Show legend Show as text

Structure type: oligomer
Compound class: lipid A

• Article ID: 119
  Trent MS, Ribeiro AA, Doerrler WT, Lin S, Cotter RJ, Raetz CRH "Accumulation of a polyisoprene-linked amino sugar in polymyxin resistant Salmonella typhimurium and Escherichia coli. Structural characterization and transfer to lipid A in the periplasm" - Journal of Biological Chemistry 276(46) (2001) 43132-43144
  

  Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-α-L-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-α-L-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-α-L-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

  Escherichia coli, lipid A, polymyxin-resistant mutants, Salmonella typhimurium

  NCBI PubMed ID: 11535605
  Publication DOI: 10.1074/jbc.M106962200
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: raetz@biochem.duke.edu
  Institutions: Department of Biochemistry and the Duke NMR Spectroscopy Center and Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710, Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA
  Methods: 13C NMR, 1H NMR, NMR-2D, DNA techniques, TLC, 31P NMR, MALDI-TOF MS, radiolabeling, NMR-1D, genetic methods, biochemical methods, radioactivity measurement
  
   
  
  Salmonella typhimurium, Escherichia coli
  CSDB ID 5231 (all data & tools)

Show legend Show as text

Structure type: monomer
Compound class: lipid A

• Article ID: 3784
  Song F, Guan Z, Raetz CR "Biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in Francisella novicida" - Biochemistry 48(6) (2009) 1173-1182
  

  Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-β-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-β-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-β-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-α-L-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-α-L-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

  Escherichia coli, lipid A, Salmonella typhimurium, Francisella tularensis, Francisella novicida, UDP-glucose, Electrospray Ionization, acetyltransferases

  NCBI PubMed ID: 19166326
  Publication DOI: 10.1021/bi802212t
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: raetz@biochem.duke.edu
  Institutions: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
  Methods: TLC, ESI-MS, genetic methods, biochemical methods, LC-ESI-MS
  
   
  
  Escherichia coli
  CSDB ID 31236 (all data & tools)

Show legend Show as text

Structure type: monomer
Compound class: lipid A
Contained glycoepitopes: IEDB_130648,IEDB_137473

• Article ID: 3784
  Song F, Guan Z, Raetz CR "Biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in Francisella novicida" - Biochemistry 48(6) (2009) 1173-1182
  

  Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-β-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-β-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-β-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-α-L-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-α-L-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

  Escherichia coli, lipid A, Salmonella typhimurium, Francisella tularensis, Francisella novicida, UDP-glucose, Electrospray Ionization, acetyltransferases

  NCBI PubMed ID: 19166326
  Publication DOI: 10.1021/bi802212t
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: raetz@biochem.duke.edu
  Institutions: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
  Methods: TLC, ESI-MS, genetic methods, biochemical methods, LC-ESI-MS
  
   
  
  Francisella novicida U112 fmlF2
  CSDB ID 31237 (all data & tools)

Show legend Show as text

Structure type: monomer
Compound class: lipid A
Contained glycoepitopes: IEDB_142488,IEDB_145000,IEDB_146664,IEDB_983931,SB_192

• Article ID: 3784
  Song F, Guan Z, Raetz CR "Biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in Francisella novicida" - Biochemistry 48(6) (2009) 1173-1182
  

  Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding paper, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-β-D-GalN. A small portion of the free lipid A of Francisella novicida is further modified with a glucose residue at position-6'. We now demonstrate that the two F. novicida homologues of Escherichia coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. On the basis of an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-β-D-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-β-D-GalN. This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-α-L-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-α-L-arabinose in polymyxin-resistant strains of E. coli and Salmonella typhimurium.

  Escherichia coli, lipid A, Salmonella typhimurium, Francisella tularensis, Francisella novicida, UDP-glucose, Electrospray Ionization, acetyltransferases

  NCBI PubMed ID: 19166326
  Publication DOI: 10.1021/bi802212t
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: raetz@biochem.duke.edu
  Institutions: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
  Methods: TLC, ESI-MS, genetic methods, biochemical methods, LC-ESI-MS
  
   
  
  Francisella novicida U112 fmlF1
  CSDB ID 31238 (all data & tools)

Show legend Show as text

Structure type: monomer
Aglycon: OPh
Contained glycoepitopes: IEDB_130648,IEDB_137473,IEDB_1391961,IEDB_141584,IEDB_885822

• Article ID: 4517
  Gao Y, Vinnikova A, Brockhausen I "Functional Identification of Bacterial Glucosyltransferase WbdN" - Book: Methods in Molecular Biology (2013) Vol. 1022, 199-214
  

  The outer membrane of gram-negative bacteria is stabilized by lipopolysaccharides (LPS). The O-antigenic polysaccharides of LPS are composed of repeating units that are exposed to and can interact with the environment. The glycosyltransferases that assemble these repeating units are encoded by the O-antigen gene cluster and utilize undecaprenol-phosphate-linked intermediates as natural acceptor substrates, and nucleotide sugars as donor substrates on the cytoplasmic face of the inner membrane. Many of the glycosyltransferase genes are known but the enzymatic functions of most of them remain to be identified. We describe here how the function of a recombinant glucosyltransferase WbdN from Escherichia coli O157 can be determined by NMR analysis of the enzyme product, using a synthetic acceptor substrate analog. A fluorescent acceptor substrate analog can be used in highly sensitive enzyme assays that allow the characterization of enzyme activity without the use of radioactive nucleotide sugar donor substrates.

  NMR, glucosyltransferase, linkage analysis, WbdN, fluorescent acceptor substrate

  NCBI PubMed ID: 23765664
  Publication DOI: 10.1007/978-1-62703-465-4_16
  Publisher: Totowa, NJ: Humana Press
  Correspondence: I. Brockhausen
  Editors: Holst O, Walker JM, Beck A
  Institutions: Department of Medicine and Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada
  Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, sugar analysis, 31P NMR, MALDI-MS, Western blotting, NMR-1D, biochemical methods, HPLC
  
   
  
  Escherichia coli O157:H7
  CSDB ID 29488 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: stewartan repeat units
Compound class: EPS
Contained glycoepitopes: IEDB_115136,IEDB_136044,IEDB_136906,IEDB_137472,IEDB_140529,IEDB_140630,IEDB_141794,IEDB_142488,IEDB_146664,IEDB_151528,IEDB_167069,IEDB_190606,IEDB_423153,IEDB_983931,SB_165,SB_166,SB_187,SB_192,SB_195,SB_7,SB_88

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Pantoea stewartii
  CSDB ID 29204 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: amylovoran repeat units
Compound class: EPS
Contained glycoepitopes: IEDB_115136,IEDB_134624,IEDB_136044,IEDB_136906,IEDB_137472,IEDB_140630,IEDB_141794,IEDB_142488,IEDB_146664,IEDB_151528,IEDB_151770,IEDB_190606,IEDB_423153,IEDB_742248,IEDB_983931,SB_163,SB_165,SB_166,SB_187,SB_192,SB_195,SB_7,SB_88

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Erwinia amylovora
  CSDB ID 29562 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-antigen repeat units
Compound class: O-antigen
Contained glycoepitopes: IEDB_130701,IEDB_135513,IEDB_136093,IEDB_136105,IEDB_136906,IEDB_137472,IEDB_137486,IEDB_141794,IEDB_144983,IEDB_145001,IEDB_151528,IEDB_152206,IEDB_190606,IEDB_225177,IEDB_885823,IEDB_983930,SB_44,SB_67,SB_7,SB_72

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Salmonella enterica B
  CSDB ID 29563 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-antigen repeat units
Compound class: O-antigen
Contained glycoepitopes: IEDB_130660,IEDB_130701,IEDB_136105,IEDB_136906,IEDB_137472,IEDB_139421,IEDB_141794,IEDB_144983,IEDB_145001,IEDB_151528,IEDB_152206,IEDB_174033,IEDB_190606,IEDB_225177,IEDB_885823,IEDB_983930,SB_44,SB_67,SB_7,SB_72

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Salmonella enterica D1
  CSDB ID 29564 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-antigen repeat units
Compound class: O-antigen
Contained glycoepitopes: IEDB_136105,IEDB_136906,IEDB_137472,IEDB_137485,IEDB_139420,IEDB_139421,IEDB_141794,IEDB_144983,IEDB_145001,IEDB_151528,IEDB_152206,IEDB_190606,IEDB_225177,IEDB_885823,IEDB_983930,SB_44,SB_7,SB_72

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Salmonella enterica D2
  CSDB ID 29565 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-antigen repeat units
Compound class: O-antigen
Contained glycoepitopes: IEDB_136105,IEDB_136906,IEDB_137472,IEDB_137485,IEDB_141794,IEDB_144983,IEDB_145001,IEDB_151528,IEDB_152206,IEDB_190606,IEDB_225177,IEDB_885823,IEDB_983930,SB_44,SB_7,SB_72

• Article ID: 4536
  Islam ST, Lam JS "Wzx flippase-mediated membrane translocation of sugar polymer precursors in bacteria" - Environmental Microbiology 15(4) (2013) 1001-1015
  

  Bacterial cell surface polysaccharides confer resistance to external stress and promote survival in biotic and abiotic environments. Glycan assembly often occurs at the periplasmic leaflet of the inner membrane (IM) from undecaprenyl pyrophosphate (UndPP)-linked polysaccharide units via the Wzx/Wzy-dependent pathway. Wzx is an integral IM protein found in Gram-negative and Gram-positive bacteria that mediates IM translocation of UndPP-linked sugar repeats from the cytoplasmic to the periplasmic leaflet; interaction of Wzx with other assembly proteins is indirectly supported by genetic evidence. Topological mapping has indicated 12 alpha-helical transmembrane segments (TMS), with the number of charged TMS residues fluctuating based on the mapping method used. A novel Wzx tertiary structure model has been built, allowing for substrate-binding or energy-coupling roles to be proposed for functionally important charged and aromatic TMS residues. It has also led to a proposed antiport-like mechanism of Wzx function. Exquisite substrate specificity of Wzx proteins was recently revealed in distinguishing between UndPP-linked substrates with identical main-chain sugar repeats, but differing in the chemical composition of a terminal sugar side-branch cap. The objective of this review is to synthesize the most up-to-date knowledge concerning Wzx flippases and to provide perspective for future investigations in this burgeoning field.

  structure, polysaccharide, interaction, Gram-negative, surface polysaccharide, Gram-positive, mapping, gram-positive bacteria, Wzx flippases, Wzx/Wzy

  NCBI PubMed ID: 23016929
  Publication DOI: 10.1111/j.1462-2920.2012.02890.x
  Journal NLM ID: 100883692
  Publisher: Blackwell Publishing
  Correspondence: Joseph S. Lam
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G 2W1
  
   
  
  Salmonella enterica E
  CSDB ID 29566 (all data & tools)

Show legend Show as text

Structure type: monomer
Trivial name: bactoprenyl diphosphate sugar
Contained glycoepitopes: IEDB_142345

• Article ID: 4582
  Mostafavi AZ, Troutman JM "Biosynthetic assembly of the Bacteroides fragilis capsular polysaccharide A precursor bactoprenyl diphosphate-linked acetamido-4-amino-6-deoxygalactopyranose" - Biochemistry 52(11) (2013) 1939-1949
  

  The sugar capsule capsular polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of multiple sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxygalactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report, we have utilized a heterologous system to reconstitute bactoprenyl diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis-encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). The function of the aminotransferase was confirmed by capillary electrophoresis and a novel high-performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of one- and two-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry. A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS and allowed HPLC-based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single-pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.

  Bacteroides fragilis, aminotransferase, caspsular polysaccharide A, biosynthesis pathway, WcfR, WcfS, acetamido-4-amino-6-deoxy-galactopyranose

  NCBI PubMed ID: 23458065
  Publication DOI: 10.1021/bi400126w
  Journal NLM ID: 0370623
  Publisher: American Chemical Society
  Correspondence: jerry.troutman@uncc.edu
  Institutions: Department of Chemistry, University of North Carolina at Charlotte , 9201 University City Boulevard, Charlotte, North Carolina 28223-0001, United States
  Methods: 1H NMR, NMR-2D, PCR, SDS-PAGE, TLC, ESI-MS, NMR-1D, genetic methods, biochemical methods, radioactivity measurement, HPLC, CE
  
   
  
  Bacteroides fragilis
  CSDB ID 29656 (all data & tools)

Show legend Show as text

Structure type: oligomer
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_135813,IEDB_135849,IEDB_136105,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_225177,IEDB_885823

• Article ID: 4900
  Mann E, Mallette E, Clarke BR, Kimber MS, Whitfield C "The Klebsiella pneumoniae O12 ATP-binding cassette (ABC) transporter recognizes the terminal residue of its O-antigen polysaccharide substrate" - Journal of Biological Chemistry 291(18) (2016) 9748-9761
  

  Export of the E. coli serotype O9a O-antigenic polysaccharide (O-PS) involves an ABC transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C-terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC33257). The O12 polysaccharide is terminated at the non-reducing end by a β-linked 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM and export is dependent on the presence of the terminal β-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype but they share only weak sequence similarity and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.

  O-antigen, Klebsiella pneumoniae, glycosylation, ABC transporter, lipopolysaccharide (LPS), glycoconjugate, membrane transport

  NCBI PubMed ID: 26934919
  Publication DOI: 10.1074/jbc.M116.719344
  Journal NLM ID: 2985121R
  WWW link: doi: 10.1074/jbc.M116.719344
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
  Methods: PCR, SDS-PAGE, DNA techniques, genetic methods, binding assays, protein detection
  
   
  
  Klebsiella pneumoniae O12, Raoultella terrigena ATCC 33257
  CSDB ID 11635 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-unit
Compound class: O-antigen
Contained glycoepitopes: IEDB_135813,IEDB_136105,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_225177,IEDB_885813,IEDB_885823

• Article ID: 5023
  Liu MA, Kenyon JJ, Lee J, Reeves PR "Rapid customised operon assembly by yeast recombinational cloning" - Applied Microbiology and Biotechnology 101(11) (2017) 4569-4580
  

  We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

  O-antigen, cloning, recombination, operon, flippase, Translocase

  NCBI PubMed ID: 28324143
  Publication DOI: 10.1007/s00253-017-8213-9
  Journal NLM ID: 8406612
  Publisher: Springer
  Correspondence: peter.reeves@sydney.edu.au
  Institutions: School of Biomedical Sciences and Infectious Diseases Program, Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Brisbane, 4001, QLD, Australia, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia
  Methods: PCR, SDS-PAGE, genetic methods, cloning, function analysis of gene clusters
  
   
  
  Escherichia coli O1A
  CSDB ID 11937 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: O-unit
Compound class: O-antigen
Contained glycoepitopes: IEDB_133754,IEDB_135813,IEDB_136105,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_225177,IEDB_885823

• Article ID: 5023
  Liu MA, Kenyon JJ, Lee J, Reeves PR "Rapid customised operon assembly by yeast recombinational cloning" - Applied Microbiology and Biotechnology 101(11) (2017) 4569-4580
  

  We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.

  O-antigen, cloning, recombination, operon, flippase, Translocase

  NCBI PubMed ID: 28324143
  Publication DOI: 10.1007/s00253-017-8213-9
  Journal NLM ID: 8406612
  Publisher: Springer
  Correspondence: peter.reeves@sydney.edu.au
  Institutions: School of Biomedical Sciences and Infectious Diseases Program, Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Brisbane, 4001, QLD, Australia, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia
  Methods: PCR, SDS-PAGE, genetic methods, cloning, function analysis of gene clusters
  
   
  
  Escherichia coli O2
  CSDB ID 12176 (all data & tools)