Cg1246, a new player in mycolic acid biosynthesis in Corynebacterium glutamicum.

Mycolic acids are key components of the complex cell envelope of Corynebacteriales. 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 Mycobacterium tuberculosis, their absence is not lethal for Corynebacterium glutamicum, which has been extensively used as a model to depict their biosynthesis. Mycolic acids are first synthesized on the cytoplasmic side of the inner membrane and transferred onto trehalose to give trehalose monomycolate (TMM). TMM is subsequently transported to the periplasm by dedicated transporters and used by mycoloyltransferase enzymes to synthesize all the other mycolate-containing compounds. Using a random transposition mutagenesis, we recently identified a new uncharacterized protein (Cg1246) involved in mycolic acid metabolism. Cg1246 belongs to the DUF402 protein family that contains some previously characterized nucleoside phosphatases. In this study, we performed a functional and structural characterization of Cg1246. We showed that absence of the protein led to a significant reduction in the pool of TMM in C. glutamicum, resulting in a decrease in all other mycolate-containing compounds. We found that, in vitro, Cg1246 has phosphatase activity on organic pyrophosphate substrates but is most likely not a nucleoside phosphatase. Using a computational approach, we identified important residues for phosphatase activity and constructed the corresponding variants in C. glutamicum. Surprisingly complementation with these non-functional proteins fully restored the defect in TMM of the Δcg1246 mutant strain, suggesting that in vivo, the phosphatase activity is not involved in mycolic acid 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. Herein, in order to avoid these time-consuming steps, we reinvestigated our previously reported tandem protocol mediated by FeCl3·6H2O, with the aim of modifying the ratio of the products to allow the challenging desymmetrization of the C2-symmetrical disaccharide trehalose. We demonstrate the usefulness of this method in providing easy access to trehalose analogues with a bioorthogonal moiety or a fluorophore in C-2, and also present their use in a one-step and two-step labeling approach, either of which can be used to study the mycomembrane in live mycobacteria.

S16 and T18 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. tuberculosis and more recently this glycosylation was characterized by mass spectrometry analysis on LppX-tb expressed and purified from Corynebacterium glutamicum. Here, using this model organism and Mycobacterium smegmatis, we show that S16 and T18 residues of LppX-tb are indeed glycosylated with several hexoses units. Interestingly this glycosylation is strictly dependent on the mannosyl transferase PMT which, in M. tuberculosis, has been reported to be crucial for virulence. Using a site directed mutagenesis approach, we were able to show that the absence of S16 and T18 glycosylation does not alter phthiocerol dimycocerosates (DIM) localization in M. tuberculosis.

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

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+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

A Method for Producing Highly Pure Magnetosomes in Large Quantity for Medical Applications Using Magnetospirillum gryphiswaldense 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 Magnetospirillum gryphiswaldense magnetotactic bacteria using minimal growth media devoid of uncharacterized and toxic products prohibited by pharmaceutical regulation, i.e., yeast extract, heavy metals different from iron, and carcinogenic, mutagenic and reprotoxic agents. This method follows two steps, during which bacteria are first pre-amplified without producing magnetosomes and are then fed with an iron source to synthesize magnetosomes, yielding, after 50 h of growth, an equivalent OD565 of ~8 and 10 mg of magnetosomes in iron per liter of growth media. Compared with magnetosomes produced in non-minimal growth media, those particles have lower concentrations in metals other than iron. Very significant reduction or disappearance in magnetosome composition of zinc, manganese, barium, and aluminum are observed. This new synthesis method paves the way towards the production of magnetosomes for medical applications.

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. In this study, we determined the crystal structure of MytA and found that its C-terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N-terminal catalytic α/ß-hydrolase domain. The LGFP motifs are composed of a 4-stranded ß-fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand-binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C-terminal LGFP domain interacts with the cell wall peptidoglycan-arabinogalactan polymer. We also show that the C-terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope.

First access to a mycolic acid-based bioorthogonal reporter for the study of the mycomembrane and mycoloyltransferases in Corynebacteria.

In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a ß-hydroxylated fatty acid and an α-branched chain similar to those of the natural mycolic acid. We demonstrate its utility for the labeling of the mycomembrane of Corynebacteria as well as for the study of mycoloyltransferases.

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, ß-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. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

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. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

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. From the analysis of the data collected as a function of temperature, a lower temperature dependence of the mobility as well as a higher rigidity of trehalose mycolates in comparison with lecithin lipids are highlighted. The present findings provide detailed molecular information which allows to go ahead in the understanding at a molecular level of the resistance to stress and antibiotics by corynebacteria and mycobacteria.

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. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate "prepore" stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ.

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. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.

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 ß-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. glutamicum, specifically abolished the O-modification of the pore-forming proteins PorA and PorH, which is critical for their biological activity. Expectedly, complementation of the cg0413 mutant with either the wild-type gene or its orthologues from Corynebacterium diphtheriae and Rhodococcus, but not Nocardia, fully restored the O-acylation of the porins. Consistently, the three-dimensional structure of CgMytC showed the presence of a unique loop that is absent from enzymes that transfer mycoloyl residues onto both trehalose and the cell wall arabinogalactan. These data suggest the implication of this structure in the enzyme specificity for protein instead of carbohydrate.

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. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown. RESULTS: In order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum Δppm1 and Δppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated N-terminal peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the Δppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX O-glycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed. CONCLUSION: Together, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled.

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. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic ß-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.

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. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.

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. To date these acids were described only as esterifying trehalose and arabinogalactan, and less frequently glycerol, important components of the highly impermeable outer barrier of Corynebacterineae. We show that the post-translational mycoloylation of PorA occurs at Ser-15 and is necessary for the pore-forming activity of C. glutamicum.

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. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal beta(1 --> 2)-linked Araf residues. Here we show that Delta aftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a Delta aftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the Delta aftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum.

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. We showed that the Rv3802 ortholog is essential in Mycobacterium smegmatis, while its Corynebacterium glutamicum ortholog, NCgl2775, is not. We provided evidence that the NCgl2775 gene is transcriptionally induced under heat stress conditions, and while the corresponding protein has no detectable activity under normal growth conditions, the increase in its expression triggers an increase in mycolic acid biosynthesis concomitant with a decrease in phospholipid content. We demonstrated that these lipid modifications are part of a larger outer membrane remodeling that occurs in response to exposure to a moderately elevated temperature (42 degrees C). In addition to showing an increase in the ratio of saturated corynomycolates to unsaturated corynomycolates, our results strongly suggested that the balance between mycolic acids and phospholipids is modified inside the outer membrane following a heat challenge. Furthermore, we showed that these lipid modifications help the bacteria to protect against heat damage. The NCgl2775 protein and its orthologs thus appear to be a protein family that plays a role in the regulation of the outer membrane lipid composition of Corynebacterineae under stress conditions. We therefore propose to name this protein family the envelope lipids regulation factor (ElrF) family.

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. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal outer membrane secretin dodecamers. It was concluded that multimerization and membrane insertion are intrinsic properties of secretin PulD that are independent of a specific membrane environment or membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and membrane insertion of a large outer membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to outer membrane proteins closely related to PulD.