title: Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen.
abstract: The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.
authors.affiliation: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA. rickp@usuhs.mil
authors.forename: Paul D
authors.initials: PD
authors.lastname: Rick
authors.forename: Kathleen
authors.initials: K
authors.lastname: Barr
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sankaran
authors.forename: Junko
authors.initials: J
authors.lastname: Kajimura
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rush
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Waechter
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008563:Membrane Lipids / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014312:Trisaccharides / Q000276:immunology / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C117254:RfbX protein, Pseudomonas; D014312:Trisaccharides; C475086:WzxE protein, E coli; C017286:enterobacterial common antigen