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Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae*

  • Darryl A. Wesener
    Footnotes
    Affiliations
    Department of Biochemistry, Madison, Wisconsin 53706
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  • Matthew R. Levengood
    Footnotes
    Affiliations
    Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Laura L. Kiessling
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706. Tel.: 608-262-0541; Fax: 608-265-0764.
    Affiliations
    Department of Biochemistry, Madison, Wisconsin 53706

    Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants R01GM055984 and R01AI063596 (to L. L. K). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Both authors contributed equally to this work.
    2 Recipient of fellowships from the National Science Foundation Graduate Research Fellowship and the National Institutes of Health Chemistry-Biology Interface Training Program Fellowship (T32 GM008505), which is supported the University of Wisconsin-Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation, for fellowships.
    3 Supported by a National Institutes of Health Postdoctoral Fellowship (F32GM089219).
Open AccessPublished:December 30, 2016DOI:https://doi.org/10.1074/jbc.M116.759340
      The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis. In each species, the galactan is constructed from uridine 5′-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

      Introduction

      Mycobacterium tuberculosis and Corynebacterium diphtheriae, the etiological agents of tuberculosis and diphtheria, respectively, are notorious members of the bacterial suborder Corynebacterineae. This taxon includes other pathogenic bacterial species, including the causative agents of leprosy, nocardiosis, and buruli ulcers. One notable feature of Corynebacterineae is the cell envelope, which has a unique composition. The Corynebacterineae cell envelope contains mycolic acids appended to branched polymers of Araf (arabinan),
      The abbreviations used are: Araf
      arabinofuranose
      FP
      fluorescence polarization
      Galf
      galactofuranose
      Galp
      galactopyranose
      mAG
      mycolyl-arabinogalactan
      UGM
      uridine 5′-diphosphate galactopyranose mutase.
      which are linked to peptidoglycan through a linear polymer of Galf (galactan) (
      • Dover L.G.
      • Cerdeño-Tárraga A.M.
      • Pallen M.J.
      • Parkhill J.
      • Besra G.S.
      Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae.
      ,
      • Crick D.C.
      • Mahapatra S.
      • Brennan P.J.
      Biosynthesis of the arabinogalactan-peptidoglycan complex of Mycobacterium tuberculosis.
      ). The macromolecular structure that extends beyond the peptidoglycan is referred to as the mAG complex. Neither the mAG nor its individual components are present in host mammals. Moreover, in M. tuberculosis the mAG complex serves as a barrier to antitubercular drugs and can modulate the human immune response in favor of bacterial immune evasion (
      • Briken V.
      • Porcelli S.A.
      • Besra G.S.
      • Kremer L.
      Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response.
      ,
      • Angala S.K.
      • Belardinelli J.M.
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      • Wheat W.H.
      • Jackson M.
      The cell envelope glycoconjugates of Mycobacterium tuberculosis.
      ). A complete understanding of mAG assembly and variation could yield novel strategies for therapeutic intervention.
      Structural features of the mycobacterial and corynebacterial cell envelopes have been characterized (Fig. 1) (
      • Daffe M.
      • Brennan P.J.
      • McNeil M.
      Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses.
      • Besra G.S.
      • Khoo K.H.
      • McNeil M.R.
      • Dell A.
      • Morris H.R.
      • Brennan P.J.
      A new interpretation of the structure of the mycolyl-arabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fast-atom bombardment mass spectrometry and 1H nuclear magnetic resonance spectroscopy.
      ,
      • Puech V.
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      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ,
      • Jankute M.
      • Grover S.
      • Birch H.L.
      • Besra G.S.
      Genetics of mycobacterial arabinogalactan and lipoarabinomannan assembly.
      ,
      • Kaur D.
      • Guerin M.E.
      • Skovierová H.
      • Brennan P.J.
      • Jackson M.
      Chapter 2: Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis.
      ,
      • Bansal-Mutalik R.
      • Nikaido H.
      Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides.
      • Zuber B.
      • Chami M.
      • Houssin C.
      • Dubochet J.
      • Griffiths G.
      • Daffé M.
      Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.
      ). The galactan is anchored to the peptidoglycan at the C-6 position of peptidoglycan muramic acid. The linker includes a phosphodiester-linked 1-α-d-N-acetylglucosamine residue with an α-substituted l-rhamnopyranosyl residue at the 3 position (Fig. 2). The 4-position of rhamnose is substituted with the galactan, a polymer of alternating β(1–5)- and β(1–6)-linked β-d-Galf residues. The enzymes the mediate galactan biosynthesis in mycobacteria have been identified. Two galactofuranosyltransferases, GlfT1 and GlfT2, generate the galactan using UDP-Galf, the activated Galf sugar donor afforded by the action of UGM (
      • Weston A.
      • Stern R.J.
      • Lee R.E.
      • Nassau P.M.
      • Monsey D.
      • Martin S.L.
      • Scherman M.S.
      • Besra G.S.
      • Duncan K.
      • McNeil M.R.
      Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues.
      ,
      • Pan F.
      • Jackson M.
      • Ma Y.
      • McNeil M.
      Cell wall core galactofuran synthesis is essential for growth of mycobacteria.
      ). GlfT1 catalyzes the addition of two to three Galf residues to the C-4 hydroxyl of l-rhamnose (
      • Mikusová K.
      • Belánová M.
      • Korduláková J.
      • Honda K.
      • McNeil M.R.
      • Mahapatra S.
      • Crick D.C.
      • Brennan P.J.
      Identification of a novel galactosyltransferase involved in biosynthesis of the mycobacterial cell wall.
      ,
      • Martinez Farias M.A.
      • Kincaid V.A.
      • Annamalai V.R.
      • Kiessling L.L.
      Isoprenoid phosphonophosphates as glycosyltransferase acceptor substrates.
      ). The galactofuranosyl polymerase GlfT2 then promotes the sequential addition of the alternating β(1–5) and β(1–6) Galf linkages (
      • Mikusová K.
      • Yagi T.
      • Stern R.
      • McNeil M.R.
      • Besra G.S.
      • Crick D.C.
      • Brennan P.J.
      Biosynthesis of the galactan component of the mycobacterial cell wall.
      • Kremer L.
      • Dover L.G.
      • Morehouse C.
      • Hitchin P.
      • Everett M.
      • Morris H.R.
      • Dell A.
      • Brennan P.J.
      • McNeil M.R.
      • Flaherty C.
      • Duncan K.
      • Besra G.S.
      Galactan biosynthesis in Mycobacterium tuberculosis: identification of a bifunctional UDP-galactofuranosyltransferase.
      ,
      • Rose N.L.
      • Completo G.C.
      • Lin S.J.
      • McNeil M.
      • Palcic M.M.
      • Lowary T.L.
      Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). After galactan polymerization, 3 α-Araf residues are added to the C-5 hydroxyl groups of the 8th, 10th, and 12th Galf residues of the galactan (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ). Arabinofuranosyltransferases elaborate these residues to append a branched Araf polysaccharide. Subsequently, multiple terminal Araf residues are acylated to form long chain mycolic acid esters. Enzymes involved in mycolic acid biosynthesis or arabinan production are the targets of clinically used antitubercular drugs (
      • Horsburgh Jr, C.R.
      • Barry 3rd, C.E.
      • Lange C.
      Treatment of tuberculosis.
      ).
      Figure thumbnail gr1
      FIGURE 1Comparative models of the structure of the mAG complex. Schematic comparison of the mAG complex from M. tuberculosis and C. diphtheriae cell walls.
      Figure thumbnail gr2
      FIGURE 2Proposed enzymatic reactions in galactan biosynthesis in C. diphtheriae.
      Although mAG biosynthesis and structure is often studied in M. tuberculosis, Corynebacterium species have recently been used as models to understand mAG assembly. Their advantages include a decreased doubling time, reduced biosafety designation, and the availability of tools for genetic manipulation (
      • Krawczyk J.
      • Kohl T.A.
      • Goesmann A.
      • Kalinowski J.
      • Baumbach J.
      From Corynebacterium glutamicum to Mycobacterium tuberculosis: towards transfers of gene regulatory networks and integrated data analyses with MycoRegNet.
      • Seidel M.
      • Alderwick L.J.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis.
      ,
      • Portevin D.
      • De Sousa-D'Auria C.
      • Houssin C.
      • Grimaldi C.
      • Chami M.
      • Daffé M.
      • Guilhot C.
      A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.
      • Seidel M.
      • Alderwick L.J.
      • Birch H.L.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Identification of a novel arabinofuranosyltransferase AftB involved in a terminal step of cell wall arabinan biosynthesis in Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis.
      ). Although most mAG biosynthetic genes are essential in M. tuberculosis (
      • Sassetti C.M.
      • Boyd D.H.
      • Rubin E.J.
      Genes required for mycobacterial growth defined by high density mutagenesis.
      ), their deletion in C. glutamicum often yields slow growing but viable mutants. For example, C. glutamicum mutants lacking AftA were recently used to determine that this arabinofuranoysltransferase appends three α(1–5)–Araf residues to the galactan to initiate arabinan biosynthesis (
      • Alderwick L.J.
      • Seidel M.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.
      ). Another C. glutamicum mutant revealed Pks13 catalyzes the final step of mycolic acid biosynthesis in corynebacteria and mycobacteria (
      • Portevin D.
      • De Sousa-D'Auria C.
      • Houssin C.
      • Grimaldi C.
      • Chami M.
      • Daffé M.
      • Guilhot C.
      A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.
      ). Unexpectedly, a C. glutamicum mutant lacking the first enzyme required for activated Araf donor sugar biosynthesis, ubiA, is viable (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ). This strain is devoid of Araf, and that it could be isolated and cultured was surprising. These examples highlight how experiments using Corynebacterium species can provide important insight into cell envelope biosynthesis within the Corynebacterineae suborder.
      Although each Corynebacterineae species possesses an mAG of similar constitution, fine structural features of the mAG can vary. The arabinan of C. diphtheriae lacks the 1,3,5-linked Araf residues that are responsible for branching, suggesting this arabinan is less complex than that of other Corynebacterineae (
      • Puech V.
      • Chami M.
      • Lemassu A.
      • Lanéelle M.A.
      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ). Accordingly, arabinan assembly in mycobacteria is mediated by a larger collection of enzymes. Six or more arabinofuranosyltransferases are involved in mycobacteria, and at least one of these enzymes is inhibited by the first-line antitubercular drug ethambutol (
      • Jankute M.
      • Cox J.A.
      • Harrison J.
      • Besra G.S.
      Assembly of the mycobacterial cell wall.
      ,
      • Telenti A.
      • Philipp W.J.
      • Sreevatsan S.
      • Bernasconi C.
      • Stockbauer K.E.
      • Wieles B.
      • Musser J.M.
      • Jacobs Jr., W.R.
      The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol.
      • Goude R.
      • Amin A.G.
      • Chatterjee D.
      • Parish T.
      The arabinosyltransferase EmbC is inhibited by ethambutol in Mycobacterium tuberculosis.
      ). Of the six mycobacterial arabinofuranosyltransferases, three belong to the Emb family of enzymes. In contrast, corynebacteria encode a single Emb homolog that is most closely related to M. tuberculosis EmbC (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ). Thus, the arabinan of C. diphtheriae is generated using fewer enzymes and is simpler than that of M. tuberculosis. The mycolic acids from Mycobacterium and Corynebacterium species also vary. Mycobacterial mycolic acids possess chains of 70–90 carbon atoms, whereas the mycolic acids of C. diphtheriae are shorter, with a chain of 30–36 carbons (
      • Puech V.
      • Chami M.
      • Lemassu A.
      • Lanéelle M.A.
      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ,
      • Minnikin D.E.
      Lipids: complex lipids, their chemistry, biosynthesis, and roles.
      ). Additionally, M. tuberculosis mycolic acids commonly contain functionalities such as cis-cyclopropane that are absent from fast-growing Mycobacterium smegmatis or corynebacterial mycolic acids. There also are differences in the galactan. Analyses of the galactan length (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ) indicate the corynebacterial galactan is shorter than that of M. tuberculosis (
      • Daffe M.
      • Brennan P.J.
      • McNeil M.
      Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses.
      ,
      • Puech V.
      • Chami M.
      • Lemassu A.
      • Lanéelle M.A.
      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ,
      • Alderwick L.J.
      • Seidel M.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.
      ,
      • Birch H.L.
      • Alderwick L.J.
      • Rittmann D.
      • Krumbach K.
      • Etterich H.
      • Grzegorzewicz A.
      • McNeil M.R.
      • Eggeling L.
      • Besra G.S.
      Identification of a terminal rhamnopyranosyltransferase (RptA) involved in Corynebacterium glutamicum cell wall biosynthesis.
      ). Understanding the source of these differences can lend insight into the molecular basis for the cell envelope properties of specific species.
      The M. tuberculosis glycosyltransferase GlfT2 (EC 2.4.1.288) is a bifunctional carbohydrate polymerase that generates the bulk of the galactan (
      • Kremer L.
      • Dover L.G.
      • Morehouse C.
      • Hitchin P.
      • Everett M.
      • Morris H.R.
      • Dell A.
      • Brennan P.J.
      • McNeil M.R.
      • Flaherty C.
      • Duncan K.
      • Besra G.S.
      Galactan biosynthesis in Mycobacterium tuberculosis: identification of a bifunctional UDP-galactofuranosyltransferase.
      ,
      • Rose N.L.
      • Completo G.C.
      • Lin S.J.
      • McNeil M.
      • Palcic M.M.
      • Lowary T.L.
      Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ,
      • Szczepina M.G.
      • Zheng R.B.
      • Completo G.C.
      • Lowary T.L.
      • Pinto B.M.
      STD-NMR studies suggest that two acceptor substrates for GlfT2, a bifunctional galactofuranosyltransferase required for the biosynthesis of Mycobacterium tuberculosis arabinogalactan, compete for the same binding site.
      ). Studies with chain-terminating glycosyl donors indicate that GlfT2 is sequence-selective, and its fidelity for forming a sequence of alternating β(1–5) and β(1–6) linkages is high (
      • Brown C.D.
      • Rusek M.S.
      • Kiessling L.L.
      Fluorosugar chain termination agents as probes of the sequence specificity of a carbohydrate polymerase.
      ,
      • Peltier P.
      • Beláňová M.
      • Dianišková P.
      • Zhou R.
      • Zheng R.B.
      • Pearcey J.A.
      • Joe M.
      • Brennan P.J.
      • Nugier-Chauvin C.
      • Ferrières V.
      • Lowary T.L.
      • Daniellou R.
      • Mikušová K.
      Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis.
      ). Within the GlfT2 polypeptide is but a single active site (
      • Szczepina M.G.
      • Zheng R.B.
      • Completo G.C.
      • Lowary T.L.
      • Pinto B.M.
      STD-NMR studies suggest that two acceptor substrates for GlfT2, a bifunctional galactofuranosyltransferase required for the biosynthesis of Mycobacterium tuberculosis arabinogalactan, compete for the same binding site.
      ,
      • May J.F.
      • Levengood M.R.
      • Splain R.A.
      • Brown C.D.
      • Kiessling L.L.
      A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site.
      ); site-directed mutagenesis indicates substitution of key amino acids abrogates the formation of both β(1–5) and β(1–6) linkages (
      • May J.F.
      • Levengood M.R.
      • Splain R.A.
      • Brown C.D.
      • Kiessling L.L.
      A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site.
      ). Experiments with isotope-labeled acceptors reveal that GlfT2 is processive (
      • Levengood M.R.
      • Splain R.A.
      • Kiessling L.L.
      Monitoring processivity and length control of a carbohydrate polymerase.
      ), and its propensity for generating alternating β(1–5) or β(1–6) Galf linkages is a consequence of processive elongation (
      • Yamatsugu K.
      • Splain R.A.
      • Kiessling L.L.
      Fidelity and promiscuity of a mycobacterial glycosyltransferase.
      ). The enzyme not only controls polymer sequence but also polymer length. In a test tube GlfT2 can generate polymers of a similar length to those obtained from cells (
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). With synthetic acceptors, the identity of the anomeric lipid was a critical determinant of product polysaccharide length (
      • Kremer L.
      • Dover L.G.
      • Morehouse C.
      • Hitchin P.
      • Everett M.
      • Morris H.R.
      • Dell A.
      • Brennan P.J.
      • McNeil M.R.
      • Flaherty C.
      • Duncan K.
      • Besra G.S.
      Galactan biosynthesis in Mycobacterium tuberculosis: identification of a bifunctional UDP-galactofuranosyltransferase.
      ,
      • Rose N.L.
      • Completo G.C.
      • Lin S.J.
      • McNeil M.
      • Palcic M.M.
      • Lowary T.L.
      Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). Acceptors with short alkyl anomeric substituents afforded only short oligomeric saccharides, whereas those with longer lipids on the acceptor afforded polysaccharide products similar to those in cells. These findings led to the proposal that M. tuberculosis GlfT2 uses the acceptor lipid as a tether and that polymer length is controlled by bivalent substrate binding (
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). The DXD motif mediates glycosyltransferase coordination to a divalent cation, and M. tuberculosis GlfT2 variants in this DXD motif afforded truncated oligosaccharide products (
      • May J.F.
      • Levengood M.R.
      • Splain R.A.
      • Brown C.D.
      • Kiessling L.L.
      A processive carbohydrate polymerase that mediates bifunctional catalysis using a single active site.
      ,
      • Wheatley R.W.
      • Zheng R.B.
      • Richards M.R.
      • Lowary T.L.
      • Ng K.K.
      Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan.
      ). Thus, GlfT2 is bifunctional with the ability to control the length of polymerized M. tuberculosis galactan. Whether these attributes of the M. tuberculosis GlfT2 are preserved in other Corynebacterineae species is unclear.
      To compare and contrast galactan biosynthesis in different species, we examined the galactan biosynthetic enzymes from C. glutamicum and C. diphtheriae NCTC 13129, including GlfT2 and UGM. The putative C. diphtheriae UGM (DIP2203) catalyzes the isomerization of UDP-Galp and UDP-Galf with an efficiency similar to other prokaryotic orthologs. This finding suggests that differences in galactan length are unlikely to arise from differences in cellular concentration of UDP-Galf. To explore the role of the galactan in cells, we employed small molecule UGM inhibitors. These compounds inhibit C. diphtheriae UGM in vitro and prevent the growth of C. glutamicum. These observations support an essential role for the galactan in corynebacteria as well as mycobacteria and suggest that the small molecule UGM inhibitors can be used to probe the cellular roles of Galf. With regard to GlfT2, our results indicate that the DIP2198 gene product is a bifunctional galactofuranosyltransferase similar to the M. tuberculosis GlfT2. The corynebacterial glycosyltransferase can elongate synthetic acceptors to afford polysaccharides commensurate in length to those isolated from corynebacteria. As with the M. tuberculosis GlfT2, the product polysaccharides attained from variants with amino acid changes in the proposed donor binding site are truncated. These findings indicate that GlfT2 orthologs have an intrinsic ability to control polysaccharide length. This length control mechanism differs from that employed in O-antigen biosynthesis in which length is controlled by an auxiliary protein (
      • Bastin D.A.
      • Stevenson G.
      • Brown P.K.
      • Haase A.
      • Reeves P.R.
      Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.
      ,
      • Woodward R.
      • Yi W.
      • Li L.
      • Zhao G.
      • Eguchi H.
      • Sridhar P.R.
      • Guo H.
      • Song J.K.
      • Motari E.
      • Cai L.
      • Kelleher P.
      • Liu X.
      • Han W.
      • Zhang W.
      • Ding Y.
      • Li M.
      • Wang P.G.
      In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ). Our investigations lay a foundation for dissecting the molecular details and mechanisms of galactan biosynthesis using genetic and chemical genetic tools.

      Discussion

      The taxon Corynebacterineae includes many bacterial species of industrial and medical importance (
      • Coyle M.B.
      • Lipsky B.A.
      Coryneform bacteria in infectious diseases: clinical and laboratory aspects.
      ,
      • Funke G.
      • von Graevenitz A.
      • Clarridge 3rd, J.E.
      • Bernard K.A.
      Clinical microbiology of coryneform bacteria.
      • Hermann T.
      Industrial production of amino acids by coryneform bacteria.
      ). Although Corynebacterineae share cell envelope features, different species occupy distinct ecological niches, and the details of their cell wall composition can vary. Survival in specific physiological environments may have been responsible for alterations in the constitution of their cell envelopes and mAG complexes. The few studies characterizing the mAG of Corynebacterineae species have uncovered structural differences (
      • Daffe M.
      • Brennan P.J.
      • McNeil M.
      Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses.
      ,
      • Puech V.
      • Chami M.
      • Lemassu A.
      • Lanéelle M.A.
      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ,
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ,
      • Daffe M.
      • McNeil M.
      • Brennan P.J.
      Major structural features of the cell wall arabinogalactans of Mycobacterium, Rhodococcus, and Nocardia spp.
      ). We probed galactan biosynthesis in corynebacteria to better understand the mechanisms by which different species assemble this fundamental component of the mAG complex. We also examined the physiological importance of the mAG using a chemical genetic approach.
      Whether disruption of galactan biosynthesis would inhibit corynebacterial growth was an open question as previous experiments have revealed that components once thought to be essential for Corynebacterineae are dispensable in C. glutamicum. Specifically, microbial genetics and chromosomal deletion experiments have shown that mycolic acids (
      • Portevin D.
      • De Sousa-D'Auria C.
      • Houssin C.
      • Grimaldi C.
      • Chami M.
      • Daffé M.
      • Guilhot C.
      A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.
      ,
      • Kacem R.
      • De Sousa-D'Auria C.
      • Tropis M.
      • Chami M.
      • Gounon P.
      • Leblon G.
      • Houssin C.
      • Daffé M.
      Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum.
      ) and the arabinan are not necessary for viability in this species (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ,
      • Alderwick L.J.
      • Seidel M.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.
      ). In contrast, attempts to isolate M. tuberculosis mutants with similar genotypes have failed (
      • Sassetti C.M.
      • Boyd D.H.
      • Rubin E.J.
      Genes required for mycobacterial growth defined by high density mutagenesis.
      ). Indeed, no M. tuberculosis strains lacking the enzymes that mediate galactan biosynthesis have been isolated, suggesting the galactan is required for M. tuberculosis viability.
      To test whether the galactan is essential for corynebacterial growth, we used a chemical biology strategy. We determined the catalytic activity of the putative C. diphtheriae UGM, DIP2203. This enzyme catalyzes the isomerization of UDP-Galp and UDP-Galf with kinetic parameters similar to those of other prokaryotic UGMs (
      • Beverley S.M.
      • Owens K.L.
      • Showalter M.
      • Griffith C.L.
      • Doering T.L.
      • Jones V.C.
      • McNeil M.R.
      Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens.
      ,
      • Wesener D.A.
      • May J.F.
      • Huffman E.M.
      • Kiessling L.L.
      UDP-galactopyranose mutase in nematodes.
      • Soltero-Higgin M.
      • Carlson E.E.
      • Gruber T.D.
      • Kiessling L.L.
      A unique catalytic mechanism for UDP-galactopyranose mutase.
      ), and the C. diphtheriae enzyme can be inhibited by small molecules developed against the M. tuberculosis UGM (
      • Dykhuizen E.C.
      • May J.F.
      • Tongpenyai A.
      • Kiessling L.L.
      Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth.
      ,
      • Kincaid V.A.
      • London N.
      • Wangkanont K.
      • Wesener D.A.
      • Marcus S.A.
      • Héroux A.
      • Nedyalkova L.
      • Talaat A.M.
      • Forest K.T.
      • Shoichet B.K.
      • Kiessling L.L.
      Virtual screening for UDP-galactopyranose mutase ligands identifies a new class of antimycobacterial agents.
      ). When our most potent small molecule inhibitor of corynebacteria UGM, compound 1, was added to C. glutamicum, corynebacterial growth was inhibited. This result suggests the galactan is essential in C. glutamicum. This finding implies that the peptidoglycan-galactan conjugate is the minimal cell envelope structure needed for viability of corynebacteria, whereas the polysaccharide emanating from the galactan, the arabinan (
      • Alderwick L.J.
      • Radmacher E.
      • Seidel M.
      • Gande R.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Sahm H.
      • Eggeling L.
      • Besra G.S.
      Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core.
      ), and the cell surface mycolic acids appear dispensable (
      • Portevin D.
      • De Sousa-D'Auria C.
      • Houssin C.
      • Grimaldi C.
      • Chami M.
      • Daffé M.
      • Guilhot C.
      A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.
      ).
      Previous cell wall analyses suggest that galactose is present at lower abundance in corynebacteria as compared with mycobacteria. This observation suggests that corynebacteria have a shorter galactan (
      • Daffe M.
      • Brennan P.J.
      • McNeil M.
      Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses.
      ,
      • Puech V.
      • Chami M.
      • Lemassu A.
      • Lanéelle M.A.
      • Schiffler B.
      • Gounon P.
      • Bayan N.
      • Benz R.
      • Daffé M.
      Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.
      ,
      • Alderwick L.J.
      • Seidel M.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.
      ,
      • Birch H.L.
      • Alderwick L.J.
      • Rittmann D.
      • Krumbach K.
      • Etterich H.
      • Grzegorzewicz A.
      • McNeil M.R.
      • Eggeling L.
      • Besra G.S.
      Identification of a terminal rhamnopyranosyltransferase (RptA) involved in Corynebacterium glutamicum cell wall biosynthesis.
      ). The in vitro carbohydrate polymerase activity of C. diphtheriae GlfT2 gives rise to shorter polymers, consistent with those observations (
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). These results indicate that the GlfT2 glycosyltransferases have an intrinsic ability to control length. Our data further suggest that galactan length can be manipulated through mutation or heterologous expression of polymerases. Thus, polysaccharide length is dictated by the enzyme's sequence.
      Several other microbial cell envelope glycopolymers possess defined length distributions. These include peptidoglycan, lipopolysaccharide (LPS) O-antigens, capsular polysaccharides, and teichoic acids (
      • Wang T.S.
      • Manning S.A.
      • Walker S.
      • Kahne D.
      Isolated peptidoglycan glycosyltransferases from different organisms produce different glycan chain lengths.
      • Glauner B.
      Separation and quantification of muropeptides with high-performance liquid chromatography.
      ,
      • Raetz C.R.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      ,
      • Yother J.
      Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation.
      • Gründling A.
      • Schneewind O.
      Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus.
      ). Efforts to elucidate how the length of these polysaccharides is controlled have revealed the importance of accessory proteins. For example, in Wzy-dependent LPS O-antigen biosynthesis product length is controlled by the polysaccharide co-polymerase protein, termed Wzz (
      • Bastin D.A.
      • Stevenson G.
      • Brown P.K.
      • Haase A.
      • Reeves P.R.
      Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.
      ,
      • Raetz C.R.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      ,
      • Paulsen I.T.
      • Beness A.M.
      • Saier M.H.
      Computer-based analyses of the protein constituents of transport systems catalyzing export of complex carbohydrates in bacteria.
      ,
      • Tocilj A.
      • Munger C.
      • Proteau A.
      • Morona R.
      • Purins L.
      • Ajamian E.
      • Wagner J.
      • Papadopoulos M.
      • Van Den Bosch L.
      • Rubinstein J.L.
      • Féthière J.
      • Matte A.
      • Cygler M.
      Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.
      ). An in vitro polymerization system was used to demonstrate that Wzy alone is sufficient for polymerization, but that addition of Wzz to the reaction can modulate the product length distribution (
      • Woodward R.
      • Yi W.
      • Li L.
      • Zhao G.
      • Eguchi H.
      • Sridhar P.R.
      • Guo H.
      • Song J.K.
      • Motari E.
      • Cai L.
      • Kelleher P.
      • Liu X.
      • Han W.
      • Zhang W.
      • Ding Y.
      • Li M.
      • Wang P.G.
      In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
      ). An LPS O-antigen assembled in an ABC transporter-dependent manner also employs an independent chain-length regulator protein. In E. coli serotype O9a O-antigen assembly, an extended coiled-coil in the chain length regulator WbdD operates as a molecular ruler to physically separate the kinase termination activity of WbdD, from the glycosylpolymerase activity of WbdA (
      • Hagelueken G.
      • Clarke B.R.
      • Huang H.
      • Tuukkanen A.
      • Danciu I.
      • Svergun D.I.
      • Hussain R.
      • Liu H.
      • Whitfield C.
      • Naismith J.H.
      A coiled-coil domain acts as a molecular ruler to regulate O-antigen chain length in lipopolysaccharide.
      ). Unlike these systems, there are no apparent accessory proteins that can modulate the length of polysaccharides generated by GlfT2. The galactan and LPS O-antigen are dissimilar in that the galactan is an essential component of the cell envelope, whereas O-antigen is not required for survival and likely evolved for host interactions (
      • Lerouge I.
      • Vanderleyden J.
      O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions.
      ). Bacteria may have evolved unique accessory proteins to alter O-antigen length as a mechanism for modulating surface antigenicity, but such a selective pressure would not be present for galactan assembly.
      The mechanisms underlying length control by GlfT2 are unknown. One possibility for the shortened polysaccharide length obtained with C. diphtheriae GlfT2 versus the M. tuberculosis ortholog is that the rate of polymerization for the former is 17-fold slower. Still, the kinetic rate alone is unlikely to be solely responsible for the divergent polymer length distributions we observed. Specifically, the tetrasaccharide acceptor gave rise to longer polysaccharides (+32 Galf residues; Fig. 5C) yet the initial velocities observed for C. diphtheriae GlfT2 remained slower (1.8 μm/min) than those obtained for M. tuberculosis enzyme (8.2 μm/min). Acceptor concentration had little effect on polysaccharide length. When the reaction was conducted at an 8-fold lower acceptor concentration (25 μm compound 4a), the longest polymer was +15 Galf residues, and a nearly identical product profile was attained. The available data indicate that the features of the acceptor and sequence of the glycosyltransferase modulate product polysaccharide length (
      • May J.F.
      • Splain R.A.
      • Brotschi C.
      • Kiessling L.L.
      A tethering mechanism for length control in a processive carbohydrate polymerization.
      ). For example, the longer polymers obtained from the tetrasaccharide versus those generated from disaccharide 4a may be due to the former binding more efficiently and remaining bound through a larger number of catalytic events. Similarly, the affinity of the glycosyl donor may influence polysaccharide product length control. These two catalytic parameters may contribute to GlfT2 length control. Moreover, the data suggest that length of polysaccharides generated by GlfT2 can be modulated through simple mutagenesis. A similar mutagenesis strategy may be useful for modulating the length of other biologically important polysaccharides. Specifically, both GlfT2 and cellulose synthase belong to the glycosyltransferase family 2 (GT-2) glycosylpolymerases (
      • Cantarel B.L.
      • Coutinho P.M.
      • Rancurel C.
      • Bernard T.
      • Lombard V.
      • Henrissat B.
      The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics.
      ), and attenuation of cellulose polymer length may be useful for the industrial production of cellulosic derived commodities such as ethanol (
      • Somerville C.
      Cellulose synthesis in higher plants.
      ).
      A mechanism for carbohydrate polymerase length control based on sugar donor binding has been suggested in an unrelated process; that is, the synthesis of Streptococcus pneumoniae type 3 capsular polysaccharide (
      • Ventura C.L.
      • Cartee R.T.
      • Forsee W.T.
      • Yother J.
      Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae.
      ). This polysaccharide is generated by a processive synthase. The enzyme uses two sugar donors, UDP-glucose and UDP-glucuronic acid, for [4)-d-Glc-β(1–3)-d-GlcUA-β(1-] polymer formation. Data suggest that the substrate concentrations and the affinity of UDP-glucuronic acid binding to the enzyme influence the product polysaccharide length (
      • Ventura C.L.
      • Cartee R.T.
      • Forsee W.T.
      • Yother J.
      Control of capsular polysaccharide chain length by UDP-sugar substrate concentrations in Streptococcus pneumoniae.
      ,
      • Forsee W.T.
      • Cartee R.T.
      • Yother J.
      A kinetic model for chain length modulation of Streptococcus pneumoniae cellubiuronan capsular polysaccharide by nucleotide sugar donor concentrations.
      ).
      The ability of GlfT2 to control polysaccharide length also could rest in an alternative motif. For example, a chain length regulatory proline motif (WPQ) was recently identified in transmembrane glycosyltransferase type 3 arabinofuranosyltransferases (
      • Seidel M.
      • Alderwick L.J.
      • Sahm H.
      • Besra G.S.
      • Eggeling L.
      Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis.
      ,
      • Berg S.
      • Starbuck J.
      • Torrelles J.B.
      • Vissa V.D.
      • Crick D.C.
      • Chatterjee D.
      • Brennan P.J.
      Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan.
      ). Lastly, an alternative hypothesis based on GlfT2 quaternary structure has been put forth (
      • Wheatley R.W.
      • Zheng R.B.
      • Richards M.R.
      • Lowary T.L.
      • Ng K.K.
      Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan.
      ). The tetrameric structure observed in the M. tuberculosis GlfT2 crystals contained a face of mostly hydrophobic and positively charged residues that the authors suggest promotes membrane association. At the center of the tetramer resides a large hollow cavity that the authors suggest functions as an area for growing galactan to be displaced into during polymerization, with the size of the cavity influencing product polymer length (
      • Wheatley R.W.
      • Zheng R.B.
      • Richards M.R.
      • Lowary T.L.
      • Ng K.K.
      Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan.
      ).
      Several of the aforementioned mechanisms may contribute to regulating galactan length. For example, the cell membrane may function in a similar role as that proposed for the GlfT2 acceptor anomeric lipid. Thus, the distance between the GlfT2 active site and membrane combined with enzyme binding to substrate would influence the length of polymeric products. We anticipate that future experiments will provide additional insight into how galactan length is controlled.

      Author Contributions

      M. R. L. and L. L. K. conceived the study. M. R. L. and D. A. W. performed the studies. DAW, M. R. L., and L. L. K. analyzed the data and wrote the manuscript.

      Acknowledgments

      MALDI-TOF mass spectrometry data were obtained at the University of Wisconsin (UW)-Madison Chemistry Instrument Center Mass Spectrometry Facility on a Bruker Ultraflex III Instrument that was supported by the National Institutes of Health (Center for Research Resources 1S10RR024601).Additional experiments were carried out at the UW-Madison Biophysics Instrumentation Facility, which is supported by UW-Madison, National Science Foundation Grant BIR-9512577 and National Institutes of Health Grant S10 RR13790. We acknowledge R. A. Splain for synthesis of the synthetic acceptor substrates used in this study and M. A. Martinez Farias for synthesis of the FP probes. We thank H. L. Hodges and A. M. Justen for helpful discussion and help preparing the manuscript.

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