26 ), whereas FAS-II comprises several distinct enzymes. It catalyzes similar types of reactions to FAS-I, but functions on acyl carrier protein (AcpM)-bound chains and is incapable of de novo synthesis. The initial substrates of FAS-II are ␤-ketoacylAcpM resulting from the condensation by mtFabH of the acylCoA products of FAS-I with malonyl-AcpM (7, 8). Following reduction by MabA, elimination of water by a yet unidentified dehydratase, and reduction by the enoyl-AcpM reductase InhA, the ␤-ketoacyl-AcpM synthases KasA and KasB catalyze further...

Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, L-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8?3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [13 C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [ 13 C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

Mycolic acids are hallmark features of the Mycobacterium tuberculosis cell wall. They are synthesized by the condensation of two fatty acids, a C 56 -64 -meromycolyl chain and a C 24 -26 -fatty acyl chain. Meromycolates are produced via the combination of type I and type II fatty acid synthases (FAS-I and FAS-II). The ␤-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-ACP. Because mtFabH represents a potential regulatory key point of the mycolic acid pathway, we investigated the hypothesis that phosphorylation of mtFabH controls its activity. Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently emerged as a major physiological mechanism of regulation in prokaryotes. We demonstrate here that mtFabH was efficiently phosphorylated in vitro by several mycobacterial STPKs, particularly by PknF and PknA, as well as in vivo in mycobacteria. Analysis of the phosphoamino acid content indicated that mtFabH was phosphorylated exclusively on threonine residues. Mass spectrometry analyses using liquid chromatography-electrospray ionization/tandem mass spectrometry identified Thr 45 as the unique phosphoacceptor. This was further supported by complete loss of PknF-or PknA-dependent phosphorylation of a mtFabH mutant. Mapping Thr 45 on the crystal structure of mtFabH illustrates that this residue is located at the entrance of the substrate channel, suggesting that the phosphate group may alter accessibility of the substrate and thus affect mtFabH enzymatic activity. A T45D mutant of mtFabH, designed to mimic constitutive phosphorylation, exhibited markedly decreased transacylation, malonyl-AcpM decarboxylation, and condensing activities compared with the wild-type protein or the T45A mutant. Together, these findings not only represent the first demonstration of phosphorylation of a ␤-ketoacyl-ACP synthase III enzyme but also indicate that phosphorylation of mtFabH inhibits its enzymatic activity, which may have important consequences in regulating mycolic acid biosynthesis.Within the infected host, Mycobacterium tuberculosis encounters numerous environmental conditions and induces or represses a number of genes for a quick adjustment to new conditions. The infection process of M. tuberculosis involves cross-talk of signals between the host and the bacterium, resulting in reprogramming of the host signaling network. Protein phosphorylation/dephosphorylation represent a central mechanism for distribution of signals to various parts of the cell, regulating growth, differentiation, mobility, and survival (1). In mycobacteria, a common signal transduction pathway is transmitted through membrane-embedded sensor kinases, enabling the pathogen to modify itself for survival in this hostile environment.Protein kinases can be classified into two families based on their similarities and enzymatic specification: (i) the histidine kinase superfamily, which relies on autophosphorylation of a conserved histidine residue ...

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall and are essential for survival. While most components of the fatty acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain a-alkyl, b-hydroxy fatty acids have been identified, the gene encoding the b-hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the M. tuberculosis gene Rv0636 as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that MSMEG1341, the Mycobacterium smegmatis homologue of Rv0636, is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of MSMEG1341. Growth of the conditional mutant in the absence of acetamide resulted in loss of mycolic acid biosynthesis and eventually loss of viability due to cell lysis. Null MSMEG1341 mutants could also be generated in a M. smegmatis strain containing an integrated copy of Rv0636, indicating that Rv0636 was the functional counterpart of MSMEG1341 in M. tuberculosis. Our results demonstrate that MSMEG1341 is an essential gene involved in mycolic acid biosynthesis and encodes the FAS-II b-hydroxyacyl-ACP dehydratase.