Acylation of glycerolipids in mycobacteria.

We report on the existence of two phosphatidic acid biosynthetic pathways in mycobacteria, a classical one wherein the acylation of the sn-1 position of glycerol-3-phosphate (G3P) precedes that of sn-2 and another wherein acylations proceed in the reverse order. Two unique acyltransferases, PlsM and PlsB2, participate in both pathways and hold the key to the unusual positional distribution of acyl chains typifying mycobacterial glycerolipids wherein unsaturated substituents principally esterify position sn-1 and palmitoyl principally occupies position sn-2. While PlsM selectively transfers a palmitoyl chain to the sn-2 position of G3P and sn-1-lysophosphatidic acid (LPA), PlsB2 preferentially transfers a stearoyl or oleoyl chain to the sn-1 position of G3P and an oleyl chain to sn-2-LPA.

Toxic effect and inability of L-homoserine to be a nitrogen source for growth of resolved by a combination of evolution engineering and omics analyses.

L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid.

Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis.

Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo.

Discovery of a novel dehydratase of the fatty acid synthase type II critical for ketomycolic acid biosynthesis and virulence of Mycobacterium tuberculosis.

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadD (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs.

Publisher Correction: Structural insights into chaperone addiction of toxin-antitoxin systems.

The original version of this Article contained errors in Figures 1 and 4. In Fig. 1b, the Mtb-SecB sequence was displayed incorrectly.

Structural insights into chaperone addiction of toxin-antitoxin systems.

SecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition.

Directed evolution of SecB chaperones toward toxin-antitoxin systems.

SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin.

Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases.

Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C-C fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates.

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism.

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo.

Chaperone addiction of toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems, in which a labile antitoxin binds and inhibits the toxin, can promote adaptation and persistence by modulating bacterial growth in response to stress. Some atypical TA systems, known as tripartite toxin-antitoxin-chaperone (TAC) modules, include a molecular chaperone that facilitates folding and protects the antitoxin from degradation. Here we use a TAC module from Mycobacterium tuberculosis as a model to investigate the molecular mechanisms by which classical TAs can become 'chaperone-addicted'.

The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis.

Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M.

The changes in mycolic acid structures caused by hadC mutation have a dramatic effect on the virulence of Mycobacterium tuberculosis.

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M.

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system.

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids.

Revisiting the assignment of Rv0241c to fatty acid synthase type II of Mycobacterium tuberculosis.

The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-II(Mt)) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-II(Mt) cycles.

The dual function of the Mycobacterium tuberculosis FadD32 required for mycolic acid biosynthesis.

Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids.