Bioorthogonal Monomycolate of Trehalose Disclosed the -Mycoloylation of Mycoloyltransferases and Other Cell Envelope Proteins in .

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism through the action of mycoloyltransferase MytC.

Cg1246, a new player in mycolic acid biosynthesis in .

Mycolic acids are key components of the complex cell envelope of . These fatty acids, conjugated to trehalose or to arabinogalactan form the backbone of the mycomembrane. While mycolic acids are essential to the survival of some species, such as , their absence is not lethal for which has been extensively used as a model to depict their biosynthesis.

Synthesis of chemical tools to label the mycomembrane of corynebacteria using modified iron(III) chloride-mediated protection of trehalose.

Trehalose-based probes are useful tools that allow the detection of the mycomembrane of mycobacteria through the metabolic labeling approach. Trehalose analogues conjugated to fluorescent probes can be used, and other probes are functionalized with a bioorthogonal chemical reporter for a two-step labeling approach. The synthesis of such trehalose-based probes mainly relies on the desymmetrization of natural trehalose using a large number of regioselective protection-deprotection steps to differentiate the eight hydroxyl groups.

S and T mannosylation sites of LppX are not essential for its activity in phthiocerol dimycocerosates localization at the surface of Mycobacterium tuberculosis.

LppX is an important virulence factor essential for surface localization of phthiocerol dimycocerosates (DIM) in Mycobacterium tuberculosis. Based on Concanavalin A recognition, M. tuberculosis LppX (LppX-tb) was initially proposed to be glycosylated in M.

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in .

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys residue.

A Method for Producing Highly Pure Magnetosomes in Large Quantity for Medical Applications Using MSR-1 Magnetotactic Bacteria Amplified in Minimal Growth Media.

We report the synthesis in large quantity of highly pure magnetosomes for medical applications. For that, magnetosomes are produced by MSR-1 magnetotactic bacteria using minimal growth media devoid of uncharacterized and toxic products prohibited by pharmaceutical regulation, i.e.

The C-terminal domain of Corynebacterium glutamicum mycoloyltransferase A is composed of five repeated motifs involved in cell wall binding and stability.

The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N-terminal domain with esterase activity and a C-terminal extension containing a conserved repeated Leu-Gly-Phe-Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function.

Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium.

Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales.

Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane.

Independent domain assembly in a trapped folding intermediate of multimeric outer membrane secretins.

The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate.

Mobility and temperature resistance of trehalose mycolates as key characteristics of the outer membrane of Mycobacterium tuberculosis.

Trehalose mycolates are fundamental characteristics of the outer membrane (mycomembrane) of Mycobacterium tuberculosis and they are supposed to play a key role in the low permeability and high resistance of mycobacteria to many antibiotics; however, still, the molecular characteristics making mycolates so effective in their biological function are not fully understood. This work aims to investigate by quasi-elastic neutron scattering the diffusive dynamical properties of trehalose mycolates in water mixtures as a function of temperature, energy and exchanged wavevector Q in order to elucidate the dynamics-function relation in the mycomembrane. A comparison with lecithin lipids in water mixtures is performed since they are considered among the most rigid and resistant lipids.

Bacterial secretins form constitutively open pores akin to general porins.

Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD.

Identification of a mycoloyl transferase selectively involved in O-acylation of polypeptides in Corynebacteriales.

We have previously described the posttranslational modification of pore-forming small proteins of Corynebacterium by mycolic acid, a very-long-chain α-alkyl and β-hydroxy fatty acid. Using a combination of chemical analyses and mass spectrometry, we identified the mycoloyl transferase (Myt) that catalyzes the transfer of the fatty acid residue to yield O-acylated polypeptides. Inactivation of corynomycoloyl transferase C (cg0413 [Corynebacterium glutamicum mytC {CgmytC}]), one of the six Cgmyt genes of C.

The ppm operon is essential for acylation and glycosylation of lipoproteins in Corynebacterium glutamicum.

Background: Due to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M.

Biochemical disclosure of the mycolate outer membrane of Corynebacterium glutamicum.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function.

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL MscL does not need the insertase YidC for insertion in vitro.

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli.

O-mycoloylated proteins from Corynebacterium: an unprecedented post-translational modification in bacteria.

O-acylation of proteins was known only in a few eukaryotic proteins but never in bacteria. We demonstrate, using a combination of protein chemistry and mass spectrometry, the occurrence of three O-acylated polypeptides in Corynebacterium glutamicum, PorA, PorH, and an unknown small protein. The three polypeptides are O-substituted by mycolic acids, long chain alpha-alkyl and beta-hydroxy fatty acids specifically produced by members of the Corynebacterineae suborder.

A deficiency in arabinogalactan biosynthesis affects Corynebacterium glutamicum mycolate outer membrane stability.

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane.

Identification of a stress-induced factor of Corynebacterineae that is involved in the regulation of the outer membrane lipid composition.

Corynebacterineae are gram-positive bacteria that possess a true outer membrane composed of mycolic acids and other lipids. Little is known concerning the modulation of mycolic acid composition and content in response to changes in the bacterial environment, especially temperature variations. To address this question, we investigated the function of the Rv3802c gene, a gene conserved in Corynebacterineae and located within a gene cluster involved in mycolic acid biosynthesis.

In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD.

Synthesis of the Klebsiella oxytoca outer membrane secretin PulD, or its membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers.

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin.

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes.

Secretins take shape.

Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins.

Structural insights into the secretin PulD and its trypsin-resistant core.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc.