Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*
| Autor: | Dennis L. Kasper, LeeAnn T. Blalock, Tae-Hyun Kim, Jessica T. Pinkham, Shite Sebastian, Robin A. Ross |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Lipopolysaccharides
Mutant Molecular Sequence Data Glycobiology and Extracellular Matrices Vaccines Attenuated Biochemistry Homology (biology) Gene Expression Regulation Fungal Amino Acid Sequence Amino Acids Site-directed mutagenesis Francisella tularensis Molecular Biology Peptide sequence Polymerase biology Cell Membrane Genetic Complementation Test O Antigens Cell Biology Periplasmic space Fusion protein Molecular biology Hexosyltransferases Codon usage bias Mutation biology.protein Biocatalysis Mutagenesis Site-Directed |
| Popis: | The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|