Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy).
Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10-13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and beta-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.
Daniels
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Department of Microbiology and Immunology, The University of Adelaide, South Australia, Australia.
Vindurampulle
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Morona
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D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D003198:Computer Simulation; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D005816:Genetic Complementation Test; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012763:Shigella flexneri / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D003062:Codon; D006602:Hexosyltransferases; C085652:O-antigen polymerase; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase