Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Mol Microbiol. 1998 Jun;28(6):1211-22. doi: 10.1046/j.1365-2958.1998.00884.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Amino Acid Sequence
  • Artificial Gene Fusion
  • Base Sequence
  • Blotting, Western
  • Codon / genetics
  • Computer Simulation
  • Cyclin-Dependent Kinases / genetics
  • Genetic Complementation Test
  • Hexosyltransferases / chemistry*
  • Hexosyltransferases / genetics*
  • Hexosyltransferases / metabolism
  • Lac Operon
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Plasmids / genetics
  • Polymerase Chain Reaction / methods
  • Protein Biosynthesis
  • Protein Structure, Secondary
  • Shigella flexneri / enzymology*
  • Shigella flexneri / genetics
  • beta-Galactosidase / metabolism

Substances

  • Codon
  • Hexosyltransferases
  • O-antigen polymerase
  • Cyclin-Dependent Kinases
  • Alkaline Phosphatase
  • beta-Galactosidase

Associated data

  • GENBANK/X71970