Abstract
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.
Highlights
New England Regional Center of Excellence Grant R01-AI47484. □S The on-line version of this article contains supplemental Table 1 and Figs. 1 and 2. 1 To whom correspondence should be addressed: 200 Longwood Ave., America
This study describes the functional and biochemical characterization of the putative Wzy in F. tularensis LVS and the identification of specific amino acid residues that play an important role in maintaining this protein’s catalytic function
The F. tularensis putative O-antigen biosynthetic gene cluster has been identified [12]; it is estimated to be ϳ17 kb in length and is predicted to contain 15 genes involved in O-antigen biosynthesis [12]
Summary
New England Regional Center of Excellence Grant R01-AI47484. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1 and 2. 1 To whom correspondence should be addressed: 200 Longwood Ave., America. The O-antigen of F. tularensis strains LVS and SchuS4 contains the rare sugars 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc) and 4,6dideoxy-4-formamido-D-glucose (Qui4NFm) as well as 2 mol of 2-acetoamido-2-deoxy-D-galacturonamide (GalNAcAN); the resulting repeat structure is 4-␣-GalNAcAN-1,4-␣GalNAcAN-1,3--QuiNAc-1,2--Qui4NFm. The three known modes of O-antigen assembly (Wzy-dependent, ABC transporter-dependent, and synthase-dependent) rely on export mechanisms and the presence of specific enzymes in a given pathway [15]. In the Wzy-dependent pathway (Fig. 1), O-repeating unit synthesis is initiated from two different classes of integral membrane proteins: N-acetylhexosamine-1-phosphate transferase (PNPT) and polyisoprenyl-phosphate hexose-1-phosphate transferase. These proteins catalyze the transfer of sugar nucleotides to undecaprenol phosphate (16 –18). This study describes the functional and biochemical characterization of the putative Wzy in F. tularensis LVS and the identification of specific amino acid residues that play an important role in maintaining this protein’s catalytic function. Two genes in the F. tularensis LVS O-antigen gene cluster encode proteins whose high degree of sequence similarity to Wzy and Wzx suggests that O-antigen is transported and polymerized via the wzy-dependent pathway
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