Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis.
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.
Kim
Tae-Hyun
TH
Department of Microbiology and Molecular Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Sebastian
Shite
S
Pinkham
Jessica T
JT
Ross
Robin A
RA
Blalock
LeeAnn T
LT
Kasper
Dennis L
DL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism; D005604:Francisella tularensis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019081:O Antigens / Q000378:metabolism; D014613:Vaccines, Attenuated
D000596:Amino Acids; D008070:Lipopolysaccharides; D019081:O Antigens; D014613:Vaccines, Attenuated; D006602:Hexosyltransferases; C085652:O-antigen polymerase